Infectious Diseases [2 ed.] 1071624644, 9781071624647

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Infectious Diseases [2 ed.]
 1071624644, 9781071624647

Table of contents :
Series Preface
Contents
About the Editor-in-Chief
About the Editor
Contributors
Infectious Diseases: Introduction
Introduction
Bibliography
Infectious Disease Modeling
Glossary
Definition of the Subject
Introduction
The SIR Model
Host Heterogeneity
Within-Host Dynamics
Multilevel Models
Future Directions
Bibliography
Primary Literature
Books and Reviews
Biological Controls and Standards for the Study and Control of Infectious Diseases
Glossary
Definition and Importance of Subject
Introduction
Emerging Infections: Immune Responses in SARS2 Vaccine Trials
Diagnostic Detection Methods: Nucleic Acid Detection and Hepatitis C
Potency of Vaccines: Inactivated Polio Vaccine (IPV)
Preparation and Establishment of International Standards
Identification of the Project
Approval of the Project by WHO
Starting Material
Filling the Vials
The Collaborative Study: Participating Laboratories
The Collaborative Study: Study Samples
The Collaborative Study: The Assays
The Collaborative Study: The Report
Future Directions
Bibliography
Next-Generation Sequencing in the Study of Infectious Diseases
Glossary
Definition of the Subject
Introduction
Sample Preprocessing
Library Preparation and Sequencing
Bioinformatics Analyses: Viroinformatics
Virus-Specific Databases
Obtaining Viral Sequences
Virome Discovery
Phylogenetic Analysis
Conclusions
Future Directions
Bibliography
Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics
Glossary
Definition of the Subject and Its Importance
Importance of Rapid Diagnosis of Infectious Diseases
Introduction
Classical Culture-Based Diagnostics
Antibody-Based Diagnostics
Molecular Diagnostics
Limitations of Classical and Conventional Diagnostics
Spectroscopy-Based Diagnostics
Infrared Spectroscopy
Raman Spectroscopy
SERS
Future Directions
Bibliography
Primary Literature
Books and Reviews
Syndromic Surveillance of Infectious Diseases
Glossary Terms
Definition of the Subject
Introduction
Data Retrieval and Analysis in Syndromic Surveillance
Applications of Syndromic Surveillance
Continuous Disease Surveillance
Active Case Finding Using Syndromic Surveillance
Situational Awareness
Detection of Infectious Diseases Among Specific Populations
Mass Gatherings
Migrant Populations
Web-Based Developments
Conclusions
Future Directions
Bibliography
Antibiotics for Emerging Pathogens
Glossary
Definition of the Subject
Introduction
Factors Contributing to Increased Antibiotic Resistance
Mechanisms by Which Microorganisms Develop Resistance to Antibiotics
Emerging Targets of Antibiotic Molecules
Microbial Fatty Acid Biosynthesis Inhibition
Isoprenoid Biosynthesis Inhibition
Cell Wall Biosynthesis Inhibition
Emerging Chemical Classes of Antibiotic Molecules
Peptidic Antibiotics
Polyketide Antibiotics
Trojan Horse Antibiotics
Future Directions
Acknowledgments
Bibliography
Climate Change Effects on Infectious Diseases
Glossary
Definition of the Subject and Its Importance
Introduction
Weather, Climate, and Disease
Spatial
Temporal-Seasonal
Temporal-Interannual
Intensity
Climate Change and Disease
Effects on Pathogens
Effects on Hosts
Effects on Vectors
Effects on Epidemiological Dynamics
Indirect Effects
Other Drivers of Disease
Climate Change and Disease in Wildlife
Wildlife Disease as a Cause of Endangerment
Wildlife Disease as a Source of Infections for Humans and Livestock
How Climate Change Can Influence Wildlife Disease
Dependency of Disease on Climate: The Example of Chytridiomycosis in Amphibians
Evidence of Climate Change´s Impact on Disease
Bluetongue
Malaria
Future Directions
Bibliography
Primary Literature
Books and Reviews
Malaria Vaccines
Glossary
Definition of the Subject
Introduction
The Malaria Life Cycle
Pathogenesis and Disease
Epidemiology
Immunity
Early Malaria Vaccines
Obstacles to Malaria Vaccines
Preerythrocytic Vaccines
Blood Stage Vaccines
Placental Malaria Vaccines
Transmission-Blocking Vaccines
Multistage, Multi-Antigen Vaccines
Whole-Organism Vaccines
Future Directions
Acknowledgments
Bibliography
Primary Literature
Books and Reviews
Sustainable Radical Cure of the Latent Malarias
Glossary
Definition of the Subject
Introduction
Malaria
Disease
Treatment
The 8-Aminoquinolines
Plasmochin
Primaquine
Tafenoquine
G6PD Deficiency as a Chemotherapeutics Problem
Military Medicine
Repurposed Tafenoquine
Beneficiaries
Technological Imperatives
Perceptions of the Malaria Problem
Future Directions
Conclusions
Bibliography
Further Reading
Waterborne Plant Viruses of Importance in Agriculture
Glossary
Definition of the Subject
Introduction
Virus Groups Transmitted Through Terrestrial and Aquatic Environments
Family Tombusviridae
Family Virgaviridae
Family Bromoviridae
Family Alphaflexiviridae
Family Solemoviridae
Future Directions
Bibliography
Water-, Sanitation-, and Hygiene-Related Diseases
Glossary and Acronyms
Definition of the Subject
Introduction
Global Guidance for the Control and Elimination of WASH-Related Diseases
Neglected Tropical Diseases
Mosquito-Borne Diseases
Progress Against Global Disease Control Targets
Bacteria: Diarrheal Diseases - Cholera and Typhoid
Control and Elimination Strategies
Treatment
Progress Towards Control and Elimination
Bacteria: Trachoma
Treatment and Elimination Strategies
Progress Towards Control and Elimination
Helminth: Guinea Worm (Dracunculiasis)
Control and Elimination Strategies
Treatment
Progress Towards Eradication
Helminth: Lymphatic Filariasis
Control and Elimination Strategies
Treatment and Morbidity Management
Progress Towards Control and Elimination
Helminth: Onchocerciasis (River Blindness)
Control and Elimination Strategies
Treatment
Progress Towards Control and Elimination
Helminth: Schistosomiasis
Control and Elimination Strategies
Treatment
Progress Towards Control and Elimination
Helminth: Soil Transmitted Helminths (Ascaris, Trichuris, Hookworm, and Strongyloides Infections)
Control and Elimination Strategies
Treatment
Progress Towards Control and Elimination
Protozoa: Malaria
Control and Elimination Strategies
Treatment
Current Prophylactics
Bed Nets
Vector Control
Progress Towards Elimination
Virus: Dengue
Control and Prevention Strategies
Treatment
Progress Towards Control and Elimination
Virus: West Nile Fever
Control Strategies
Treatment
Progress Towards Control
Virus: Yellow Fever
Control and Elimination Strategies
Treatment
Progress Towards Control and Elimination
Future Directions
Bibliography
HIV/AIDS Global Epidemic
Glossary
Definition of the Problem
Introduction
Basic Virology of HIV
HIV and the Cause of AIDS
Epidemiology of the Global HIV Pandemic
Eastern and Southern Africa
Asia and Pacific
Eastern Europe and Central Asia
The Caribbean
Latin America
Western and Central Europe and North America
Middle East and North Africa
Prevention of HIV
WHO´s 90-90-90 Recommendations
Genetic Diversity of HIV
Treatment
HIV Reservoirs and Cure Research
HIV Vaccine Strategies
Future Directions
Bibliography
Polio and Its Epidemiology
Glossary
A Brief Definition of Polio and Its Importance
Introduction
The Epidemiology of Polio
Structural and Functional Organization of the Poliovirus Genome
Poliovirus Infections in Cells, Individuals, and Populations
Poliovirus Infections at the Level of the Host Cell
Poliovirus Infections at the Level of the Individual Host
Poliovirus Infections in Populations
Future Directions: The Endgame Stage of Eradication and Sustainability Afterward
Concluding Statement
Bibliography
Tuberculosis, Epidemiology of
Glossary
Definition of the Subject and Its Importance
Introduction
Basic and Descriptive Epidemiology of Tuberculosis
Epidemiological Indicators
Etiologic Epidemiology of Tuberculosis
Natural History
Exposure
Infection
Disease
Mortality
Tuberculosis/HIV Coinfection
Drug-Resistant Tuberculosis
Control of Tuberculosis
Global Epidemiological Situation
Incidence
Prevalence
Mortality
Case Notifications
Treatment Outcomes
Future Directions
Bibliography
Paleopathology of Infectious Human Diseases
Glossary
Definition and Importance of the Subject
Introductory Discussion
M. tuberculosis
Future Directions
Bibliography
Avian Oncogenic and Immunosuppressive Viruses
Glossary
Definition of the Subject
Avian Tumors: Viral and Non-viral Etiology
The Avian Oncogenic Viruses
The Immunosuppressive Viruses and Their Impact in Commercial Flocks
The Diversity of Clinical Signs Prior to Tumor Formation
Differential Diagnosis Between Tumors Caused by Avian Oncogenic Viruses
Control by Vaccination
The Oncogene Carried by MDV
Multiple Virus Infections: Clinical Manifestations
Multiple Virus Infections as Revealed by Differential Molecular Diagnosis
Multiple Virus Infections: Intracellular Consequences
Molecular Recombination Between Viruses of the Same Species
Molecular Interactions Between RNA Viruses
Molecular Interactions Between DNA Viruses
Molecular Interactions Between DNA and RNA Viruses
Herpes and Retrovirus Interactions In Vitro and In Vivo
In Vitro Studies
In Vivo Studies: The Complexity of In Vivo Systems Versus In Vitro Systems
Fowlpox Viruses and Retroviruses
Summary and Future Directions
Bibliography
Salmonella in Poultry and Other Birds
Glossary
Definition of the Subject
Introduction
History
Description
An Overview of Salmonella Typing: Taxonomy and Public Health Perspectives
The Salmonella Genome
Nomenclature and Serotyping
Detection of Salmonella in Clinical Samples, Animal Feces, and Environmental Samples
Origin of Salmonella Infection
Salmonella in Laying Hen Flocks
Sampling for Salmonella in Laying Houses
Matrix of Sampling: Feces, Dust
Salmonella Enteritidis in Chicks
Salmonella Enteritidis in Adult Chickens
Salmonella in Table Eggs and the Effect of Temperature
Salmonella enterica in Chicken Meat
Control of Salmonella in Poultry
From Poultry Salmonellosis to Human Salmonellosis: This Is a Food-Borne Human Disease Rather than a Poultry Disease
Salmonella Prevalence in Israel Poultry Farms
Salmonella in Wild Birds of Israel
Pathogenesis of Nontyphoidal Salmonella
Clinical and Pathological Signs of Nontyphoidal Salmonella in Birds
Fowl Typhoid
Salmonella Genetics
Core Genome and Mobile Elements
Antimicrobial Resistance
Genotypic and Sequence-Based Typing
Molecular Mechanisms of Infection, Cellular Pathogenesis, and Virulence Factors
Future Directions: Prospective Approaches in Salmonella Control
Bibliography
Further Reading: Books and Reviews
Additional Information for Molecular Mechanisms of Infection Found in:
Campylobacter in Poultry and Other Birds
Glossary
Definition of the Subject
Introduction
History and Classification
Growth Requirements
Incidence and Distribution
Horizontal Transmission
Vertical Transmission
Incubation Period
Clinical Signs and Pathology
Pathogenesis
Diagnosis
Culture-Based Isolation and Detection Methods
Immunological Methods for the Detection of Campylobacter
Nucleic Acid-Based Diagnostic Methods
Campylobacter in Israel Poultry Farms
Intervention Strategies, Prospective Approaches, and Future Directions
Bibliography
Newcastle Disease Virus
Glossary
A Brief Definition of NDV and Its Importance
Introduction
Structural and Functional Organization of the NDV Genome
Phylogenetic Classification
NDV Infection at the Level of the Host
Viral Infection
Active Immunity
Passive Immunity
NDV Infection in Bird Populations
NDV Diagnosis
NDV Prevention
Vaccines and Vaccination
Biosecurity and Eradication
NDV as an Oncolytic Agent
Future Directions
Bibliography
Vector-Borne Diseases in Ruminants
Glossary
Definition of the Subject
Introduction
Tick-Borne Diseases
Introduction
Protozoan Parasites
Bacterial Pathogens
Viral Pathogens
Crimean-Congo Hemorrhagic Fever (CCHF)
Mosquito-Borne Diseases
Introduction
Rift Valley Fever (RVF)
Bovine Ephemeral Fever (BEF)
Culicoides (Biting Midges)-Borne Diseases
Introduction
Bluetongue
Epizootic Hemorrhagic Disease (EHD)
Simbu Serogroup Viruses
Tsetse Flies
Introduction
Trypanosomosis
Arthropod-Borne Helminthic Infections
Introduction
Dicrocoelium Spp.
Parafilaria Bovicola
Thelazia Spp.
Prevention and Control of Vector-Borne Disease in Ruminants
Conclusions and Future Directions
Bibliography
Index

Citation preview

Encyclopedia of Sustainability Science and Technology Series Editor-in-Chief: Robert A. Meyers

Lester M. Shulman Editor

Infectious Diseases Second Edition

A Volume in the Encyclopedia of Sustainability Science and Technology, Second Edition

Encyclopedia of Sustainability Science and Technology Series Editor-in-Chief Robert A. Meyers

The Encyclopedia of Sustainability Science and Technology series (ESST) addresses the grand challenge for science and engineering today. It provides unprecedented, peer-reviewed coverage in more than 600 separate articles comprising 20 topical volumes, incorporating many updates from the first edition as well as new articles. ESST establishes a foundation for the many sustainability and policy evaluations being performed in institutions worldwide. An indispensable resource for scientists and engineers in developing new technologies and for applying existing technologies to sustainability, the Encyclopedia of Sustainability Science and Technology series is presented at the university and professional level needed for scientists, engineers, and their students to support real progress in sustainability science and technology. Although the emphasis is on science and technology rather than policy, the Encyclopedia of Sustainability Science and Technology series is also a comprehensive and authoritative resource for policy makers who want to understand the scope of research and development and how these bottom-up innovations map on to the sustainability challenge.

Lester M. Shulman Editor

Infectious Diseases Second Edition A Volume in the Encyclopedia of Sustainability Science and Technology, Second Edition

With 91 Figures and 29 Tables

Editor Lester M. Shulman Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine Tel Aviv University Tel Aviv, Israel Central Virology Laboratory, Laboratory of Environmental Virology at Sheba Medical Center; Retired Public Health Services, Israel Ministry of Health Tel Aviv, Israel

ISSN 2629-2378 ISSN 2629-2386 (electronic) Encyclopedia of Sustainability Science and Technology Series ISBN 978-1-0716-2462-3 ISBN 978-1-0716-2463-0 (eBook) https://doi.org/10.1007/978-1-0716-2463-0 © Springer Science+Business Media, LLC, part of Springer Nature 2013, 2023 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Series Preface

Our nearly 1000-member team recognizes that all elements of sustainability science and technology continue to advance as does our understanding of the needs for water, clean air, food, energy, and health and the relation of every single aspect of this vast body of knowledge to climate change. Our Encyclopedia content is at a level for university students, professors, engineers, and other practicing professionals. It is gratifying for our team to note that our online First Edition has been heavily utilized as evidenced by over 500,000 downloads which of course is in addition to scientists’ utilization of the print Encyclopedia and print single volumes. Now we are pleased to have a Living Reference available on-line, which assures the sustainability community that we are providing the latest peer-reviewed content covering the science and technology of the sustainability of the earth and we are also publishing the content as a series of individual topical books for easy use by those with an interest in particular subjects. Our team covers the physical, chemical, and biological processes and measurements that underlie the earth system including pollution and remediation, health and climate change and we comprehensively cover every energy and environment technology as well as all types of food production, water, transportation, and the sustainable built environment. Our team members include two Nobel Prize winners (McFaddden and Fischlin), two former Directors of the NSF (Colwell and Killeen), and the Chief Scientist of the Rocky Mountain Institute (Lovins). And our more than 50 eminent section editors and now book editors assure the quality of our selected authors and their review presentations. The extent of our coverage clearly sets our project apart from other publications which now exist, both in extent and depth. In fact, current compendia of the science and technology of several of these topics do not presently exist, and yet the content is crucial to any evaluation and planning for the sustainability of the earth. It is important to note that the emphasis of our project is on science and technology and not on policy and positions. Rather, policy makers will use our presentations to evaluate sustainability options. Vital scientific issues include: human and animal ecological support systems; energy supply and effects; the planet’s climate system; systems of agriculture, industry, forestry, and fisheries and the ocean, fresh water and human communities, waste disposal, transportation, and the built environment as well as infectious diseases and the various systems on which they depend; and the balance of all of these with sustainability. In this context, sustainability is a characteristic of a v

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Series Preface

process or state that can be maintained at a certain level indefinitely even as global population increases from 7.7 billion in 2021 to 9.7 billion by 2050 and 11 billion by around 2100 (UN Estimate). This population growth is expected mostly in Africa and India and Southeast Asia, and the hope for decreasing malnutrition coupled with better diet, decreasing poverty, and an increase in wealth implies something like a 50% increase in food demand by as early as 2030. But the UN forecasts that if recent trends continue, the number of people affected by hunger will surpass 840 million by 2030. If the world economy grows at an average of a nominal 3% per annum, there will be more than a doubling in size by 2050. This means that global production of concrete, steel, and other metals as well as plastics, which presently make up nearly a third of all greenhouse emissions, will also likely double. The building of new or expanded cities, bridges, roads, dams, and manufacturing plants and the construction of sea walls and pumping capacity for climate adaptation as well as air conditioning will be required. This means that there are going to be some real problems in reducing or more likely even slowing the growth of greenhouse gas emissions as well as for energy, agriculture, and water. It is increasingly clear that conflicting demands among biofuels, food crops, forestation, and environmental protection will be difficult to reconcile. The “green revolution” was heavily dependent on fertilizers which are manufactured using increasingly expensive and diminishing reserves of fossil fuels. In addition, about 70% of available freshwater is used for agriculture. Clearly, many natural resources will either become depleted or scarce relative to population. All of this highlights the need for the technology and science presented in this book series as well as truly major increases in research and development. Palm Desert, CA, USA December 2022

Robert A. Meyers, Ph.D. Editor-in-Chief

Contents

Infectious Diseases: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Lester M. Shulman

1

Infectious Disease Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angela R. McLean

9

Biological Controls and Standards for the Study and Control of Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Philip David Minor

21

Next-Generation Sequencing in the Study of Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neta S. Zuckerman and Lester M. Shulman

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Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jeremy D. Driskell and Ralph A. Tripp

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Syndromic Surveillance of Infectious Diseases . . . . . . . . . . . . . . . . Aharona Glatman-Freedman and Zalman Kaufman

75

Antibiotics for Emerging Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . Vinayak Agarwal and Satish K. Nair

83

Climate Change Effects on Infectious Diseases . . . . . . . . . . . . . . . . Matthew Baylis and Claire Risley

99

Malaria Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Matthew B. Laurens and Christopher V. Plowe Sustainable Radical Cure of the Latent Malarias . . . . . . . . . . . . . . 155 J. Kevin Baird Waterborne Plant Viruses of Importance in Agriculture . . . . . . . . 175 Walter Q. Betancourt Water-, Sanitation-, and Hygiene-Related Diseases . . . . . . . . . . . . . 189 Y. Velleman, L. Blair, F. Fleming, and A. Fenwick HIV/AIDS Global Epidemic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Phyllis J. Kanki and Catherine K. Koofhethile vii

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Contents

Polio and Its Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Lester M. Shulman Tuberculosis, Epidemiology of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Giovanni Sotgiu, Matteo Zignol, and Mario C. Raviglione Paleopathology of Infectious Human Diseases . . . . . . . . . . . . . . . . . 347 M. Spigelman Avian Oncogenic and Immunosuppressive Viruses . . . . . . . . . . . . . 363 Irit Davidson Salmonella in Poultry and Other Birds . . . . . . . . . . . . . . . . . . . . . . 383 Avishai Lublin and Yigal Farnoushi Campylobacter in Poultry and Other Birds . . . . . . . . . . . . . . . . . . . 417 Avishai Lublin and Yigal Farnoushi Newcastle Disease Virus Ruth Haddas

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

Vector-Borne Diseases in Ruminants . . . . . . . . . . . . . . . . . . . . . . . . 441 Adi Behar, Daniel Yasur-Landau, and Monica Leszkowicz-Mazuz Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

About the Editor-in-Chief

Robert A. Meyers Dr. Meyers was manager of Energy and Environmental Projects at TRW (now Northrop Grumman) in Redondo Beach, CA, and is now president of RAMTECH Limited. He is coinventor of the Gravimelt process for desulfurization and demineralization of coal for air pollution and water pollution control and was manager of the Department of Energy project, leading to the construction and successful operation of a first-of-akind Gravimelt Process Integrated Test Plant. Dr. Meyers is the inventor of and was project manager for the DoE-sponsored Magnetohydrodynamics Seed Regeneration Project, which has resulted in the construction and successful operation of a pilot plant for production of potassium formate, a chemical utilized for plasma electricity generation and air pollution control. He also managed TRW efforts in magnetohydrodynamics electricity generating combustor and plasma channel development. Dr. Meyers managed the pilot-scale DoE project for determining the hydrodynamics of synthetic fuels. He is a coinventor of several thermo-oxidative stable polymers which have achieved commercial success such as the GE PEI, Upjohn Polyimides, and Rhone-Poulenc bismaleimide resins. He has also managed projects for photochemistry, chemical lasers, flue gas scrubbing, oil shale analysis and refining, petroleum analysis and refining, global change measurement from space satellites, analysis and mitigation (carbon dioxide and ozone), hydrometallurgical refining, soil and hazardous waste remediation, novel polymers synthesis, modeling of the economics of space transportation systems, space rigidizable structures, and chemiluminescence-based devices. ix

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About the Editor-in-Chief

Dr. Meyers is a senior member of the American Institute of Chemical Engineers, member of the American Physical Society, member of the American Chemical Society, and served on the UCLA Chemistry Department Advisory Board. He was a member of the joint USA-Russia working group on air pollution control and the EPA-sponsored Waste Reduction Institute for Scientists and Engineers. Dr. Meyers has more than 20 patents and 50 technical papers in the fields of photochemistry, pollution control, inorganic reactions, organic reactions, luminescence phenomena, and polymers. He has published in primary literature journals including Science and the Journal of the American Chemical Society and is listed in Who’s Who in America and Who’s Who in the World. Dr. Meyers’ scientific achievements have been reviewed in feature articles in the popular press in publications such as The New York Times Science Supplement and The Wall Street Journal as well as more specialized publications such as Chemical Engineering and Coal Age. A public service film was produced by the Environmental Protection Agency on Dr. Meyers’ chemical desulfurization invention for air pollution control. Dr. Meyers is the author or Editor-in-Chief of a wide range of technical books, including the Handbook of Chemical Production Processes, Handbook of Synfuels Technology, Handbook of Petroleum Refining Processes now in its 4th Edition, the Handbook of Petrochemical Production Processes (McGraw-Hill) now in a 2nd edition, and the Handbook of Energy Technology and Economics, published by John Wiley & Sons; Coal Structure, published by Academic Press; and Coal Desulfurization as well as the Coal Handbook published by Marcel Dekker. He served as chairman of the Advisory Board for A Guide to Nuclear Power Technology, published by John Wiley & Sons, which won the Association of American Publishers Award as the best book in technology and engineering. He also served as Editor-in-Chief of three editions of the Elsevier Encyclopedia of Physical Science and Technology. Most recently, Dr. Meyers serves as Editor-in-Chief of the Encyclopedia of Analytical Chemistry as well as

About the Editor-in-Chief

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Reviews in Cell Biology and Molecular Medicine and a book series of the same name both published by John Wiley & Sons. In addition, Dr. Meyers currently serves as Editor-in-Chief of two Springer Nature book series, Encyclopedia of Complexity and Systems Science Series and Encyclopedia of Sustainability Science and Technology Series.

About the Editor

Lester M. Shulman Assoc. Prof. Lester M. Shulman (BSc, Yale University; MA, Brandeis University; PhD Weizmann Institute of Science) has published 94 peer-reviewed manuscripts, 8 case reports, 12 book chapters, and 11 review articles. He conducted basic research on anti-viral and anti-cancer activities of cytokines at the Weizmann Institute of Science (1984–1989); investigated causes of acute renal failure as Head of the Nephrology Laboratory at Sheba Medical Center, Tel Hashomer, Israel (1990–1993); and then studied molecular epidemiology of viruses at the Israeli Public Health Services Central Virology Laboratory at the Sheba Medical Center, where he headed the Environmental Virology Laboratory and the Israel National Center for Viral Gastroenteritis (1993–2015). His main interest included the study of the molecular biology of enteric viruses (polioviruses; non-polio enteroviruses; persistent poliovirus infections in immunedeficient individuals, rotaviruses, noroviruses; and vaccine efficacy) and wastewater-based epidemiology (environmental surveillance for polioviruses, studying the virome during recovery of wastewater for use in agriculture and recreation). As a leading researcher in the field, he participated in international meetings establishing WHO standard guidelines for environmental surveillance for poliovirus. He taught Clinical Virology at the School of International Health and Medicine, Ben Gurion University, Beersheba, Israel (2000–2010), and Clinical Virology and Epidemiology at the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel (2008–2015).

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About the Editor

Since retirement in 2015, he has conducted annual reviews of supplementary surveillance for polioviruses conducted by each of the 53 countries in the WHO European Region for WHO/EURO (2014–2018), edited the 5th Edition of the Global Polio Laboratory manual for the WHO (Geneva, Switzerland), and participated in the UNESCO Global Water Pathogen Project, coauthoring a chapter on Polio and other Enteroviruses. He has also been a Contributing Investigator and consultant for BARD (Application of metagenomic approaches to examine the virome in recycled waters for safe and sustainable reuse in irrigation), WHO (PAM – a novel Polio Antigen Microarray assay for profiling personalized immune histories of exposure to polioviruses), and CDC (Assessment of SARS-CoV-2 Viability and Persistence in Sewage Samples across the Unites States using in vitro cell culture and molecular methods) projects. In 2022, he joined the WHO and Bill and Melinda Gates nOPV2 Project as Consultant and Technical Writer in general and for the Genetic Characterization Sub Group in particular. Assoc. Prof. Shulman is Editor of this Infectious Disease Volume, 2nd Edition, for The Springer Encyclopedia of Sustainability Science and Technology, and is author of a chapter on Polio and its Epidemiology and coauthor of a chapter on Next Generation Sequencing in the Study of Infectious Diseases in this volume.

Contributors

Vinayak Agarwal Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA J. Kevin Baird Eijkman-Oxford Clinical Research Unit, Eijkman Institute of Molecular Biology, Jakarta, Indonesia Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK Matthew Baylis LUCINDA Group, Institute of Infection and Global Health, University of Liverpool, Neston, UK Adi Behar Division of Parasitology, Kimron Veterinary Institute, Israeli Veterinary Services and Animal Health, Bet Dagan, Israel Walter Q. Betancourt Water and Energy Sustainable Technology Center, University of Arizona, Tucson, AZ, USA L. Blair Department for Programmes, SCI Foundation, London, UK Irit Davidson Avian Diseases, Kimron Veterinary Institute, Bet Dagan, Israel Jeremy D. Driskell Department of Infectious Diseases, College of Veterinary Medicine, Animal Health Research Center, University of Georgia, Athens, GA, USA Yigal Farnoushi Avian Diseases, Kimron Veterinary Institute, POB 12, Bet Dagan, Israel A. Fenwick Faculty of Medicine, School of Public Health, Imperial College London, London, UK F. Fleming Department for Monitoring, Evaluation and Research, SCI Foundation, London, UK Aharona Glatman-Freedman Israel Center for Disease Control, Israel Ministry of Health, Ramat Gan, Israel Department of Epidemiology and Preventive Medicine, School of Public Health, Tel Aviv University Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel xv

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Ruth Haddas Avian Diseases Division, Kimron Veterinary Institute, BeitDagan, Israel Phyllis J. Kanki Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Zalman Kaufman Israel Center for Disease Control, Israel Ministry of Health, Ramat Gan, Israel Catherine K. Koofhethile Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Matthew B. Laurens Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA Monica Leszkowicz-Mazuz Division of Parasitology, Kimron Veterinary Institute, Israeli Veterinary Services and Animal Health, Bet Dagan, Israel Avishai Lublin Avian Diseases, Kimron Veterinary Institute, POB 12, Bet Dagan, Israel Angela R. McLean Zoology Department, Institute of Emerging Infections, University of Oxford, Oxford, UK Philip David Minor National Institute for Biological Standards and Control (retired), St. Albans, Hertfordshire, UK Satish K. Nair Department of Biochemistry and Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Christopher V. Plowe Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA Mario C. Raviglione TB Department (STB), HIV/AIDS, TB & Malaria Cluster (HTM), World Health Organization, Geneva, Switzerland Claire Risley LUCINDA Group, Institute of Infection and Global Health, University of Liverpool, Neston, UK Lester M. Shulman Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Central Virology Laboratory, Laboratory of Environmental Virology at Sheba Medical Center; Retired, Public Health Services, Israel Ministry of Health, Tel Aviv, Israel Giovanni Sotgiu Epidemiology and Biostatistics Unit, Department of Biomedical Sciences, University of Sassari, Sassari, Italy

Contributors

Contributors

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M. Spigelman The Kuvin Center for the Study of Infectious and Tropical Diseases and Ancient DNA, Hadassah Medical School, The Hebrew University, Jerusalem, Israel Division of Infection and Immunity, Centre of Clinical Microbiology Royal Free hospital UCL London, London, UK Department of History and Archaeology, Macquarie University, Sydney, NSW, Australia Ralph A. Tripp Department of Infectious Diseases, College of Veterinary Medicine, Animal Health Research Center, University of Georgia, Athens, GA, USA Y. Velleman Department for Policy and Communications, SCI Foundation, London, UK Daniel Yasur-Landau Division of Parasitology, Kimron Veterinary Institute, Israeli Veterinary Services and Animal Health, Bet Dagan, Israel Matteo Zignol TB Department (STB), HIV/AIDS, TB & Malaria Cluster (HTM), World Health Organization, Geneva, Switzerland Neta S. Zuckerman Bioinformatics and Metagenomics, Central Virology Laboratory, Public Health Services, Israel Ministry of Health, Tel-Hashomer, Israel

Infectious Diseases: Introduction Lester M. Shulman Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Central Virology Laboratory, Laboratory of Environmental Virology at Sheba Medical Center; Retired, Public Health Services, Israel Ministry of Health, Tel Aviv, Israel

Article Outline Introduction Bibliography

Introduction Critical sustainability issues occur in a context that is exceedingly complex, constituting a dynamic, multivariable universe involving multiple social, political, economic, and cultural aspects interacting among themselves, across sectors, and with the physical and non-human living parts of the world. [1]

At first glance a volume on Infectious Diseases in an Encyclopedia of Sustainability Science and Technology (ESST) appears to be an oxymoron since our goal is to overcome problems caused by infectious diseases, not to sustain the transmissible pathogenic agents that cause the disease and interfere with the sustainability and quality of human life and its continued survival within the Earth’s closed ecosystem. Sustainability refers to an interaction of a population and the carrying capacity of its environment that results in a stable but dynamic equilibrium [1] that constantly needs to readjust as both are continually changing. Therefore, sustainability in the context of this volume refers to the goal of identifying and characterizing the pathogen or pathogens causing the

infectious disease, and then carrying out health policies to control the disease caused by the pathogen, eliminate the disease caused by the pathogen, eliminate the infection caused by the pathogen, eradicate the pathogen, and finally to cause the extinction of the pathogen (see Table 1 for a definition of these terms). Thus, when we present our current state of knowledge about specific infectious diseases and the pathogens that cause them, we are addressing the needs of health care providers, policy makers, scientists, engineers, and the general public to make informed decisions. When studying infectious diseases, we need to consider the presence and transmission of the pathogenic agent within subregions of countries, entire countries, larger geographical regions, and all of the regions of the world during pandemics, since infectious disease burdens vary [2]. Examples of the ability of infectious diseases in one country to affect neighboring countries or be spread by international travel is covered in a number of chapters in this volume. Wood et al., concluded from their study of the drivers of infectious diseases in 60 intermediate sized countries, that “infectious disease burden correlate with human demography and economics, whereas associations with environmental and biotic factors was less clear.” How can we measure the impact of infectious diseases? Disability adjusted life years (DALYs) is one commonly used method to measure the impact of a human disease by adjusting the number of years lived with a disease by the severity of the disease and the number of years lost to premature mortality [3]. Unfortunately, the data needed to calculate DALYs is frequently incomplete [2, 4]. The causes of death are usually classified into three broad categories: Class 1, Infections diseases and maternal, perinatal, and nutritional conditions; Class 2, non-communicable diseases; and Class 3, injuries [4]. Thus the reports for deaths from Class 1 causes is a combined value including deaths due to sub-Class 1A, infectious

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_1104 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2022 https://doi.org/10.1007/978-1-4939-2493-6_1104-1

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Infectious Diseases: Introduction

Infectious Diseases: Introduction, Table 1 Stages in disease elimination. WR Dowdle [16, 17] Term Control of the disease

Elimination of the disease

Elimination of the infection

Eradication of the pathogen Extinction of the pathogen Reemergence

Definition “The reduction of disease incidence, prevalence, morbidity or mortality to a locally acceptable level as a result of deliberate efforts. Continued intervention measures are required to maintain the reduction.” Control may or may not be related to global targets set by the WHO “Reduction to zero of the incidence of a specified disease in a defined geographical area as a result of deliberate efforts. Continued intervention measures are required” “Reduction to zero of the incidence of infection caused by a specific agent in a defined geographical area as a result of deliberate efforts. Continued measures to prevent reestablishment of transmission are required” and elimination must be validated (Table 2) “Permanent reduction to zero of the worldwide incidence of infection caused by a specific agent as a result of deliberate efforts. Intervention measures are no longer needed. “This process needs to be certified “The specific infectious agent no longer exists in nature or in the laboratory.” Extinction may occur as a result of a public health campaign or through a natural event The reintroduction of an infectious pathogen from an existing external reservoir or from a laboratory (stocks or de novo synthesized simple genomes; See Racaniello et al. [18]) into a geographic area where it had previously been eliminated or eradicated

and parasitic diseases and sub-Class 1B, respiratory infections, with death due to sub-Class 1C, maternal conditions; sub-Class 1D, perinatal conditions; and sub-Class 1E, nutritional conditions, making it difficult to calculate the burden of infectious diseases alone. See Michaud [4] for a listing of the infectious diseases included in sub-Classes 1A and 1B. In 2001, it was estimated that infectious diseases accounted for 26% of the 56.2 million people who died and that most of these deaths due to infectious diseases were in developing regions [4]. Economic factors are important for determining disease burdens [2, 5]. Five of the ten leading causes of death in low- and middle-income countries were lower respiratory infections; HIV/AIDS (see the chapter on ▶ “HIV/AIDS Global Epidemic” by Phyllis Kanki and C. Koofhethiule); diarrheal diseases; tuberculosis (see the chapters on ▶ “Tuberculosis, Epidemiology of” by G. Sotgiu et al., and ▶ “Paleopathology of

Example Diarrheal diseases

Neonatal tetanus

Measles, poliomyelitis

Smallpox (humans); Wild type 2 and 3 polioviruses (humans); and rinderpest (ruminants) None

Wild and circulating vaccine-derived polioviruses

Infectious Human Diseases” by M. Spigelman); and malaria (see chapters on ▶ “Malaria Vaccines” by M. Laurens et al., ▶ “Water-, Sanitation-, and Hygiene-Related Diseases” by Velleman et al., and ▶ “Sustainable Radical Cure of the Latent Malarias” by Kevin Baird). In 2001, sub-Class 1A and 1B infectious diseases were responsible for 413 million lost DALYs [4]. Ranking of infectious diseases among the different causes of death was projected to change considerably between 1990 and 2020 [4]. The infectious disease burden for populations living in countries or within countries in extreme poverty is particularly dire compared to populations in high-income countries [5]. “It is essential to include the burden of nonfatal health outcomes in the global assessment of health problems because it notoriously shifts the ranking of health policy” [4]. An added confounding factor is that different infectious diseases are studied at different levels of intensity. An interesting analysis by Furuse [6] measured the burden-adjusted

Infectious Diseases: Introduction Infectious Diseases: Introduction, Table 2 Principles for standard operating procedures for validation of elimination of an infectious disease and/or eradication of a pathogen [17]

1 2 3 4 5

Principles contained in the Standard Operating Procedures Preparation of dossiers for validation, verification, or certification Submission and assessment of dossiers for validation, verification, or certification Feedback on dossiers for validation, verification, or certification Acknowledgement of validation, verification, or certification Activities after validation, verification, or certification (post-elimination surveillance for the disease according to its epidemiological characteristics

research intensity for different diseases (e.g., the number of publications on a specific disease) to determine which infectious diseases were neglected by researchers. The explosion of prepublications and published articles addressing the current zoonotic COVID-19/SARS 2 virus caused pandemic was not included in this study. The pandemic has caused a major revision in the ranking of health policy, has had tremendous economic effects due to closures and shelter in place policies in many countries, and will have additional impact as long-term effects after recovery from infection become apparent. One subset of infectious diseases is vaccine preventable infectious diseases. In 2018, 700,000 children died from vaccine-preventable infectious diseases out of the 5.3 million children under the age of 5 years who were estimated to have died from all causes [7]. One of the major issues contributing to this was economic, e.g., the cost and availability of vaccines rather than vaccine acceptance. Economics factors also disproportionately affect disease burden where effective treatments are available (for example, see chapter ▶ “Sustainable Radical Cure of the Latent Malarias” by Kevin Baird). Vaccines and available treatments have already substantially reduced DALYs for a number of infectious diseases [5]. These diseases include diarrheal

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diseases, lower respiratory infections, malaria, polio, and HIV/AIDS. Zoonotic infectious diseases are diseases transferred from non-human animal reservoirs to humans. Zoonotic infectious diseases by definition affect both humans and animals. For zoonotic diseases where one of the animal hosts is livestock, the burden to human life expressed in DALYs should also contain a component related to the animal disease burden. The suggested new measure, the zDALY, includes a measure of time lost to society by making it the equivalent of time needed to earn the income to recover the financial loss [8]. This calculation takes into account the disproportionate burden for the same disease in low- and high-income countries. The global burden of animal diseases (GBAD) can be measured in terms of the economic impact and/or decrease in productivity of the animal after infection with the pathogen [9, 10]. This, in term, is a function of disease frequency, morbidity and mortality and their effect on efforts to respond to the disease and any effects on human health. Key factors for measurement GBAD include disease classification (including case definition and its applicability in the field), data collection, disease losses, animal expenditure, sustainability and equitability (determining impacts in low -income countries comparable to impacts in high-income countries), and the need to relate to these classifications in a socioeconomic context. Disproportionality in effects between high- and low-income countries, the socioeconomic context, can be partially accounted for if the measure of time lost to society is made equivalent to time needed to earn the income to recover the financial loss (see zDALY, above). In 2017, Huntington et al., [11] estimated that the animal health products market was 24 Billion US dollars or approximately 2.5% of the 7.8 trillion US dollars spent globally for human health products. In their analysis they emphasized the disproportional importance of livestock to the small-holder farmer, stating that “for these vulnerable people, poor animal health leads directly to poverty and malnutrition, exposure to zoonotic disease risk, poor health, and reduced welfare.”

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The chapter “Infections Disease Modeling” by Angela McLean introduces the readers to methods for mathematically describing host to host transmission of infectious diseases and the pathogens that cause them, the course of infection within a host, and models that take into account both. One important indicator for the ability of an infectious agent to spread is the basic reproductive number, R0, which is the number of secondary infections that result during the time course of an infection assuming that the population in contact with the infected host is naive (has no immunity to the pathogen through previous natural infection or through deliberate medical intervention (vaccination or preventive treatments). If R0 is below 1, transmission of the disease winds down, whereas the higher it is above 1, the faster it will spread throughout a naïve population. The models take into account changes in the immune status within an infected host and of the population as disease spreads and/or as preventive measures are taken. It is very important to clearly define and understand what epidemiological question is being asked when fitting data into a model designed to provide answer for designing evidence-based intervention strategies. It is critical to have the tools that can provide the answer to determine the number of uninfected hosts in a population the number of asymptomatic infected hosts that do not show or that do not yet show symptoms, and the number of infected individuals who develop symptoms Two examples were knowledge about the number of asymptomatic infections was/is critical in relation to enforcing quarantines to break the chain of transmission of a pathogen, especially when encountering a newly emerging pathogen with many unknown characteristics, are the failure of a quarantine imposed in New York in 1916 in response to one of the first major outbreaks of poliomyelitis [12] in the US and the current pandemic COVID-19 today where for both, many asymptomatic and highly infectious cases were missed. These illustrate the importance to knowing the number of infected individuals capable of transmitting an infectious disease. This information can be obtained providing that there are sensitive tools for detection of the pathogen that can be employed to test

Infectious Diseases: Introduction

individuals in a representative portion of the population or the entire population of individuals for example through use of environmental surveillance or syndrome-based surveillance. The next chapters describe some of the methods available for obtaining the epidemiological information required for modeling infectious diseases. New technology is discussed in chapters on ▶ “Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics” by Driskell and Tripp, ▶ “Syndromic Surveillance of Infectious Diseases” by A. Freedman and Z. Kaufman, and ▶ “Next-Generation Sequencing in the Study of Infectious Diseases” by N. Zuckerman and L. Shulman. The chapter on ▶ “Next-Generation Sequencing in the Study of Infectious Diseases” describes the use of next generation sequencing (NGS) to study infectious viral diseases. This chapter describes the latest technology for obtaining long partial or entire genomic sequences of viruses (WGS) from viruses isolated from clinical or environmental samples or directly from nucleic acids extracted from these samples. Millions of short sequences generated by NGS sequence platforms are assembled into longer sequences using bioinformatic programs that assemble overlapping short sequences into longer sequences de novo or by comparison with viral sequences previously reported to public sequence databases. Complete genomes enrich molecular epidemiological analysis of evolutionary changes that occur during viral infections of a single cell, a single organism, or during the chain of organism-to organism endemic transmissions or transmission during outbreaks. De novo assembly can identify novel viruses since it is estimated that less than 1% of the viruses on our planed have been identified (for examples see reviews by Virgen [13], PaezEspino et al. [14], and Meier-Kolthoff [15]). This group of chapters concludes with a chapter on ▶ “Biological Controls and Standards for the Study and Control of Infectious Diseases” by P. Minor, since comparison of results from different laboratories requires development, verification, and use of universally available standards, international standards, and controls. “Accurate measurement is required for clinical studies, and

Infectious Diseases: Introduction

is likely to involve a biological parameter such as infectivity that is not readily expressed in physical terms. Where this is the case a biologically active preparation may be produced to act as the reference to be included in assays. Production of the highest order, primary biological references of this type is the remit of WHO and they are used to calibrate secondary references in an established hierarchy of measurement.” Surveillance is important for detecting the presence of infectious agents, for monitoring transmission of the infectious agents, and for monitoring the effectiveness of intervention in individuals and in populations. It is possible to determine the health status of an individual when a clinical sample from the individual is tested. Environmental surveillance is an example of a cost-effective method for studying whether there are infected individuals in a population, however it does not identify the infected host or hosts. Examples of environmental surveillance are presented in the chapter ▶ “Polio and Its Epidemiology” by L. Shulman. The chapter ▶ “Syndromic Surveillance of Infectious Diseases” by A. Freedman and Z. Kaufman, describes a more indirect population-targeted surveillance method based on detecting pathogen-caused changes rather than the pathogen itself. Syndrome surveillance uses medical data from different sources to monitor disease trends and to detect disease outbreaks when upward shifts in number and/or frequency of “events” exceed baseline levels. The data (specific clinical data, nonspecific clinical data, administrative data, and web-based data) may be manually or automatically retrieved in real-time, near real time, or retrospectively, from electronic databases or with considerably more effort from non-electronic sources. The collected data is then screened using selected syndromes, diagnoses, or key terms. Results may be used (1) for continuous Disease Surveillance, (2) active case finding, (3) situational awareness (following the course and progress of an outbreak), and (4) detection of infectious diseases among specific populations or sub-populations. Safe reuse of water recovered from sewage for recreational purposes and for agriculture is assuming increasing importance for

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sustainability as natural sources of water for these purposes diminish. The chapter, by W. Betancourt ▶ “Waterborne Plant Viruses of Importance in Agriculture” describes the use of environmental surveillance to detect viruses of agricultural importance in water recovered from sewage by wastewater treatment plants and to measure the reduction in titers during the purification process. Interestingly, the immediate source of the pathogens may be either the agricultural hosts of the pathogens and/or the humans that consume the agricultural products. The reader may find additional information on survivability of pathogens in wastewater and their removal by wastewater treatment plants on line at https://www.waterpathogens.org/, the home of the Global Water Pathogens Project (GWPP), “a knowledge resource on pathogens supporting sanitation and safe water and promoting quantitative information via monitoring of sewage, fecal sludges and freshwaters to inform public health measures.” Environmental surveillance for human poliovirus is covered in the chapter on ▶ “Polio and Its Epidemiology.” Environmental surveillance works best for catchment populations served by centralized sewage systems and for pathogens that are excreted into sewage. One of the main advantages of environmental surveillance is that test results indicate whether there are infected individuals in the catchment population that is upstream of the collection site. Environmental surveillance is cost- and labor-effective compared to testing all individuals, or only symptomatic individuals, since only a single assay is needed to detect whether one or more individuals in the population are infected with a given pathogen. Disadvantages, in addition to not indicating which individual or individuals are infected, are the many environmental factors that need to be taken into account between the point of excretion and collection and the quality of laboratory procedures for recovery and identification. Negative results may indicate that the pathogen is absent or present at levels below the limits of detection and quantification. A number of chapters include detailed characterizations of specific pathogens, describing how they

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reproduce, how they are transmitted, and what means are available to control, eliminate, or eradicate them. Infectious diseases that affect livestock and plants used for food are discussed in addition to infectious disease agents infecting humans since we must also consider their effect on the livestock and plants when discussing sustainability of humans in our ecosystem. Additionally, livestock may serve as hosts for pathogens that can directly infect humans or under specific rare conditions be transmitted to humans (zoonosis), and they may serve as one of the hosts for vector borne disease pathogens that include humans in their life cycle. Each chapter on specific pathogens includes a discussion on treatment and control. This is the main theme in chapter on ▶ “Antibiotics for Emerging Pathogens” by Agarwal and Nair, ▶ “Malaria Vaccines” by Laurens et al., and ▶ “Sustainable Radical Cure of the Latent Malarias,” by Kevin Baird. Antibiotics for emerging pathogens describes the use of these organic small molecules to treat infections caused by pathogenic bacteria and the development of resistance to these compounds by the microorganisms that they target. Two chapters deal with treating and preventing malarial infections. The first by Plow ▶ “Malaria Vaccines” describes the efforts to develop vaccines that would reduce the 229 million annual cases and 409,000 annual deaths caused by malaria parasites. The second by Baird, “Sustainable Radical Cure of the Latent Malarias,” describes efforts to cure latent cure malarial infections and the technical character of the obstacles and the ways and means by which sustainable approaches to overcoming them have evaded us. The chapter points out an important practical and ethical consideration, namely, the urgency and the character of its technological solution is determined in part or whole by how a particular medical problem is perceived and managed and by whom it is perceived and managed. Viral pathogens infecting humans are discussed in chapters ▶ “HIV/AIDS Global Epidemic” by Phyllis Kanki and C. Koofhethiule, ▶ “Polio and Its Epidemiology” by Lester Shulman, ▶ “Water-, Sanitation-, and Hygiene-

Infectious Diseases: Introduction

Related Diseases” by Yael Bellman et al., and ▶ “Paleopathology of Infectious Human Diseases” by Mark. Spigelman. Non-viral organisms infecting humans are discussed in chapters on ▶ “Water-, Sanitation-, and Hygiene-Related Diseases” by Yael Bellman et al.; ▶ “Tuberculosis, Epidemiology of” by Sotgiu et al., Malarial Vaccines by Mathew Laurens et al., ▶ “Sustainable Radical Cure of the Latent Malarias” by Kevin Baird, and ▶ “Paleopathology of Infectious Human Diseases” by Mark. Spigelman. HIV/AIDS, identified in the early 1980s is a global pandemic that has affected 10s of millions of people. At the end of 2019, 38 million HIVinfected individuals were alive. The chapter discusses the epidemiology of the global HIV pandemic and the disproportionally higher incidence in Eastern and Southern Africa and in Asia. Infection is lifelong. Tuberculosis is one of the opportunistic infections that can occur in people infected with HIV. Effective methods for therapy have reduced mortality, however the chapter underscores the need to develop effective methods to prevent and/or or cure infections. Some of the major difficulties that need to be overcome are the integration of viral genetic material into the human genome and the major deterioration of multiple components of the human immune system that occur as a consequence of infection. The chapter on polio starts with a physical characterization of polioviruses and the pathological effects caused by poliovirus infections most relevant to understanding the epidemiology of polio, the disease. Unlike HIV infections, poliovirus infections are of short duration, however the damage caused to motor neurons in some infections can lead to permanent lifelong paralysis and even death. The global efforts to eradicate poliomyelitis and the viral agent causing the disease under the Global Poliomyelitis Eradication Initiative declared by the World Health Assembly in 1988 are presented to indicate some of the difficulties that have hampered achieving eradication. Despite these difficulties, two of the three endemic wild serotypes, types 2 and 3, have been eradicated. One difficulty in eradication of the disease caused by these two serotypes in contradistinction to eradication of these endogenous

Infectious Diseases: Introduction

serotypes has been the relatively rare instances when progeny of types 1, 2, and/or 3, replicationcompetent, vaccine strains in “live” poliovirus vaccines regain neurovirulence during replication in vaccinees or their contacts and by themselves cause outbreaks. Future directions needed to achieve and sustain eradication and prevent reemergence include the development of a new generation of “live” polio vaccines that are just entering use. The chapter, ▶ “Tuberculosis, Epidemiology of” by G. Sotgiu et al., discusses efforts for control/eradication of tuberculosis, a disease caused by bacteria belonging to the Mycobacterium tuberculosis complex. Tuberculosis is curable by treatment by Combination chemotherapy, was used between 1995 and 2009 to successfully treat 41 million tuberculosis patients and prevent six million deaths. The emergence of drug and multi-drug resistant strains, documented since the introduction of treatment, is one major difficulty that needs to be overcome in order to transition from control to eradication. The chapter on ▶ “Paleopathology of Infectious Human Diseases” by M. Spigelman describes ancient DNA research, the use of cutting-edge technological approaches that allow us to identify the origins, extent including epidemics, and types of ancient microbial diseases (including tuberculosis) that were so important in determining the course of human history and human evolution by sequencing DNA retrieved from preserved ancient genetic material. Pathogens infecting non-human hosts are discussed in chapters on ▶ “Avian Oncogenic and Immunosuppressive Viruses” by Irit Davidson, ▶ “Newcastle Disease Virus” by Ruth Hadas., ▶ “Campylobacter in Poultry and Other Birds,” and ▶ “Salmonella in Poultry and Other Birds” by Avishai Lublin et al. and ▶ “VectorBorne Diseases in Ruminants” by Adi Bachar et al. Chapters ▶ “Avian Oncogenic and Immunosuppressive Viruses” and ▶ “Newcastle Disease Virus” describe economically significant viral pathogens that affect the poultry industry and influence its sustainability. Moreover, study of some of these viruses provides scientific insights into aspects of viral tumorigenesis that cannot ethically be tested in humans. Bacterial infections

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of poultry are covered in the chapters on ▶ “Campylobacter in Poultry and Other Birds” and ▶ “Salmonella in Poultry and Other Birds.” Avian hosts are an important reservoir for thermophilic Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli. Poultry flocks are often colonized with high numbers of the organism in their intestinal tract. Although the majority of Campylobacter species are usually harmless for poultry, it is a leading cause of food-borne gastroenteritis in humans worldwide and contaminated chicken meat is known as the primary source of human infection.” The second leading bacterial cause of gastroenteritis in humans is Salmonella. Twenty percent of world poultry products are contaminated with Salmonella. While Salmonella infections in humans are usually self-limiting, in some cases they can cause severe infections requiring antibiotic treatment and hospitalization and may even result in death. Chapter ▶ “Vector-Borne Diseases in Ruminants” covers “the ability of a pathogen to infect, replicate in and be transmitted by vector species into a new host, the genetics of both pathogen and vector, the virulence of the pathogen, immune status and genetics of the host(s) which are all crucial in determining whether an infection develops, remains, spreads or disappears and whether the host animal once infected, can circumvent the disease, becomes sick or a subclinical carrier, and if it recovers or dies.” Vector borne diseases result from interactions between an infectious agent, the host, the vectors that transmit the infectious agent, and the environment. The ability of the pathogen to infect, replicate in and be transmitted by the vector species, the genetic characteristics of the pathogen and of the host, and the immune status of the host determine whether the host becomes infected, becomes an asymptomatic or symptomatic carrier of the pathogen, and/or whether it recovers or dies. Vector borne diseases of humans are presented in the chapter on ▶ “Water-, Sanitation-, and Hygiene-Related Diseases” by Velleman et al. Vector borne diseases affecting ruminants is addressed in the chapter on ▶ “Vector-Borne Diseases in Ruminants” by Adi Bachar.

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Finally, factors, such as climate change and deforestation affect ecological balances and can affect the geographical regions where diseases occur and/or lead to emergence of new diseases as addressed in the chapter on ▶ “Climate Change Effects on Infectious Diseases” by Bayliss et al. As time passes and climate-driven change including those relating to infections disease become more obvious, public awareness of the urgent need to reverse the human contribution driving climate change before the changes become irreversible has assumed increasing urgency and increasing political and financial backing.

Infectious Diseases: Introduction

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5 years of age: Implications for COVID-19 vaccination. How can we do better? Allergy Asthma Proc 42(5):378–385 Torgerson PR, Ruegg S, Devleesschauwer B, AbelaRidder B, Havelaar AH, Shaw APM, Rushton J, Speybroeck N (2018) zDALY: an adjusted indicator to estimate the burden of zoonotic diseases. One Health 5:40–45 Rushton J, Bruce M, Bellet C, Torgerson P, Shaw A, Marsh T, Pigott D, Stone M, Pinto J, Mesenhowski S, Wood P (2018) Initiation of Global Burden of Animal Diseases programme. Lancet 392(10147): 538–540 Rushton J, Huntington B, Gilbert W, Herrero M, Torgerson PR, Shaw APM, Bruce M, Marsh TL, Pendell DL, Bernardo TM, Stacey D, Grace D, Watkins K, Bondad-Reantaso M, Devleesschauwer B, Pigott DM, Stone M, Mesenhowski S (2021) Roll-out of the Global Burden of Animal Diseases programme. Lancet 397(10279):1045–1046 Huntington B, Bernardo TM, Bondad-Reantaso M, Bruce M, Devleesschauwer B, Gilbert W, Grace D, Havelaar A, Herrero M, Marsh TL, Mesenhowski S, Pendell D, Pigott D, Shaw AP, Stacey D, Stone M, Torgerson P, Watkins K, Wieland B, Rushton J (2021) Global Burden of Animal Diseases: a novel approach to understanding and managing disease in livestock and aquaculture. Rev Sci Tech 40(2): 567–584 Williams G (2013) Paralysed with fear: the story of polio. Palgrave Macmillan, Houndsmills/Basingstoke Virgin HW (2014) The virome in mammalian physiology and disease. Cell 157(1):142–150 Paez-Espino D, Eloe-Fadrosh EA, Pavlopoulos GA, Thomas AD, Huntemann M, Mikhailova N, Rubin E, Ivanova NN, Kyrpides NC (2016) Uncovering Earth’s virome. Nature 536(7617):425–430 Meier-Kolthoff JP, Uchiyama J, Yahara H, PaezEspino D, Yahara K (2018) Investigation of recombination-intense viral groups and their genes in the Earth’s virome. Sci Rep 8(1):11496 Dowdle WR (1998) The principles of disease elimination and eradication. Bull World Health Organ 76(Suppl 2):22–25 World Health O (2016) Generic framework for control, elimination and eradication of neglected tropical diseases. World Health Organization, Geneva, p 2016 Racaniello VR, Baltimore D (1981) Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214(4523):916–919

Infectious Disease Modeling Angela R. McLean Zoology Department, Institute of Emerging Infections, University of Oxford, Oxford, UK

Article Outline Glossary Definition of the Subject Introduction The SIR Model Host Heterogeneity Within-Host Dynamics Multilevel Models Future Directions Bibliography

“mathematical epidemiology.” A growing body of work considers the spread of infection within an individual, often with a particular focus on interactions between the infectious agent and the host’s immune responses. Such models are sometimes grouped together as “within-host models.” Most recently, new models have been developed that consider host–pathogen interactions at two levels simultaneously: both withinhost dynamics and between-host transmissions. Infectious disease models vary widely in their complexity, in their attempts to refer to data from real-life infections and in their focus on problems of an applied or more fundamental nature. This entry will focus on simpler models tightly tied to data and aimed at addressing welldefined practical problems.

Introduction Glossary Basic reproductive number A summary parameter that encapsulates the infectiousness of an infectious agent circulating in a population of hosts. Host An organism that acts as the environment within which an infectious agent replicates. Infectious agent A microorganism that replicates inside another organism. Pathogen An infectious agent that damages its host. Variant One of several types of an infectious agent, often closely related to and sometimes evolved from other variants under consideration.

Definition of the Subject Infectious disease models are mathematical descriptions of the spread of infection. The majority of infectious disease models consider the spread of infection from one host to another and are sometimes grouped together as

Why is it that smallpox was eradicated in 1979 [1] but measles, once scheduled for eradication by the year 2000, still kills over a hundred thousand children each year [2]? Both diseases can be prevented with cheap, safe, and effective vaccines which probably induce lifelong immunity, and neither virus has an environmental or animal reservoir. One way to address this question is to consider the comparative ease of spread of the two infections. A useful parameter that summarizes this ease of spread is the “basic reproductive number” always denoted as R0. The definition of the basic reproductive number is the number of secondary infections caused during the entire duration of one infection if all contacts are susceptible (i.e., can be infected). The concept has widespread currency in the literature on infectious disease models with varying degrees of affection [3]. There is no question that it has been a useful, simple rule of thumb for characterizing how easily an infection can spread [4]. Furthermore, the simplest of calculations relate R0 to the degree of intervention

© Springer Science+Business Media, LLC 2012 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_539 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media LLC, 2012 https://doi.org/10.1007/978-1-4419-0851-3_539

9

10

Infectious Disease Modeling

needed to bring an infection under control and, eventually, eradicate it. The relationship between the basic reproductive number and disease control arises from the simple fact that if each infectious person causes less than one secondary case, then the number of infections must fall. If it is always true, even when there is no infection circulating, that each new case causes less than one secondary case, then the infection will die out. This simple observation leads to a straightforward calculation for the proportion of a population that must be vaccinated in order to achieve eradication, pc: pc ¼ 1 

1 R0

ð1Þ

This relationship arises from the fact that if (R0 – 1) out of the R0 people a case might have infected have been vaccinated, then each case, in a population vaccinated to that degree, will cause less than one secondary case. For example, say R0 ¼ 10, if 9/10 of the population are successfully immune following vaccination, then infection cannot spread. Thus, in general, pc ¼ (R0 – 1)/R0, as stated in Eq. 1. If the fraction of the population that are successfully vaccinated is greater than pc, then each case will cause, on average, less than one case, and infection cannot spread. Comparing estimated values for R0 for measles and smallpox and inferred values for the proportion that need to be vaccinated to ensure eradication (Table 1) leads to a simple answer to the question why has smallpox been eradicated, but not measles? Smallpox, with a basic reproductive number around three, was eradicated with vaccination coverage of around 67%. The higher R0 for measles, nearer to 15, requires vaccination coverage close to 95% to ensure eradication. Many parts of the world remain unable to achieve such

high coverage; measles remains suppressed by tremendous efforts at vaccination but is not yet eradicated. These calculations are so straightforward that they can be made without recourse to any formal modeling. However, embedding these ideas inside a formal modeling framework has proven very useful. The next section describes the simplest applicable model.

The SIR Model The “plain vanilla” model of mathematical epidemiology is called the SIR model because it splits the host population into three groups: • The susceptible (S) can be infected if exposed • The infectious (I) are both infected and infectious to others • The recovered (R) are no longer infectious and are immune to further infection The SIR model’s structure then consists of a set of assumptions about how people flow into, out of, and between these three groups. Those assumptions can be represented graphically as in Fig. 1. The assumptions of the SIR model with vaccination are the following: People are born at a constant rate B, and a proportion p of them are vaccinated at birth. Vaccinated newborns are immune for life and so they join the recovered class. Unvaccinated newborns enter the

pB vaccinated births

Infectious Disease Modeling, Table 1 The basic reproductive number, R0, and the critical vaccination proportion for eradication, pc, for measles and smallpox Infection Smallpox Measles Measles

Place India India UK

Time 1970s 1970s 1960s

R0 3 15 15

pc (%) 67 93 93

βIS

(1−p)B unvaccinated births

S

infection

μS death

γI I

recovery

μI death

R μR death

Infectious Disease Modeling, Fig. 1 The SIR model in graphical form. The host population is divided into three groups, and transitions of people between those groups are described. Those transitions represent the five processes: birth, vaccination, death, infection, and recovery

Infectious Disease Modeling

11

difference equations, ordinary differential equations, or stochastic differential equations. The difference equation form is as follows: Sðt þ 1Þ ¼ SðtÞ þ ð1  pÞB  bIðtÞSðtÞ  mSðtÞ

Rðt þ 1Þ ¼ RðtÞ þ pB þ gIðtÞ  mRðtÞ

ð4Þ

90000

160

80000

140

70000 I S R

120 100

60000 50000

80

40000

60

30000

40

20000

20

10000 0 10

a

20

30

40

50

Time in years 180

1.8

160

1.6

140

1.4 None

1.2

50% 100

70%

1

80

90%

0.8

60

0.6

40

0.4

20

0.2

0

0 0

10

20 30 Time in years

40

50

Axis title

120

Number susceptible or recovered

This difference equation form is particularly easy to handle numerically and can be straightforwardly solved in a spreadsheet. Figure 2a shows the

180

0

b

ð2Þ

Iðt þ 1Þ ¼ IðtÞ þ bIðtÞSðtÞ  gIðtÞ  mIðtÞ ð3Þ

0

Number of infectious people

Infectious Disease Modeling, Fig. 2 Numerical solutions to the SIR difference equation model. Infection circulates in a population of 100,000 individuals, with an expectation of life at birth of 50 years. The infectious period is 1 week, and the basic reproductive number is 5. This gives the following model parameters: B ¼ 38 per week, m ¼ 0.00385 per person per week,? ¼ 1 per person per week, and ß ¼ 0.00005 per infected per susceptible per week. (a) shows damped oscillations in all three classes after an initial perturbation of 20% of the susceptible class into the recovered class. In (b), vaccination of 50%, 70%, or 90% of newborns is introduced at time 10 years. With R0 ¼ 5, the critical vaccination proportion pc ¼ 0.8. Vaccination coverage above this level (at 90%) leads to eradication

Number of infectious people

susceptible class. Susceptibles are infected at a per capita rate proportional to I, the number of infectious people in the population. This gives rise to a transfer from the susceptible to the infectious class at rate ßIS. Susceptibles are also subject to a per capita background death rate m. Infectious people recover into the recovered class I at per capita rate? or die at the per capita background death rate m. Recovered individuals are immune for the rest of their lives, so the only exit from the recovered class is at the per capita background death rate m. These assumptions can be written in several different forms of equations, for example,

12

solutions to Eqs. 2, 3, and 4 over 50 years with a 1-week timestep. Parameters are set so that an infection with a basic reproductive number of 5 and a 1-week duration of infection is spreading in a population of 100,000 individuals. The figure illustrates how this model shows damped oscillations towards a stable state. The same is true for the ODE version of this model. This model is useful for understanding the impact of vaccination. In Fig. 2b, the solutions to Eqs. 2, 3, and 4 are shown when vaccination at birth is introduced 10 years into the model run. With a basic reproductive number of 5, Eq. 1 tells us that vaccination of over 80% of newborns will lead to eradication. This is exemplified in the pink line where 90% vaccination leads to no further cases. Vaccination coverage below this threshold value reduces the numbers of cases and increases the inter-epidemic period but does not lead to eradication. Notice the very long inter-epidemic period at 70% vaccination. This phenomen occurs when vaccine coverage levels are close to but do not achieve the critical coverage level. Under these circumstances, it takes a very long time to accumulate enough susceptibles to trigger the first epidemic after vaccination is introduced. It may therefore appear as though eradication has been achieved even though vaccination coverage is below the critical level. This phenomenon, named “the honeymoon period,” [5] was first described in modeling studies and later identified in field data [6]. The ability to identify target vaccination levels predicted to lead to disease eradication has been widely influential in policy circles [7]. Models with the same fundamental structure as the SIR model are used to set targets for vaccination coverage in many settings [8]. Similar models are also used to understand the likely impact of different interventions of other sorts, for example, drug treatment [9] or measures for social distancing [10]. However, models for informing policy need to explore more of the wrinkles and complexities of the real world than are acknowledged in the simple equations of the SIR model. The next section describes some of the types of host heterogeneity that

Infectious Disease Modeling

have been explored in making versions of the SIR model that aim to be better representations of the real world.

Host Heterogeneity There are many aspects of host heterogeneity that have bearing on the transmission and impact of infections. Two of the most important are host age and spatial distribution. In this section, the modeling of these two types of host heterogeneity is introduced with reference to two specific infections: rubella and foot-and-mouth disease. Rubella is a directly transmitted viral infection that usually causes mild disease when contracted during childhood. However, infection of a woman during early pregnancy can lead to serious birth defects for her unborn child. The set of consequent conditions is labeled “congenital rubella syndrome” or CRS. Because vaccination acts to extend the time between epidemics (Fig. 1b), it also acts to increase the average age at infection. This sets up a complex trade-off when introducing rubella vaccination to a community. On the one hand, vaccinated girls are protected from catching rubella at any age, but on the other hand, the girls who remain unvaccinated are likely to catch rubella when they are older, more likely to be in their childbearing years and so at greater risk of CRS. This means that vaccination with low coverage can actually lead to more CRS, and only when coverage levels get above a certain level do the benefits of vaccinating the community outweigh the costs. Calculating where that level lies then becomes an important public health question. Because age is such an important component of the risks associated with rubella infection, models of this system need to take account of host age. The relevant versions of Eqs. 2, 3, and 4 are difference equations with two independent variables, age (a) and time (t): Sða þ 1, t þ 1Þ ¼ Sða, tÞ  Sða, tÞ½Sa0 bða, a0 ÞIða, tÞ  mðaÞSða, tÞ

ð5Þ

Infectious Disease Modeling

13

Iða þ 1, t þ 1Þ ¼ Iða, tÞ þ Sða, tÞ½Sa0 bða, a0 ÞIða, tÞ gðaÞIða, tÞ  mðaÞIða, tÞ

ð6Þ Rða þ 1, t þ 1Þ ¼ Rða, tÞ þ gðaÞ Iða, tÞ  mðaÞ Rða, tÞ

ð7Þ

Notice how these equations, by taking account of age as well as time, allow consideration of several different kinds of age dependence. Firstly, Eq. 6 calculates the number of cases of infection of a given age over time. Since the main consideration in balancing up the pros and cons of rubella vaccination is the number of cases in women of childbearing age, this is an essential model output. Secondly, the per capita rate at which susceptibles become infected depends on their age and on the age of all the infected people. This model is thus able to take account of the complexities of family, school, and working life which drive people of different ages to agedependent patterns of mixing. Thirdly, the recovery rate?(a) and, more importantly, the background death rate m(a) can both be made to depend on age. Since a fixed per capita death rate is a particularly bad approximation of human survival, this is another important advance on models without age structure. Models with age structure akin to that presented in Eqs. 5, 6, and 7 have been essential components of the planning of rubella vaccination strategies around the world [11, 12]. A model as simple as these equations would never be used for formulating policy; furthermore, most agestructured models use the continuous time and age versions and so have the structure of partial differential equations. Nevertheless, Eqs. 5, 6, and 7 illustrate the fundamentals of how to include age in an epidemiological model. The spatial distribution of hosts is another important aspect of their heterogeneity. If the units of infection are sessile (e.g., plants), the assumption that all hosts are equally likely to contact each other becomes particularly egregious and models that acknowledge the spatial location of hosts more important. One example of units of

infection that do not move is farms. If trade between farms has been halted because of a disease outbreak, then disease transmission between farms is likely to be strongly dependent upon their location. This was the case during the 2001 footand-mouth disease epidemic in the UK, and spatial models of that epidemic are nice examples of how to explicitly include the distance between hosts in a model epidemic. On February 19, 2001, a vet in Essex reported suspected cases of foot-and-mouth disease (FMD) in pigs he had inspected at an abattoir. FMD is a highly infectious viral disease of cloven-hoofed animals. Because of its economic and welfare implications for livestock, FMD had been eradicated from Western Europe. The FMD outbreak that unfolded in the UK over the ensuing months had a huge impact with millions of farm animals killed and major economic impact in the countryside as tourism was virtually shut down. There was heated debate about the best way to control the spread of infection from farm to farm. FMD virus is so very infectious that no attempt was made to control its spread within a farm. Once infection of livestock on a farm was detected, all susceptible animals were slaughtered. Mathematical models of the spread of this epidemic thus treat each farm as a unit of infection, and, as before, farms can be categorized as susceptible, infectious, etc. The best of these models [13] keeps track of every single farm in the United Kingdom, characterizing farms by their location and the number of sheep and cattle they hold. The model classifies farms into four groups: susceptible, incubating, infectious, or slaughtered. As in all epidemic models, the heart of the model is the per capita rate at which susceptible farms become infected – the so-called force of infection. Because this FMD model is an individual-based, stochastic simulation, it is not possible to write out its equations in a simple form as before, but the probability of infection for a single farm can easily be written. Suppose all farms in the UK are listed and indexed with i. Then pi, the probability that an individual farm i becomes infected during one unit of time, is:

14

Infectious Disease Modeling

pi ¼ bi Sall

infectious farmsj tj K

dij



ð8Þ

where ßi is the susceptibility of farm i, determined by the number of sheep and cows it holds; tj is the infectiousness of farm j, also determined by the number of sheep and cows it holds; and K (dij) is a function of the distance between the pair of farms i and j which determines how quickly infectiousness falls off with increasing distance. K is known as the “infection kernel.” In the FMD example, the infection kernel was estimated from contact tracing data on farms that were sources of infection and their secondary cases. This observed relationship shows a very sharp falling off of infectiousness, with a farm just 2 km distant being less than tenfold as infectious to a susceptible farm than one that is adjacent. This section describes just two of the possible heterogeneities that are often included when making models of the spread of epidemics. There is almost no end to how complex an epidemiological model can become. However, it is very easy for complex models to outstrip the data available to calculate their parameters. In some cases, this can mean that models become black boxes concealing ill-informed guesswork, rather than prisms unveiling the implications of well-sourced and well-understood data.

understanding why these vaccines failed is a major research priority [17]. A quantitative description of the interaction between HIV and host immune cells would be an asset to such understanding. For one component of host immunity – the cytotoxic T cell (CTL) response – such a description can be derived. The question is how effective are host CTL responses at killing HIVinfected cells? Not how many CTLs are present, nor which cytokines they secrete, but how fast do they kill HIV-infected cells? During the course of a single infection, HIV evolves to escape from the selection pressure imposed by host CTLs [18]. In this process, new HIV variants emerge that are not recognized by the host CTLs. These variants are called “CTL escape mutants.” These CTL escape mutants can be seen to grow out in hosts who mount relevant CTL responses (Fig. 3) and to revert in hosts who do not. The rate of reversion in hosts without relevant CTL responses reflects the underlying fitness cost of the mutation. The rate of outgrowth in hosts who do mount relevant CTL responses is a balance between the efficacy of those responses and the fitness cost of the mutations. These costs and benefits need to be examined in the context of the underlying rate of turnover of HIV-infected

120

Within-Host Dynamics

100 % escape mutant

Mathematical models can also be used to investigate the dynamics of events that unfold within infected hosts. In these models, the units of study are often infected cells and immune cells responding to infection. As with epidemiological models, there is a wide range of modeling styles: Some models detail many different interacting components; others make a virtue of parsimony in their description of within-host interactions. In this section, a simple model of the within-host evolution of HIV is used to illustrate how pared-down, within-host models of infection can address important practical questions. Several trials of prophylactic HIV vaccines have shown little or no effect [14–16], and

80 60 40 20 0 0

500

1000

1500

2000

2500

Time (days)

Infectious Disease Modeling, Fig. 3 The outgrowth of HIV CTL escape mutants through time. Data sets from three different patients (reviewed in [19]) are shown as red, brown, and yellow symbols. Equation 12 is fitted to these data, yielding rates of outgrowth, c  (a  â), of 0.048 (red), 0.012 (brown), and 0.006 (yellow)

Infectious Disease Modeling

15

cells. All this can be represented in a two-line mathematical model [19]. Let x be the number of host cells infected with “wild-type” virus – that is, virus that can be recognized by the relevant host CTL responses. Let y be the number of host cells infected with escape mutant virus. The model then consists of a pair of ordinary differential equations describing the growth rate of each population of infected cells. The wild-type population grows at rate a, is killed by the CTL response in question at rate c, and is killed by all other processes at rate b. The escape mutant population grows at rate â (â < a, reflecting the underlying fitness cost of the mutation) and is killed by all other processes at rate b. Escape-mutant-infected cells are not killed by the CTL response in question because of the presence of the escape mutation in the viral genome. These assumptions give rise to the following pair of linear ordinary differential equations: x0 ¼ ax  bx  cx

ð9Þ

y0 ¼ ^ay  by

ð10Þ

The observed quantity, call it p, is the fraction of virus that is of the escape mutant type; p ¼ y/(x þ y). Simple application of the quotient rule for differentiation yields the single differential equation p0 ¼ ðc  ða  ^aÞÞpð1  pÞ

ð11Þ

with solution: p ¼ ðk exp ððc  ða  ^aÞÞtÞ þ 1Þ1

ð12Þ

where for p0, the fraction escaped at time 0: k¼

ð 1  p0 Þ p0

ð13Þ

It is straightforward to fit the analytic expression (12) to data on the outgrowth of escape mutants to obtain estimates of the quantity c  (a  â). Figure 3 shows fitted curves with estimates of c  (a  â) of 0.048, 0.012, and 0.006. The quantity of interest is the parameter c – the

rate at which CTL kills cells infected with wildtype virus. Fortunately, independent estimates of the fitness cost of the escape mutation (a  â) are available. The median of several such observations yields (a  â) ¼ 0.005 [19]. Taken together and combined with further data, the inference is that on average, a single CTL response kills infected cells at rate 0.02 per day. The half-life of an HIV-infected cell is about 1 day. This figure was itself derived from the application of elegantly simple models to data on the post-treatment dynamics of HIV [20, 21]. If a single CTL response kills infected cells at rate 0.02 per day and their overall death rate is one, then just 2% of the death of infected cells can be attributed to killing by one CTL response. Patients will typically mount many responses – but probably not more than a dozen. This analysis shows that even though CTL responses are effective enough to drive viral evolution, they are, in quantitative terms, very weak. A vaccine to protect against HIV infection would have to elicit immune responses that are manyfold stronger than the natural responses detected in ongoing infection. This simple, model-based observation greatly helps understand why the vaccines trialed so far have failed.

Multilevel Models The models discussed so far deal either with events inside individuals or with transmission amongst individuals (people or farms) in a population. Some questions require simultaneous consideration of events at both levels of organization. This is particularly true for questions about the evolution of infectious agents as their evolution proceeds within individual hosts, but they are also transmitted between hosts. Models that capture events at both the within-host and between-host levels are fairly recent additions to the literature on infectious disease modeling. Here, they are illustrated with two examples, a set of models that consider the emergence of a zoonotic infection in humans and a model of the within-host evolution and between-host transmission of HIV.

16

Emerging infections are a continuing threat to human well-being. The pandemics of SARS in 2003 and H1N1 swine flu in 2009 illustrated how quickly a new infectious agent spreads around the world. Neither of these was as devastating as some predicted, but the continuing pandemic of HIV is ample proof that emerging infectious diseases can have devastating consequences for human communities. Many novel emerging infections arise as zoonoses – that is, infections that cross from animals into humans [22]. To become a successful emerging infection of humans – that is, one that spreads widely amongst people – is a multi-step process [23]. First, the pathogen must cross the species barrier into people, then it must transmit between people, and finally, it must transmit efficiently enough that epidemics arise. This latter step amounts to having a basic reproductive number, R0, that is greater than 1. The emerging infections mentioned already, SARS, swine flu, and HIV, have transited all these steps. But there are other zoonoses that transmit to humans without emerging as epidemics or pandemics. For example, simian foamy virus, a retrovirus that is endemic in most old-world primates [24], can be detected in people who work with primates [25] or hunt them [26]. There is no record of any human-to-human transmission, implying that this zoonosis only completes the first step in becoming an emerging infection. Other infections, whilst spreading from person to person, still do not cause epidemics because that spread is insufficiently efficient. An example of such an infection is the newly discovered arenavirus from Southern Africa called “Lujo virus” [27]. This virus caused a small outbreak in the autumn of 2008. Very dramatically, four out of the five known cases died, but with five cases and just four transmission events, the basic reproductive number stayed below one, and there was no epidemic. Acquiring R0 > 1 is thus an important threshold that zoonoses must breach before they can become emerging infections. Antia and colleagues [28] developed an elegant model of the within-host evolution and between-host transmission of a zoonotic infection that initially has R0 < 1, but through within-host adaptation in

Infectious Disease Modeling

humans can evolve to become efficient enough at transmitting from one human to another that R0 increases above 1 and epidemics become possible. They developed a multi-type branching process model of the transmission and evolution of a zoonosis. They found that the probability of emergence depends very strongly on the basic reproductive number of the pathogen as it crosses into humans. This is because, even when R0 < 1, short chains of transmission are still possible (as exemplified with Lujo virus described above). During ongoing infections in humans, the zoonosis has opportunities to evolve towards higher transmissibility. The higher its initial R0, the more opportunities there are for such ongoing evolution and hence for emergence. This model of the emergence of a novel infection has been extended by other authors to address questions about the interpretation of surveillance data [29] and the role of host heterogeneity in the process of emergence [30]. These extensions confirm the original finding that the transmission efficiency (R0) of the introduced variant (and any intermediate variants) is a very important driver of the probability of emergence. Kubiak and colleagues explored the emergence of a novel infection in populations split into several communities, with commuters acting to join those communities together. They found that most communities are sufficiently interconnected to show no effect of spatial distribution on the emergence process, even a small number of commuters being sufficient to successfully transmit any novel pathogen between settlements. Thus, although many zoonotic events happen in isolated parts of the world, unless they are really cut off from urban centers, that isolation offers little barrier to the transmission of newly emerged infections. HIV emerged as a human infection sometime during the end of the 1800s and the early 1900s [31]. It was only recognized as a new human infection in the 1980s when cases of immunodeficiency in young Americans were unusual enough to warrant investigation [32]. As discussed above, during the course of a single infection, HIV is able to adapt to escape from the selection pressures imposed by its host’s immune response. HIV variants that cannot be recognized

Infectious Disease Modeling

17

by current host CTLs are termed “CTL escape mutants.” These mutants yield important information about the strength of the immune responses that they evade. However, since they were shown to transmit from one host to another, their status has been raised to potential drivers of evolutionary change across the global HIV pandemic [33, 34]. Different hosts respond to different parts of HIV’s proteins (known as epitopes). For CTL responses, it is the host class 1 human leukocyte antigen (HLA) type that determines which epitopes are recognized. When CTL escape mutants are transmitted into a host who does not make immune response to that epitope, the mutations are no longer advantageous, and the virus can revert to the wild type [35]. Global change in the prevalence of CTL escape mutants is therefore driven by three parallel processes: the selection of escape mutants in some hosts, transmission between hosts, and reversion of escape mutants in other hosts. Once again, this is a process that takes place across multiple levels of organization, evolution and reversion of escape mutations

within infected hosts, and transmission between hosts. Fryer and colleagues [36] developed a multilevel model of the three processes of within-host evolution, within-host reversion, and betweenhost transmission. The model is a version of the so-called SI model which is a simplified version of the SIR model presented above which does not allow recovery. The model allows heterogeneity in hosts and in the infecting virus so that there are hosts who do and do not mount immune responses to a given epitope and there are viruses that do and do not have escape mutations in that epitope. This model is represented in Fig. 4. As in the SIR model described in section “The SIR Model,” the rate at which susceptibles become infected is determined by the number of infectious people present. However, in this model, because it represents the spread of a sexually transmitted disease, it is the proportion of hosts who are infectious that drives new infections. Furthermore, there are now two virus types circulating – wild type and escape mutant. Within-host adaptation allows hosts who do mount immune responses to the epitope to

β(IIR+INIR)SIR/N W

births

W

(μ+α)IIR W death

escape φIIR W

SIR death

IIR W

IIR E

μSIR

(μ+α)IIR E

death

β(IIR +INIR)SIR/N E E infection

β(IIR+INIR)SNIR/N W W

births

SNIR death

(μ+α)INIR W death

ψINIR reversion E

μSNIR β(IIR +INIR)SNIR/N E E

Infectious Disease Modeling, Figure 4 A model of the within-host evolution and between-host transmission of HIV escape mutants [36]. Hosts are divided into two types: immune responders (superscript IR) and nonimmune responders (superscript NIR). There are also two variants of virus, wild type (subscript WT) and escape mutant (subscript E). Hosts are either susceptible, S, or infectious, I, and the type of virus with which they are

INIR W

INIR E

(μ+α)INIR E death

infected is denoted by the subscript. Rates of infection are determined by the number of people infectious with each virus type. Immune responding hosts infected with the wild-type virus drive immune escape at per capita rate?, whilst nonimmune responding hosts infected with escape mutant virus drive reversion at per capita rate? All hosts are prone to per capita death rate m, and infected hosts have an additional death rate α

18

Infectious Disease Modeling

drive the evolution of escape mutants, and conversely, hosts who do not mount such responses can drive the reversion of escape mutant viruses back to the wild type. This model’s behavior is easy to understand. The total numbers of susceptible and infectious people simply follow the well-characterized SI model. Figure 5a shows total cases through time. The total epidemic goes through three phases: an initial exponential growth, a saturation phase, and then settling to a long-term equilibrium. Figure 5b shows the proportion of all cases that are escape mutants through time. Not surprisingly, faster escape rates and slower reversion rates lead to higher prevalence of escape mutants. Less intuitive are the following characteristics of Fig. 5b. Whilst the epidemic is in its exponential growth phase, so long as reversion rates are reasonably

Proprtion of infections with escape

120000 100000 80000 60000 40000 20000 0 0

50

a

100

150

200

Time (years) 1

Proprtion of infections with escape

Infectious Disease Modeling, Fig. 5 Predictions of a model of within-host evolution and between-host transmission of HIV. (a) shows total numbers of susceptible (blue) and infectious (red) people through time. (b) shows the proportion of infections that are with escape mutant virus for a range of escape and reversion rates. The mean times to escape and reversion for each curve are as follows: red – escape 1 month, reversion never; brown – escape 5 years, reversion never; yellow – escape 5 years, reversion 50 years; pink – escape 1 year, reversion 10 years; green – escape 5 years, reversion 10 years; mauve – escape 1 year, reversion 1 year

fast (say once in 10 years or faster), the prevalence of escape is expected to stabilize quite quickly. However, this is not a long-term equilibrium, and as the total epidemic turns over, the escape prevalence shifts again. For an epitope that escapes fast but reverts at an intermediate rate, this leads to a substantial drop in the prevalence of escape. Secondly, fixation of escape variants only occurs if they never revert, and even then fixation takes a very long time – much longer than it takes for the underlying epidemic to equilibrate. Thirdly, the predicted dynamics and equilibrium are very sensitive to the reversion rate when that is slow. Notice the big difference, in the long term, between no reversion (brown line) and average time to reversion of 50 years (yellow line). As well as predicting the future spread of escape mutations for different rates of escape

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

b

50

100 Time (years)

150

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Infectious Disease Modeling

and reversion, this model can be used to infer escape and reversion rates from data on their current prevalence. This exercise reveals a surprisingly slow average rate of escape. Across 26 different epitopes, the median time to escape was over 8 years. There is close agreement between rates of escape inferred using this model and those estimated from a longitudinal cohort study. These slow rates are in marked contrast to the general impression given by a large number of case reports in which escape is described as occurring during the first year of infection. However, a collection of case reports is a poor basis upon which to estimate an average rate of escape. These are just two examples from the new family of infectious disease models that encapsulate processes at multiple levels of organization. As data on pathogen evolution continues to accrue, this approach will doubtlessly continue to yield new insights.

Future Directions It seems likely that infectious diseases will continue to trouble both individuals and communities. Whilst technological advances in new drugs, new vaccines, and better methods for surveillance will undoubtedly assist with the control of infection, several trends in society pull in the opposite direction. Chief amongst these is a growing population, and second is increasing population density as more and more people live in towns and cities. What can infectious disease modeling do to help? Models can help in two different ways. The first is to assist the understanding of systems that are intrinsically complicated. Many different interacting populations, events that occur on multiple timescales, and systems with multiple levels of organization can all be better understood when appropriate models are used as an organizing principle and a tool for formal analysis. Sometimes, the problem is that there is not enough data. A systematic description can be very revealing in searching for which new data are most needed. There are also situations where the

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problem is a deluge of data. In these circumstances, well-constructed models provide a useful organizing scheme with which to interrogate those data. The second use of models is as representations of well-understood systems used as tools for comparing different intervention strategies. The model of the farm-to-farm spread of FMD described at section “Host Heterogeneity” is a fine example of this use of modeling. It includes enough detail to be a useful tool for comparing different interventions, but is still firmly rooted in available data so does not rest on large numbers of untested assumptions.

Bibliography Primary Literature 1. Fenner F (1982) A successful eradication campaign. Global eradication of smallpox. Rev Infect Dis 4(5): 916–930 2. Moss WJ, Griffin DE (2006) Global measles elimination. Nat Rev Microbiol 4:900–908 3. Farrington CP, Kanaan MN, Gay NJ (2001) Estimation of the basic reproduction number for infectious diseases from age-stratified serological survey data. J R Stat Soc Ser C Appl Stat 50:251–292 4. Anderson RM, May RM (1991) Infectious diseases of humans. Oxford University Press, Oxford 5. McLean AR, Anderson RM (1988) Measles in developing countries. Part II: the predicted impact of mass vaccination. Epidemiol Infect 100:419–442 6. McLean AR (1995) After the honeymoon in measles control. Lancet 345(8945):272 7. Babad HE et al (1995) Predicting the impact of measles vaccination in England and Wales: model validation and analysis of policy options. Epidemiol Infect 114:319–344 8. McLean AR (1992) Mathematical modelling of the immunisation of populations. Rev Med Virol 2: 141–152 9. Arinaminpathy N, McLean AR (2009) Logistics for control for an influenza pandemic. Epidemics 1(2): 83–88 10. Longini IM et al (2005) Containing pandemic influenza at the source. Science 309(5737): 1083–1087 11. Anderson RM, Grenfell BT (1986) Quantitative investigations of different vaccination policies for the control of congenital rubella syndrome (CRS) in the United Kingdom. J Hyg 96:305–333. Cambridge University Press 12. Metcalf CJE et al (2010) Rubella metapopulation dynamics and importance of spatial coupling to the

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Infectious Disease Modeling risk of congenital rubella syndrome in Peru. J R Soc Interface 8(56):369–376 Keeling MJ et al (2001) Dynamics of the 2001 UK foot and mouth epidemic: stochastic dispersal in a heterogeneous landscape. Science 294(5543): 813–817 Flynn NM et al (2005) Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis 191(5):654–665 Buchbinder SP et al (2008) Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebocontrolled, test-of-concept trial. Lancet 372(9653): 1881–1893 Rerks-Ngarm S et al (2009) Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 361(23):2209–2220. Epub 2009 Oct 20 Council of the Global HIV Vaccine Enterprise (2010) The 2010 scientific strategic plan of the global HIV vaccine enterprise. Nat Med 16(9):981–989 Phillips RE et al (1991) Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature 354:453–459 Asquith B et al (2006) Inefficient cytotoxic T lymphocyte-mediated killing of HIV-1-infected cells in vivo. PLoS Biol 4:e90 Wei X et al (1995) Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373(6510): 117–122 Ho DD et al (1995) Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373(6510):123–126 Woolhouse MEJ (2002) Population biology of emerging and re-emerging pathogens. Trends Microbiol 10(10):s3–s7 Wolfe ND et al (2007) Origins of major human infectious diseases. Nature 447:279–283 Meiering CD, Linial ML (2001) Historical perspective of foamy virus epidemiology and infection. Clin Microbiol Rev 14:165–176 Switzer WM et al (2004) Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates. J Virol 78(6):2780–2789

26. Wolfe ND et al (2004) Naturally acquired simian retrovirus infections in central African hunters. Lancet 363:932–937 27. Briese T et al (2009) Genetic detection and characterization of Lujo virus, a new hemorrhagic fever–associated arenavirus from Southern Africa. PLoS Pathog 5(5):e1000455. https://doi.org/10.1371/journal.ppat. 1000455 28. Antia R et al (2004) The role of evolution in the emergence of infectious diseases. Nature 426:658–661 29. Arinaminpathy N, McLean AR (2009) Evolution and emergence of novel human infections. Proc R Soc B 273:3075–3083 30. Kubiak R et al (2010) Insights into the evolution and emergence of a novel infectious disease. PLoS Comput Biol 6(9):e10000947 31. Worobey M et al (2008) Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960. Nature 455(7213):661–664 32. CDC (1981) Kaposi’s sarcoma and Pneumocystis pneumonia among homosexual men – New York City and California. MMWR 30:305–308 33. Kawashima Y et al (2009) Adaptation of HIV-1 to human leukocyte antigen class I. Nature 458:641–645 34. Goulder PJ et al (2001) Evolution and transmission of stable CTL escape mutations in HIV infection. Nature 412:334–338 35. Leslie AJ et al (2004) HIV evolution: CTL escape mutation and reversion after transmission. Nat Med 10:282–289 36. Fryer HR et al (2010) Modelling the evolution and spread of HIV immune escape mutants. PLoS Pathog 6(11):e1001196

Books and Reviews Anderson RM, May RM (1991) Infectious diseases of humans. Oxford University Press, Oxford Keeling MJ, Rohani R (2008) Modeling infectious diseases in humans and animals. Princeton University Press, Princeton Nowak MA, May RM (2000) Virus dynamics. Oxford University Press, Oxford

Biological Controls and Standards for the Study and Control of Infectious Diseases Philip David Minor National Institute for Biological Standards and Control (retired), St. Albans, Hertfordshire, UK

Article Outline Glossary Definition and Importance of Subject Introduction Emerging Infections: Immune Responses in SARS2 Vaccine Trials Diagnostic Detection Methods: Nucleic Acid Detection and Hepatitis C Potency of Vaccines: Inactivated Polio Vaccine (IPV) Preparation and Establishment of International Standards The Collaborative Study: Participating Laboratories The Collaborative Study: Study Samples The Collaborative Study: The Assays The Collaborative Study: The Report Future Directions Bibliography

Glossary BIPM Bureau Internationale de Poids et Mesure; agency responsible for the implementation of the SI system COP Correlate of Protection ECBS Expert Committee on Biological Standards of WHO ELISA Enzyme-Linked Immunosorbent Assay HCV Hepatitis C virus International standard Material distributed in identical vials shown in a collaborative study to improve the agreement between assays and

established by the Expert Committee on Biological Standards and Control of WHO. The international standard is the primary reference to which all other reference materials are ultimately calibrated. International unit The international unit is defined as a fraction of the biological activity in a vial of the international standard. It is arbitrarily assigned by ECBS and bears no relationship to the unit assigned to any other standard except under specific circumstance, for instance where a replacement standard is produced. IPV Inactivated Polio Vaccine; nonreplicating polio vaccine ISO International Organization for standardization NGO Nongovernmental Organization NIBSC National Institute of Biological Standards and Control, currently a branch of the UK regulatory authority, responsible for production of most of the international standards. NIFDC The Chinese regulatory agency with a strong interest in standardization PCR Polymerase Chain Reaction PEI Paul Ehrlich Institute; the German regulatory agency with a strong interest in standardization SARS2 virus Serious Acute Respiratory Syndrome 2 virus Secondary standard Material calibrated against the international standard SI International system of measurement sIPV IPV made from the attenuated vaccine strains of polio derived by Albert Sabin SoGAT Committee for the Standardization of Genome Amplification Technology WHO World Health Organization

Definition and Importance of Subject The measurement of parameters is central to science and critical to the accurate diagnosis, treatment, and prophylaxis of disease. All measurements are made

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_1108 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2022 https://doi.org/10.1007/978-1-4939-2493-6_1108-1

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Biological Controls and Standards for the Study and Control of Infectious Diseases

relative to a standard which ideally is defined in terms of universal physical constants as in the SI system of measurement (for example, the unit of mass is defined in terms of Planck’s constant). Biological activity is difficult or impossible to define in these terms and measurements are best made relative to a physical reference material with biological activity established by the World Health Organization. The use of this conventional calibrator helps to relate measurements and harmonize assays and so make medical treatments better controlled.

Introduction Biological standardization originated when Paul Ehrlich developed reference materials for antitoxins against diphtheria and tetanus at the start of the twentieth century. Antitoxins for clinical use were prepared by inoculation of horses with amounts of the relevant toxin sufficient to raise an immune response but not to kill the animal; as this was a difficult balance to strike the quality of the product was variable and some batches were completely inactive. An assay based on lethal challenge of mice was developed and allowed a quantitative assessment of the potency of a preparation but the reproducibility of the test method was poor, varying with the batch of mice and the execution of the procedure in ways that could not be controlled precisely. Thus a reference

preparation that was known to be active was included in each test run, to show that the assay was behaving as expected, and to give a measure of activity against which the potency of the unknown in the assay run could be expressed and the dosage decided [1]. This remains the easiest way to standardize biological assays for most practical purposes today. Most laboratories include a run control in an assay to show that the procedure has been carried out correctly and that the results from the control are within the expected limits, or to get a quantitative measure of the potency of the test item. International standards are a more formal and widely accepted version of this approach and are developed in accordance with a formal and rigorous process under the auspices of WHO. They act as the primary references against which other materials may be calibrated, so enabling comparisons between assays and between laboratories to standardize medical practice. The hierarchy of references for biological standardization is shown in Table 1 and pictorially in Fig. 1. Examples of the kinds of product involved are given in Table 2. The preparation of international biological reference materials is among the responsibilities of WHO as laid down by the United Nations. Most are developed by the National Institute of Biological Standards and Control (NIBSC) in the UK [2] which is one of several WHO collaborating centers involved in standardization work.

Biological Controls and Standards for the Study and Control of Infectious Diseases, Table 1 Hierarchy of biological standards Material International standard

Nature Primary reference material

Secondary reference

Established nationally or by pharmacopoeias Established in house The analyte

Working reagent Patient sample or medical material

Provenance Highest-order reference material, established by the WHO through the Expert Committee on Biological Standardization Calibrated against the international reference

Comments To be used to calibrate lower-order references. The physical standard is assigned an arbitrary unitage and is intended to last 10–15 years Used as a calibrant or working reagent

May be calibrated against a secondary reagent

Used in routine assays

Biological Controls and Standards for the Study and Control of Infectious Diseases

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Biological Controls and Standards for the Study and Control of Infectious Diseases, Fig. 1 Hierarchy of analytical reference materials Biological Controls and Standards for the Study and Control of Infectious Diseases, Table 2 Examples of international reference materials; see NIBSC website (nibsc.org) and catalogue for further information Purpose Diagnostic

Antigen (hepatitis B, HIV)

Nucleic acid (HCV, HIV, and B19)

Therapeutic

Blood products (factor VIII)

Vaccines

Polio potency reagents for IPV, live measles vaccines

Hormones (insulin, growth hormone, erythropoietin) Polio vaccine in vivo virulence references, MAPREC

Most physical measurements are based on the SI system, where the units are now derived from universal constants. For example, the kilogram is now defined by reference to Planck’s constant rather than, as previously, a physical artifact stored in the Bureau International des Poids et Mesure (BIPM) in Paris [3]. Other base units, such as the meter and second, are similarly defined in terms of universal constants. Having defined the unit, it is realized by a closely defined reference method which gives an answer within certain narrow limits of uncertainty; the reference method is the crucial element in the process. In contrast, in biological systems there is no reference method as it is not clear what the key physical parameter to be measured may be. In fact, the key property to be measured is a biological activity of some sort and the assays are calibrated against a physical reference artifact such as an

Antibody (SARS2, measles, and polio) Cytokines (IL6, TNF, and GMCSF)

antigen which is known to have the desired biological activity. The primary reference will eventually be exhausted and require replacement, with unavoidable errors in calibration between the original and subsequent materials. The measured parameter is also inevitably hard to define in precise physical terms. The biological measurement system is empirical and arises from practical need. It focuses on the parameter of interest, namely the biological activity that requires measurement. Certain exhaustively characterized biological medicines such as insulin are now assayed and dosed in SI units, i.e., micrograms. Others such as live attenuated vaccines are not, as the potency is linked to the biological property of infectivity and not to an SI unit such as mass. Others again are the subject of unresolved vigorous arguments. For example, immunoglobulins are nonliving proteins and should therefore be amenable to measurement

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in mass units, and some choose to express immunoglobulin potency in micrograms. However, the important features are the biological properties (binding, neutralization, etc.) which arise from the nature of the mixture of the range of individual molecules present. Different mixtures of antibody molecules with different avidities for different epitopes can lead to the same biological property or properties, and it is probably the biological properties that are of specific interest rather than the mass of protein. Some feel that immunoglobulins should be assayed as biological materials including the use of reference materials, other as physical entities measurable in SI units by a reference method. The debate continues. An important factor is whether the use of a reference material helps harmonize results and facilitate comparisons. Three examples follow.

Emerging Infections: Immune Responses in SARS2 Vaccine Trials The way in which reference materials could help clarify issues in emerging infections is illustrated by the SARS2 pandemic. The SARS2 virus is a human coronavirus distinct from other human corona viruses in its effects. The SARS1 and MERS viruses are highly pathogenic but not highly transmissible, while conversely the coronaviruses that cause the common cold are readily transmitted but not highly pathogenic. SARS2 emerged in late 2019 and is both highly infectious and pathogenic. Vaccine development began early and proved very successful with a great range of platforms being explored at unprecedented speed. They included nucleic acid vaccines (both RNA and DNA), vectored vaccines such as adeno virus vectors of different sorts, classical inactivated vaccines, and vaccines based on the expression of viral proteins in a variety of systems. Most involved the spike protein (S) of the virus, but this included the S1 component of the S protein on its own, the entire S protein, the receptor binding domain (RBD) located in the S1 component, and versions of the protein (or the gene encoding it) modified to improve stability, tolerability, or other features. Novel adjuvants were included in some cases.

Many platforms were novel with little previous experience of the performance to be expected in routine human immunization programs. The range of immunogens and presentations tested was very broad and the immune responses generated would be expected to be similarly very wide ranging in quantity and quality [4–11]. The vaccines were evaluated in small clinical trials, initially for safety but eventually for clinical efficacy in Phase 3 trials. As efficacy trials need to be very large to achieve the appropriate power and are difficult to design and implement, methods for assessing immune responses as surrogates of effectiveness were also used. The importance of immune correlates of protection (COP) increases as the pandemic proceeds and many individuals have been infected and may therefore already be wholly or partially immune. The assays of immune response also covered a very wide range of techniques [4–11]. Neutralization assays included neutralization of wild-type virus, of pseudoviruses in which a laboratory virus such as vesicular stomatitis virus is modified so that it expresses the antigen from the virus of interest and can be neutralized by antibodies against it, and SARS viruses genetically modified to include targets that express a gene such as luciferase, giving a readout in easily automated quantifiable form rather than the more laborintensive plaque assays or assays of cytopathic effect. Different detailed protocols were used, for example, measuring endpoints as 50% endpoint dilution titers, 90% endpoint titers, or total neutralization and the assays used a range of target cells. It is extremely difficult to relate the results in one system to those in another although easy to conclude that an immune response has been induced. Binding assays such as ELISA were also used as they are easy to perform and to automate, giving high throughput and reproducibility. The details of the available assays were widely different. Examples of the variation between assays include the target antigen adsorbed to the ELISA plate to which the specific patient antibodies will bind, which could be the entire S protein, a modified stabilized version of it, the S1 subunit which contains the region involved in virus adsorption to

Biological Controls and Standards for the Study and Control of Infectious Diseases

the cell, or a truncated stabilized version of it, and the receptor binding domain (RBD) itself which was of variable length depending on the particular laboratory. Some assays used the ability of antibodies to inhibit binding of the RBD to its human receptor ACE2 as a read out rather than measuring binding of the antibody to the antigen directly. The format of the assays varied; the reagents used in the assays were variable and the details of the incubation depended on the protocol developed. The particular subclass of immunoglobulin molecules detected could also be assay specific. The overall pattern is therefore one of chaotic complexity. While in some examples the differences in assays must have made major quantitative difference in the outcome, the general extent of the effects is not known and is of limited interest as the principle read out of the efficacy trials is to establish the degree of protection from disease. As the trials become harder to carry out because of the decreasing number of nonexposed, nonimmunized individuals, an immune correlate of protection (COP) becomes more desirable. Consequently, the objective is to try and relate the magnitude of the immune response to protection for the different vaccines and to compare the results of different clinical trials. Many groups sought to normalize their results by including a panel of convalescent human sera or plasma. In general, different panels were used so while the use of a panel made the results from different trials comparable to a degree (assuming that the convalescent serum panels were roughly the same) uncertainty remained. An international standard was prepared under the auspices of WHO at the National Institute of Biological Standards and Control in the UK with the intention that it should provide a single reference point for secondary reference materials and for all assays so that they may be compared whatever other references were included (Table 1). The assumption is that such international reference materials are sufficiently like the materials to be assayed to improve comparability while acknowledging that they cannot be identical to all samples that are received [12]. The extent to which they are formally and philosophically appropriate is a matter of heated debate. The criterion employed in this

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chapter is that if expressing results as a potency relative to that of the reference improves agreement between the results of different assays, the reference performs a necessary and valuable function.

Diagnostic Detection Methods: Nucleic Acid Detection and Hepatitis C The detection of hepatitis C by nucleic acid detection by PCR initially followed a course analogous to that described for the SARS2 antibody standard. At the start there was a great deal of heterogeneity and discrepancy in results but this has gradually resolved itself thanks in part to the use and development of reference materials and discussions among the various stakeholders. Various forms of hepatitis were recognized in the twentieth century. Hepatitis A is caused by a picornavirus spread by the fecal oral route, while hepatitis B is caused by a hepadnavirus spread by exchange of bodily fluids such as blood and by sexual contact. Once these two forms had been recognized and tests were available, it was clear that other forms also occurred. They were collectively termed non-A non-B hepatitis, one of which was blood borne and is now known as hepatitis C. Infections of recipients of blood products such as factor VIII used in treatment of hemophiliacs were very common if not universal, and transfusion transmitted infections occurred at high frequency, but the disease was wrongly thought to be mild. In fact, it is one of the most common causes of liver failure and hepatic cancer in the long term, setting up chronic infections. The infectious agent was discovered in the early 1990s by molecular means as it was not possible to culture the virus in vitro. Tests for specific antibodies were developed, followed by tests based on PCR to detect the presence of the virus nucleic acid. Results of PCR tests were often expressed in copies or genome copies and it was easy to take the view that the PCR tests were precisely and accurately quantitative and essentially chemical in nature. In fact, the situation was far more complicated and the genome copy of one laboratory was not necessarily the same as that of another. The technical detail of the assays including the nucleic

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Biological Controls and Standards for the Study and Control of Infectious Diseases

acid extraction methods and the regions of the genome targeted were different and other technical details of the assays varied. Genome or PCR detectable copies and infectious virus are not equivalent although they would be expected to correlate as the PCR assays detect only a small region of a genome that may be fragmented or overrepresented, or detectable with different sensitivities. Secondly even if the assay detected whole genomes, it does not follow that the nucleic acid is present in an infectious virus or that if it was, the virus would successfully infect a susceptible cell. Finally there are now known to be many strains and six distinct genotypes of hepatitis C and an assay that detects one genotype is not necessarily as sensitive in the detection of others. The distribution of the genotypes varies globally so that this factor could be important. Standardization of the assay results was a matter of great clinical significance for recipients of blood and blood products to minimize or prevent iatrogenic infections. There was no reference assay method that would give the “true” result or any criterion for what a “true” result meant. The situation was therefore ripe for biological standardization. Blood products such as clotting factors for the treatment of hemophilia may be made by recombinant expression or by fractionation of plasma from blood donors. The fractionated blood products derive from large pools of donations (in excess of 10000) so that an infected donor may contaminate many recipients and infection of recipients was a regular occurrence in the 1990s. Regulators and manufacturers adopted measures that included selection of donors, testing of individual donors and donations for markers of infection, testing of the pools before fractionation, and assessing the processes used for their ability to remove or inactivate viruses if they were present. In the 1990s pools were tested by the UK regulatory laboratory (NIBSC) who found 5% of them positive for HCV sequences, and a broadly similar selection of pools were tested by the German regulatory laboratory, the Paul Ehrlich Institute, who found 30% contained HCV sequences [13–16]. The different assays used were likely to be a factor in the discrepancy although the sample set was different [14].

Should similar issues arise between manufacturers and regulators, for instance if the manufacturer finds a pool negative but the regulator does not, the materials derived from it could not be released to the market. Products would be wasted and there would be major disruption of the supply of products which are lifesaving. Harmonization was essential. The first reagent that was widely used to compare and harmonize assays was a genotype 3 plasma and the results varied greatly depending on the nature of the assays which were mostly developed against genotype 1. The development of the first WHO international standard which was a genotype 1–contaminated plasma has been described [13] and it greatly improved agreement between assays when results were expressed as a potency relative to the standard rather than as genome equivalents or copies. Use of the reference began a gradual harmonization of assays including the region targeted by the PCR, the extraction methods, and other steps in the process such that currently agreement between assays is excellent. NIBSC and SANQUIN from the Netherlands also established SoGAT, a committee for the Standardization of Genome Amplification Technologies where diagnostic manufacturers, plasma fractionators, and regulators discussed issues and reached a consensus on their resolution (2, and follow the links to SoGAT). Currently there are very few transmissions of hepatitis C in Europe and the USA. The success of hepatitis C standardization raised a number of issues. Firstly the standard is a physical material used in the assays and therefore finite. At some stage it will be used up and therefore need to be replaced, and if the sensitivity and performance of the commercial tests used are expressed in international units, it will be necessary to calibrate the replacement as accurately as possible against the first. This is the aim of studies of replacements but as it is thermodynamically impossible to make the second and subsequent standards identical to the first, there will always be an uncertainty associated with the calibration. If the assays are relatively imprecise the uncertainty can be lost in the uncertainty of the assay, and these are exactly the kind of assays for which

Biological Controls and Standards for the Study and Control of Infectious Diseases

the references are intended and most useful. As precision improves, as it has with the HCV assays, replacement of the reference becomes more of an issue, and it might be reasonable to attempt to apply SI principles including reference assays to the problem. It is, however, easy to lose sight of the fact that the short sequence of the HCV genome which is detected and quantified in current PCR assays is really a surrogate of the main point of interest which is the infectious nature of the plasma preparation. It is also striking that the development of the HCV assays and the role of standardization have parallels with the SARS2 antibody issues described earlier.

Potency of Vaccines: Inactivated Polio Vaccine (IPV) The eradication of poliomyelitis is at an advanced stage and changing circumstances require changes in the vaccines used [17] Inactivated polio vaccine (IPV) was introduced in the 1950s and initially consisted of polio virus infected tissue culture medium treated with formalin under carefully controlled conditions so that the infectivity was lost but the immunogenicity was retained. A potency assay was required to assign the dose because it was to be expected that immunogenic material would be lost in the processing (which included filtration to remove cell debris and clumps of virus), and the formalin treatment itself which was quite likely to modify the viral proteins [18] and affect the immunogenicity of the viral particles. As the key property of interest is the ability of the vaccine to induce an immune response in recipients, the first assays focused on immunogenicity of the batches in animals, including monkeys, guinea pigs, and chickens. While the tests demonstrated immunogenicity, they were less satisfactory as quantitative tests to decide the dosage required. The rat immunogenicity test was developed by van Steenis and is quantitative [19]. However, because of variability between batches of rats and the conduct of the tests it is essential to include a reference material in each test as a basis for measuring potency and

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demonstrating the satisfactory nature of the vaccine under test. Tests involving animals are expensive, hard to reproduce, and increasingly unacceptable on ethical grounds. Consequently, there is pressure to replace them with in vitro alternatives, and for IPV this means using an assay for antigenicity, using the reaction of the vaccine with antibodies in an ELISA format. Poliovirus exists in at least two different antigenic forms, the D antigen predominantly associated with the infectious particle and the C antigen predominantly associated with the empty capsid or heated and inactivated virus [17, 18]. Antibodies to D antigen predominate in sera taken from patients in the convalescent recovery phase of the disease while antibodies to C antigen predominate in acute phase sera. It was therefore reasonable to assume that antibodies against the D antigen were protective, so that the D antigen was the active ingredient of the vaccine and the potency of the vaccine could be expressed in terms of its D antigen content. Antigenicity is the ability of a material to react with an antibody; immunogenicity is the ability of a material to induce antibodies. While it is likely that a treatment that affects one will also affect the other, particularly for a labile material like polio, they are not identical. The readout in most current formats of D antigen assays is a measure of the amount of antibody binding to the antigen (IPV) but the virus is a complex structure with many epitopes. Some epitopes on polio virus are known to be modified after the formalin treatment required to produce IPV [18]. The composition of the antibody preparation and the production process therefore affect the results obtained. For example, a monoclonal antibody might be used to detect the antigen. If the epitope to which the antibody is directed is affected by the formalin treatment it is likely to react less well than a monoclonal antibody against an unmodified site and therefore give a lower figure for antigen content. It is not clear what the “correct” figure would be in these circumstances and the use of antigen assays as a substitute for immunogen assays is only justified as a surrogate of potency for a product of consistent properties with a validated clinical history. In practice, most

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Biological Controls and Standards for the Study and Control of Infectious Diseases

IPV was produced by a few manufacturers from the same three strains (one for each serotype) using essentially the same production process, so that use of the antigen assay to harmonize potency assays was appropriate because of the great similarity of the products. The rat immunogenicity assay was retained as it seemed to reflect immunogenicity in human recipients. The situation changed with the success of the program for the eradication of wild-type polio [17]. The manufacture of classical IPV from wild-type viruses involves the growth of large amounts of virus which would be a potential source of reintroduction of the disease and it was considered that manufacture of IPV from the live attenuated Sabin vaccine strains would be significantly safer. Manufacture of IPV from the Sabin strains (sIPV) was therefore encouraged although they are antigenically slightly different from the strains used in most classical IPV production. The attempts so far to clarify and harmonize the assay of sIPV by developing reference materials illustrate how complex the measurement of vaccine potency can be. It became clear that as well as the antigenic properties, the immunogenicity of the strains was also different; in particular the type 2 strain of Sabin vaccine was about tenfold less immunogenic on the basis of mass, infectivity, or D antigen content than the classical strains. This is true in both animal models and clinical trials in human subjects, and it shows that D antigen content is not simply related to immunogen content. Strains differ in their specific immunogenicity per D antigen unit. The assay of D antigen content was also complicated, but in a different way. Measurement of vaccine potency is necessary to ensure that products from different manufacturers are comparable and immunogenic doses of vaccine are given. The initial focus has been on in vitro assays, involving ELISA assays of various formats, using different antibodies and different in-house technical details. The hope was that the reference materials used to assay classical IPV (consisting of preparations of classical IPV and relevant antibodies for capture and detection in ELISA) would work equally well for Sabinbased IPV (sIPV). Preliminary studies showed

that in assays of different products using in-house methods the use of the existing reagents did not improve agreement to the degree expected and it was concluded that sIPV-specific reagents were required. A collaborative study including sIPV from different manufacturers was set up; participants were to assay the materials using the existing classical IPV reference and their in-house assay methods and a common method using monoclonal antibodies and reagents which were supplied [20]. The results showed that the common method gave better agreement as might have been expected and one of the candidate sIPV preparations was established as a reference with D antigen content expressed in Sabin D antigen units distinct from the previous wild-type D antigen units. The situation remains complex. The sIPV products are clearly different in how they react and it is not clear how significant this is. An in vivo study of immunogenicity is planned. This raises an entirely different set of issues. At the time of writing it is striking that even for a virus like polio where antigenic variation within a serotype is not a major feature and immunity to one strain confers immunity to all strains of that serotype changing the strain in the vaccine has a profound effect on all methods for assessing potency.

Preparation and Establishment of International Standards The international standard (IS) for antibodies to SARS2 is a pool of serum or plasma from human subjects [12]. Serum is preferable as it is the most likely analyte in the assays that will use the reference and the principle of comparing like with like as closely as possible is fundamental to the process of standardization. A pool of samples is used for practical reasons as samples are likely to be available only in relatively small amounts, but there is also the theoretical advantage that the preparation will be more likely to contain representatives of all specificities if it is derived from many individuals. Many materials have been produced for other analytes where a biological activity is measured or the assay includes a complex biological process.

Biological Controls and Standards for the Study and Control of Infectious Diseases

Examples include, as above, vaccines, antigens, and nucleic acids used in diagnostic procedures. The details of the development are published in each case but the general principles of the formal process are summarized here. Details of the procedure to be followed in developing an international standard are given in WHO guidelines [21]. Identification of the Project A number of laboratories and institutes are classified as World Health Organization collaborating centers for specific topics, among them collaborating centers for biological standardization. This includes the National Institute of Biological Standards and Control (now a branch of the Medicines and Healthcare Regulatory Agency) in the UK which prepares, stores, and dispatches most, but not all, of the international references used worldwide. There is continuing in-depth discussion among the agencies with an interest in biological standardization to review progress and the need for novel references. NIBSC has an extensive back catalogue and part of the function is to evaluate the use of the materials in the catalogue and replace them as they become exhausted if they are still required. This would be the case for many of the references relevant to vaccines of all types, including potency standards and reagents for classical inactivated and live vaccines which will be replaced as a matter of course as needed. In all cases the lead is taken by a WHO Collaborating Center for Biological Standardization. An example comes from the developing use of the live attenuated Sabin vaccine strains for the manufacture of inactivated polio vaccines rather than the classical strains that have been used for over 50 years. As described above, studies were required to see if existing reference materials for use in potency determination were suitable for the new strains and to develop new references when it was shown that they were needed. The need for new references may be raised with a collaborating center by WHO, NGOs such as the Bill and Melinda Gates Foundation or PATH, by CEPI (for new and emerging infections such as Ebola Zika or SARS-2), or any of the collaborating centers or manufacturers. While many projects are initiated from Europe or the USA this is increasingly not the case. For

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instance, international references for Enterovirus 71 to help standardize assays of serum antibodies and vaccines were developed as the result of an initiative by the Chinese regulatory laboratory and Collaborating Center (NIFDC) in response to a clear need identified in China and East Asian countries that was not present in Europe or the USA where EV71 is less of an issue at present. The portfolio of International Standards for EV71 now available through WHO is the result of a joint program between NIFDC and NIBSC. Except under special circumstances such as response to the covid pandemic, preparation of international references typically takes two years. It requires a great deal of financial input including manpower and capital equipment including adequate reliable storage space where the references may be kept safely for a decade or more. Approval of the Project by WHO The Expert Committee on Biological Standardization (ECBS) of WHO was established in 1947 to respond to the remit for biological standardization given to WHO by the United Nations. A proposal is submitted to ECBS by the collaborating center leading the project which summarizes the need for the reference and how it will be made, the likely demand, the source of the material to be used, and setting out a timetable for various steps in the process including filling the vials, completing the collaborative study, and submitting the results to ECBS with a final proposal for establishment. If the proposal is approved, ECBS will receive updates on progress as required, particularly if problems are anticipated. For example, sourcing material for the development of a reference material for Ebola antibodies was made difficult by other possible uses for convalescent plasma or serum, such as clinical treatment of cases. Starting Material The reference is a physical standard where supplies are eventually exhausted and as replacing it is a major project and creates problems of calibration, the batch size is usually large, (typically 2000–10000 vials) with the intention that it should meet the expected demand for 10–15

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years. It can be difficult to obtain sufficient material. It is possible to include materials from alternative sources in the eventual collaborative study to see if they work well enough to be used. In preparing antibody standards for the Ebola outbreak one material in the study was human immunoglobulin prepared from transgenic cows carrying human antibody genes which was included for comparison with more conventional samples. Monoclonal antibodies are often proposed as standards for serological assays and have been included in some studies. In general, they have not proved useful in improving agreement between assays, possibly because of the limited range of molecules involved compared to conventional sera. The reference should be as similar as possible to the material to be analyzed and this must be recognized in sourcing the reference material. In the Ebola outbreak serum could be sought from repatriated health care workers in the USA or UK who may have had many medical interventions compared to convalescent patients from the afflicted countries, who would be the patients of particular concern. In preparing the SARS2 reference serum material could have been obtained from vaccine trials or convalescents, where the responses would be different. It is possible to explore and identify possible differences between materials in the collaborative study where laboratories test the same samples and this can provide useful information. Apart from the serological references, references for vaccines are of particular value to ensure the harmonization of tests for immunogen and antigen content. The vaccine should be typical of those available and use of the reference ensures that vaccines from different sources are comparable and therefore likely to produce the same effect. This is not yet an option in SARS2 because of the diversity of vaccines and platforms explored. The material to be used will be evaluated in the laboratory leading the study for its suitability in whatever assays are available. Sometimes it may be useful to conduct a preliminary collaborative study among an additional two or three laboratories to see if the material is likely to be suitable.

An important criterion is that the material be safe. Thus serum should be free of the statutory blood-borne viruses such as HIV HCV and HBV, and if a pathogen such as Ebola or SARS is to be used as a reference for antigen or nucleic acid assays, it must be presented in a way that renders it nonhazardous. Viruses may be inactivated for antigen assays or nucleic acid expressed in safe vectors. Filling the Vials The reference material should be obtained, stored, and processed by the lead laboratory under a suitable formally accredited quality management system. It should be filled into stoppered vials or glass-sealed ampoules preferably in specialized purpose designed facilities. In many cases the material is lyophilized as this makes the material more stable and it can be shipped at room temperature. However, this means that a precise quantity of material must be put into each container and the lyophilized material must be reconstituted in a precise specified volume to give the correct concentration. The variation of the fill volume required to do this properly is less than 1% and this is more precise than can be achieved by most commercial vaccine filling machines. After lyophilization the ampoules are flushed with inert gas and sealed to prevent oxidation and improve stability. Stoppered vials can allow such gaseous exchange and are therefore not usually the container of choice for lyophilized materials. Containers are typically stored at –20ºC before dispatch. The freeze-drying process is highly technical in nature and the filling and lyophilization of international standards is very specialized. If the reference is not lyophilized the precision of fill required is irrelevant as the concentration of the reagent does not depend on a reconstitution volume. On the other hand, the filled vials will have to be stored under conditions where the contents are adequately stable and this usually but not always means low temperatures(–80 ºC or below). Shipping must be at low temperatures, making it expensive and vulnerable to breaks in the cold chain. Liquid fills are therefore not so desirable as lyophilized materials when it comes to the international references.

Biological Controls and Standards for the Study and Control of Infectious Diseases

It must be shown that the processing of the material to give the final filled vial does not affect the activity of the material in an unacceptable way, so that there are always developmental steps before the main fill to establish the correct parameters. An additional requirement is to show that the reference has an acceptable stability under the conditions of storage. This will involve testing containers taken in real time under the conditions of storage, usually –20 ºC, but also accelerated degradation studies where containers are stored at elevated temperatures (such as 4, 20, and 37 ºC) and samples taken at shorter intervals. Stability is then predicted by the Arrhenius equation. This process gives an indication of stability but is not necessarily reliable for a complex analyte, and it must be supplemented by real-time studies of some sort, including feedback from users. Details of the procedures involved in producing a filled batch are given in [21] and are most relevant for the specialized laboratory that produces the filled containers.

The Collaborative Study: Participating Laboratories The main objective of a collaborative study of a biological reference is to demonstrate that it is suitable for the calibration of other materials, whether they are secondary references or material such as vaccines or sera where it is necessary to know the potency of the material for analytical or clinical purposes. There are many nuances to this primary aim; for instance, the study may show that different assays are in fact measuring different aspects of the materials tested, or that the reference material is not typical, or that the analytes are so variable that no material will be suitable. The intention is to assign a potency to the reference in the light of the collaborative study and to demonstrate that expressing results as potencies relative to the reference improves agreement between assays to an extent that makes it worthwhile. This may be within a laboratory, between laboratories or between assays. If successful, the use of a

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potency will allow comparison of results in diagnosis or dosing in clinical use. For laboratory studies to produce a certified reference material under the SI system the laboratories must be selected extremely carefully, and qualified and accredited to appropriate ISO standards. Only one or maybe two laboratories will be involved and the reference method is specified precisely. In contrast for biological standards the intention is to involve many laboratories, typically 5–25, who will eventually have an interest in using the reference. Participating laboratories should be competent, but not necessarily formally accredited to a quality system. The assays used are not specified by the study coordinator in detail and should be already established in the participating laboratories. The assumption is that they will differ from laboratory to laboratory but that as no assay is necessarily more accurate, precise, or correct than any other the best final estimate will be a composite of the results of all of them. The study can therefore lead to conclusions on the nature of the assays used and the extent to which they are fit for purpose. Laboratories will be invited to take part and will include manufacturers, regulatory laboratories, public health laboratories, and others. It is advantageous to have good geographical representation as this helps the global acceptance and use of the references giving greater global harmonization, and there is now a good distribution of competent laboratories around the world. The entire process is based on consensus.

The Collaborative Study: Study Samples The samples are sent to participating laboratories as a coded panel, which includes the candidate reference and other materials. As a minimum, duplicates of the candidate reference will be included but the nature of the panel varies with the intended purpose of the reference. If a vaccine is the target the panel may include vaccines from different manufacturers, low potency or failed batches, alternative candidate references, and possibly (as for the IPV study) vaccines made from

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different seed strains. For a serological reference, materials from different sources such as convalescent sera from different locations or vaccine trials, or samples of high or low potency may be included. It is important that the number of samples is limited given the amount of work involved. Some guidance may be given on the suitable dilution range of the samples. Supplementary studies may be needed to assess factors such as commutability which is the extent to which the reference is truly able to represent the real analytes. The samples will be assayed blind by participating laboratories by whatever method or methods they wish to use, as notified to the study coordinator. Usually three assays will be performed on each sample and the inclusion of blinded duplicates allows the true variability of the assay in a laboratory to be assessed when samples are retested. This gives a figure for intra-laboratory variation to be compared with inter-laboratory variation.

The Collaborative Study: The Assays The fundamental principle of the study is that the assays used in the study are not specified by the organizer, but that they should be listed in the report. This makes it possible to see whether different assays give different results, for example, whether neutralization assays for viral antibody levels relate to titers obtained by ELISA, and how close the relationship is. Statistical validity is a crucial aspect of the studies, which must be designed with the collaboration of a biostatistician. The assays should allow assessment of their validity; for example, a dilution series of two samples for comparison should give curves that are parallel and linear within acceptable statistical limits which will require definition. The results of the assays should be returned to the study coordinator as raw data to allow them to be analyzed by a common statistical approach, but the participating laboratories should also submit their own analysis to confirm that similar conclusions are being drawn.

The Collaborative Study: The Report The study organizer will draft a report of the study, how it was carried out, the results, and the analysis and conclusions with a recommendation where relevant that the material be established as a reference material and the potency to be assigned. This is then circulated to participants for comment and amendment as required. Once agreement is reached the report is submitted to WHO for consideration by the Expert Committee on Standardization, and circulation to their expert panel for comment. The submission will then be discussed by WHO and if appropriate the material will be established as an international standard with an arbitrary assigned unitage. This may not be straightforward. In the instance of the reference for SARS2 antibodies the data were submitted to the WHO Expert Committee on Biological Standardization (ECBS) and the IS established as part of the formal process as the first International Standard antibody for neutralizing antibodies against SARS 2 with an assigned potency of 1300 international units (IU) per vial. The samples in the study were also assayed by in vitro binding assays of various formats. This raises the issue of how the different assays relate to each other and to protection. In the early days of standardization of antibody measurements, most assays were based on virus neutralization, or an assay such as hemagglutination inhibition (HI) that was considered likely to be related to a biological readout. Neutralization assays are mechanistically very complex and poorly understood virologically. However, the technical readout is fairly simple, involving inhibition of a cytopathic effect by the antibody, or some other more sophisticated evidence of infection, for example, where the challenge virus has been manipulated to contain a marker gene such as luciferase where the measurement of expression can be automated easily. The use of a reference control for variation in the execution of the assay improves agreement within and between laboratories and is widely accepted. It was therefore reasonable to use the same reference materials to improve agreement between

Biological Controls and Standards for the Study and Control of Infectious Diseases

binding assays such as ELISA and to assume that the results were linked to the neutralization titers, so that if a neutralizing antibody titer was shown to be protective, the same titer in IU as measured by ELISA would also be protective. This is not necessarily true. Not all binding antibodies are neutralizing, not all neutralizing antibodies are detected by binding assays and not all antibodies are protective, although at a population level there is usually a correlation. The issue reached a crisis in rubella where pregnant women are tested for antibodies to see if they needed a vaccination to ensure protection. Protective levels for an individual were expressed in IU and depending on the assay used vaccination might be needed or not, which is clearly unacceptable. Consequently, a view has developed that international references for antibody assays in their current form should only apply to neutralization or other strictly biological assays and not to ELISA or binding assays. Data from binding assays were produced in the same collaborative study that established the neutralizing antibody International Standard for SARS2 and were reviewed. They showed that the use of a common reference previously assigned a unitage for neutralization assays improved agreement between assays to such an extent that ECBS assigned potencies (in binding assay units or BAU rather than international units). The first observation is that the same IS improved agreement between assays within a category (e.g., anti-RBD IgG assays) and between categories (e.g., neutralizing antibodies, antiRBD antibodies, and anti-N antibodies). Not only are the activities different, as for the neutralizing activity and the anti-RBD binding assays, but in the instance of the anti-N IgG and anti-S IgG they are against different proteins. This is probably a result of the nature of the samples, which were all postinfection convalescent plasma. All antibodies were induced by infection with the whole virus and the spectrum of antibodies might be expected to be reasonably comprehensive. The same would not be true for postvaccination sera. Moreover, depending on the vaccine antigen the spectrum of antibodies would be different; a vaccine based on RBD would not be expected to induce antibodies to

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parts of the S1 or S proteins outside the RBD. Consequently, the use of the reference for binding assays if it is acceptable at all must be approached with care and the data interpreted with caution and a clear view of what is being measured. Having said that the observation from phase 3 clinical trials is that protection correlates with a general level of seropositivity at roughly the same titer for different vaccines. It would be wrong to regard this as a hard, definite level of protective antibody in an individual but it seems to be an indication, and a suggestion of the strength of antibody response required to have the desired effect. The reference may be useful, but it must not be misleading.

Future Directions There are many niche reference materials where a need remains, although several are also disestablished every year as they become surplus to requirements. This will remain the case. New reference materials will be required as new issues arise. For example, over the past few years a number of infections have emerged or acquired greater significance, including Ebola which caused an unprecedented outbreak in Western Africa; Zika, which caused unexpected clinical issues in Brazil and elsewhere; and SARS2 whose emergence as a major concern for world health was unexpected. In addition, novel complex treatments such as cell and gene therapies are developing and are clearly biological treatments in the classical sense that could benefit from the approaches described here. New technologies such as next generation sequencing will have a major impact on the diagnosis of disease and the control of live attenuated vaccine, perhaps obviating the need for animal testing; the control and standardization of these methods is already being addressed but will require major further efforts. In addition, as understanding of the activities being measured increases it is likely that assays will cease to be biological in the sense used here and will fall under the remit of the SI. This has already happened for insulin, which is now a well-

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characterized product with dosing expressed essentially in micrograms and it is predictable for diagnostics such as PCR for hepatitis C. The field continues to move and adapt to novel developing circumstances and needs.

Bibliography 1. Mattiuzzo G, Bentley EM, Page M (2019) The role of reference materials in the research and development of diagnostic tools and treatments for haemorrhagic fever viruses. Viruses 11:781. https://doi.org/10.3390/ v11090781 2. https://nibsc.org 3. www.bipm.org 4. Zhu F-C, Li Y-H, Guan X-H et al (2020) Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a doseescalation, open-label, non-randomised, first-inhuman trial. Lancet 395(10240):1845–1854. https:// doi.org/10.1016/S0140-6736(20)31208-3 5. Mulligan MJ, Lyke KE, Kitchin N et al (2020) Phase 1/2 study to describe the safety and immunogenicity of a COVID-19 RNA vaccine candidate (BNT162b1) in adults 18 to 55 years of age: interim report. Nature. https://doi.org/10.1038/s41586-020-2639-4 6. Folegatti PM, Ewer KJ, Aley PK et al (2020) Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial Oxford corona virus ph1 trial 20th July https://doi. org/10.1016/S0140-6736(20)31604-4 7. Zhu F-C, Guan X-H, Li Y-H et al (2020) Immunogenicity and safety of a recombinant adenovirus type-5vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebocontrolled, phase 2 trial Cansino corona virus ph2 trial 20th July. https://doi.org/10.1016/S01406736(20)31605-6 8. Xia S, Duan K, Zhang Y et al (2020) Effect of an inactivated vaccine against SARS-CoV-2 on safety and immunogenicity outcomes: interim analysis of 2 randomized clinical trials. JAMA. https://doi.org/ 10.1001/jama.2020.15543 9. Anderson EJ, Rouphael NG, Widge AT et al (2020) Safety and immunogenicity of SARS-CoV-2 mRNA-1273 vaccine in older adults. https://doi.org/ 10.1056/NEJMoa2028436

10. Jackson LA, Anderson EJ, Rouphael NG et al (2020) An mRNA vaccine against SARS-CoV-2 – preliminary report. N Engl J Med 383:1920–1931. https://doi.org/10.1056/NEJMoa2022483 11. The Oxford Covid Vaccine Clinical Trial Group (2020) Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Published Online December 8, 2020. https://doi.org/10.1016/S0140-6736(20) 32661-1 12. Knezevic I, Page M, Minor PD et al (2021) WHO International Standard for evaluation of the antibody response to COVID-19 vaccines: call for urgent action by the scientific community. Lancet Microbe. https:// doi.org/10.1016/S2666-5247(21)00266-4 13. Saldanha J, Lelie N, Heath A, The WHO Collaborative Study Group (1999) Establishment of the first International Standard for nucleic acid amplification technology (NAT)assays for HCV RNA. Vox Sang 76: 149–158 14. Saldanha J (1999) Standardisation of nucleic acid amplification techniques (NAT) for the detection of viral contamination of blood and blood products. Cryo-Letters 20:147–154 15. Saldanha J (1999) Standardisation progress report. Biologicals 27:285–289 16. Saldanha J, Heath A, Lelie N et al (2000) Calibrationof HCV working reagents for NAT assays against HCV International Standard. Vox Sang 78:217–224 17. Minor PD (2012) The polio eradication programme and issues of the endgame. J Gen Virol 93:457–474 18. Ferguson M, Wood DJ, Minor PD (1993) Antigenic structure of poliovirus in inactivated vaccines. J Gen Virol 74:685–690 19. Van Steenis G, van Wezel AL, Sekhuis VM (1981) Potency testing of killed polio vaccine in rats. In: International symposium on reassessment of inactivated poliomyelitis vaccine, Bilthoven 1980, Dev Biol Stand 47:119–128 (S. Karger, Basel 1981) 20. Laura Crawt L, Atkinson E, Tedcastle A et al (2018) WHO/BS/2018.2338 Report on the WHO collaborative study to establish the 1st International Standard for Sabin inactivated polio vaccine (sIPV). https://www.who.int/publications/m/item/who-bs2018.2338 21. World Health Organization (2006) Annex 2 Recommendations for the preparation, characterization and establishment of international and other biological reference standards (revised 2004) WHO Technical Report Series, No. 932, 75-131

Next-Generation Sequencing in the Study of Infectious Diseases Neta S. Zuckerman1 and Lester M. Shulman2,3 1 Bioinformatics and Metagenomics, Central Virology Laboratory, Public Health Services, Israel Ministry of Health, Tel-Hashomer, Israel 2 Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 3 Central Virology Laboratory, Laboratory of Environmental Virology at Sheba Medical Center; Retired, Public Health Services, Israel Ministry of Health, Tel Aviv, Israel

Article Outline Glossary Definition of the Subject Introduction Sample Preprocessing Library Preparation and Sequencing Bioinformatics Analyses: Viroinformatics Conclusions Future Directions Bibliography

Virome The collection of all viruses in a specific habitat or ecosystem. It includes eukaryotic viruses, viruses that infect eukaryotic cells; bacteriophages, viruses that infect bacteria or archaea; and human endogenous retroviruses (HERV), e.g., integrated viral elements in the human genome that closely resemble and could be derived from retroviruses, or their equivalent in nonhuman species

Definition of the Subject Viruses are an important member of the microbiome and have been shown to have an impact on human health and disease. Next-generation sequencing (NGS) is one of the major tools used to study the molecular basis of viruses and their composition in a specific habitat of ecosystem of interest. Starting with the most basic “ingredient” of any research study, a sample, this entry will familiarize the reader with the various molecular approaches to enrich viruses in that sample, the different library preparation and sequencing technologies to obtain viral genetic sequences from that sample, and the numerous bioinformatics algorithms and types of analyses that exist to analyze and make sense of all the information contained within those sequences.

Glossary Introduction Bioinformatics An interdisciplinary approach for analyzing, interpreting, and assigning biological annotations to sequence data Metagenomics Sequencing and analyzing all the genetic material in the sample of interest Microbiome (microbiota) The collection of all microorganisms that live in a specific habitat or ecosystem Next-generation sequencing A high-throughput, parallel method to sequence DNA/RNA fragments

Living, self-replicating organisms and infectious disease-causing organisms have existed side by side for millions of years. Just as these organisms evolve, so have our tools for understanding the molecular basis for their evolution. One of tools underlying the biggest advancements achieved in understanding the molecular basis of evolution is our ability to sequence nucleic acids that make up the genomes of such organisms and the downstream nucleic acids that participate in

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_1090 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2020 https://doi.org/10.1007/978-1-4939-2493-6_1090-1

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determining their phenotypes. These technological advancements have gone hand in hand with fundamental changes in our perception and understanding of what constitutes a complex living organism such as a human being, for example. One of the fundamental changes in perception is that the complex living organism is really a host, carrying a collection of symbiotic mutual (both benefit), commensal (one benefits without affecting the other), and parasitic (one benefits at the expense of the other) organisms. Indeed, the quantity of microbial cells present in a human body was recently estimated at the ratio of 1.3:1 for microbial to human cells [1], with bacterial cells containing many more genes compared to the human genome [2], and the human genome was estimated to be composed of up to 8% of sequences with viral origin [3]. Another fundamental perceptual change is a transition from thinking in terms of a single agent causing a single infectious disease to the concept that the ability of an infectious agent to cause a disease (its phenotype) depends not only on the genotype of the agent and its host but is also moderated by the collection of genotypes and interactions of the phenotypes of all the organisms living in it at the time of infection. In fact, these organisms sharing our body do not only affect in the body’s reaction to infectious diseases but also have a role in regulating fundamental elements such as our mood, cognition, or pain [4]. The first scientific evidence of a bacteria being a part of a human system was observed by the pediatrician Theodor Escherich in mid-1880, in the intestinal flora of children suffering from diarrhea and in healthy children; this bacteria was later named Escherichia coli [5]. Since this discovery, countless studies have investigated the role of bacteria in human health and disease. However, the term “microbiome” was first coined by Joshua Lederberg in 2001 and referred to the collection of microorganisms that “literally share our body space and have been all but ignored as determinants of health and disease” [6]. The microbiome (or microbiota, terms which are often used interchangeably in the literature) is the collection of all microorganisms that live in a specific habitat or ecosystem, for example, the human body, and is

composed of bacteria, archaea, viruses, parasites, and fungi. With the realization that the microbiome holds vital roles in many functions of the human body, numerous worldwide projects were established aiming to understand these roles and how they impact human health. Since then, the microbiome has been shown to be beneficial in allergy prevention, increased gut absorption, and reducing infection and even involved in reducing the risk for cancer [4]. The microbiome has been also linked to diseases such as inflammation and cancer, bowel diseases, obesity, arthritis, and autism, and these findings greatly contributed to better stratification of patients and novel disease biomarkers [2]. Despite extensive research efforts in all fronts of the microbiome realm, the most studied member of the microbiome to date are bacteria, mainly due to the development of standardized methods that have helped in exploring this community. Specifically, the usage of 16S ribosomal RNA (a highly conserved gene that is widely present in all bacterial species) as a biomarker has enabled the development of specialized tools and pipelines to characterize bacterial communities in various health and disease conditions [7]. In recent years, efforts are also directed toward investigating the viral part of the microbiome. The term “virome” is used to refer to the collection of all viruses and includes eukaryotic viruses that infect eukaryotic cells, bacteriophages that infect bacteria or archaea, and human endogenous retroviruses (HERV), which are viral elements in the human genome that closely resemble and can be derived from retroviruses [3]. Viruses are among the fastest mutating elements, and the global virome is estimated to contain the most members of all communities in the global biome, with ~1031 members of our planet’s virome, ~1.5 million mammalian and waterfowl viruses, and more than 260 viruses that can infect human cells [8–10]. Viral genomes can consist of DNA and/or RNA that is single or double stranded and can even include regions of both [11]. Although viruses are the most abundant entity on earth, challenges in their detection, isolation, and classification underlie the paucity of data collected on them so far, with only ~2200

Next-Generation Sequencing in the Study of Infectious Diseases

genomes deposited in public repositories such as NCBI (National Center for Biotechnology Information) [10]. In contrast to bacteria, viral communities are more challenging to characterize, as there are no phylogenetically conserved genes that are common to all viruses. Thus, metagenomics, i.e., sequencing all the genetic material in the sample of interest, is becoming the preferred approach to study the virome. The composition of the human gut virome, for example, which is under extensive research in recent years, is precipitately being revealed. There are ~1015 DNA and RNA viruses in the gut microbiome, outnumbering bacterial cells [12]. Most of the viruses in the human gut have DNA genomes with 99% of them bacteriophage and the remaining 1%, eukaryotic viruses [11, 13]. Of those with RNA genomes, 97% in the healthy gut were pathogenic plant viruses and the remaining 3% animal viruses [11, 14]. The gut virome was found to be a key player in shaping the gut microbiome; however, its roles in human health and disease remain largely unexplored. In the classic view, viral infections were asymptomatic or symptomatic with a specific virus causing the specific disease. Recent discoveries have expanded the way we view effects of viral infection by correlating presence of viruses with changes outside of this simple paradigm, including regulation of host physiology, participation in dynamic regulation of the immune system and microbiota homeostasis, protection against invading microorganisms, regulation of autoimmune responses, and influencing systemic infections [11, 12, 15, 16]. Viruses have coevolved with humans and their microbiome, and their interactions with the microbiome play a role not only in the setting of disease but also in human health. Indeed, many eukaryotic viruses rarely cause disease and/or remain dormant within the human host. Viruses can modulate the composition of bacterial and archaeal host populations (direct interaction) or act as vectors of genetic elements that alter microbial host genotypes and phenotypes by horizontal gene transfer which may change the homeostatic balance (indirect interaction) [17]. The diversity of the microbiome may also facilitate changes in host

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ranges of viruses [18], with the key concept being that the human host and its microbiome are in a homeostatic balance that may be altered by viral infections and which may determine susceptibility and response to infections and other diseases [15, 16]. Enteric viruses, for example, were reported to recapitulate the beneficial effects of commensal bacteria by modulating the innate and adaptive immunity of the host [19, 20]. Acute rotavirus gastroenteritis was shown to radically change the composition of the gut microbiome, which in turn can affect the composition of the gut virome [12]. In the setting of diseases, viral infections are also implicated as inducers of cancer and other related disorders. In soft tissue Kaposi sarcoma, for example, the etiology was linked to Kaposi’s sarcomaassociated herpesvirus (KSHV), also known as human herpesvirus 8 [21]. KSHV was also shown to be the causative agent of primary effusion lymphoma and the plasma cell variant of multicentric Castleman’s disease [22]. Besides KSHV, six additional oncoviruses were thus far identified as collectively responsible for 12–15% of cancers in humans [23] – Epstein-Barr virus, the first discovered oncovirus, is associated with several types of lymphomas [24], human T-lymphotropic virus type 1 has been shown to cause adult T-cell leukemia/ lymphoma [25], chronic infection with hepatitis B virus accounts for at least 50% of hepatocellular carcinomas worldwide [26], hepatitis C virus is one of the major etiologic agents of liver cancer [27], human papillomavirus is associated with cervix and many other cancer types [28], and finally Merkel cell polyomavirus is associated with the rare and aggressive type of skin cancer Merkel cell lymphoma [29]. Zoonotic viruses are transferred by animals or insects to humans and are responsible for a myriad of human diseases, some of which are life-threatening like Ebola virus. Changing climate and expansion of humans into areas of high population diversity increase the chances for emergence of such viruses, with an estimate of 600,000 to 800,000 unknown viruses that have a zoonotic potential for infecting humans [30]. Livestock are similarly at risk. Transmissibility of the emerging viruses may result in large outbreaks and even pandemics causing high economic damage and immense health burdens [31].

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Next-Generation Sequencing in the Study of Infectious Diseases

Since the development of the first-generation Sanger sequencer by Fredrick Sanger and colleagues in 1977, two additional sequencing “generations” have been developed to date. All three generations of sequencing approaches have profoundly advanced our knowledge of infectious diseases. A quick search on the Internet reveals the many studies where first- and especially second- and third-generation sequencing techniques have been used for basic and populationscale genome research: to study human, animal, and plant genetics; to deepen our understanding of the microbiome; to perform environmental research; to study tumors and tumorigenesis; for transcriptome analysis; for single-cell studies; for population-scale genomics; for clinical research; for clinical diagnosis; for rapid identification of pathogens; for surveillance and outbreak investigation; for insights into paths of transmission including animal and insect vectors and zoonosis; for risk analysis for diseases susceptibility, pathogen virulence, and antibiotic and antiviral resistance and spread between strains and species; to characterize the evolutionary relationships to ancient genomes (termed paleobiology); and by combinatorial analysis to enhance our understanding of how genetic differences affect health and disease. Finally, to quote Metzker on the potential for next-generation sequencing (NGS), “one’s imagination being the primary limitation to its use” [32]. This era of technological advancements enables us to make headway in many areas of research, revealing novel data which were not possible to obtain otherwise. The continuous development of NGS and its gradual introduction

into all areas of research and essentially into public health laboratories and application to clinical aspects drive molecular biologists into educating themselves about these new technologies and even get involved with bioinformatics and data analysis. Indeed, nowadays many biologists are required to learn such skills, and many institutions offer formal training, as the field matures rapidly. In this entry, you will find description of the different approaches, methodologies, and pipelines utilized to explore the virome. Starting with the most basic “ingredient” of any research study, a sample, this entry will familiarize you with the various molecular approaches to enrich viruses in that sample, the different library preparation and sequencing technologies to obtain viral genetic sequences from that sample, and the numerous bioinformatics algorithms and types of analyses that exist to analyze and make sense of all the information contained within those sequences. Figure 1 summarizes these steps which are described in detail in the following sections.

Sample Preprocessing Traditionally, the virus to be sequenced is extracted from a clinical or environmental sample and inoculated in an appropriate cell line. Prior to sequencing, the cultured virus is extracted from the culture or culture supernatant via centrifugation or high-density gradient [33, 34]. This and similar approaches provide a considerable source of enriched viruses for molecular characterization, serological studies, and neutralization tests. However, in addition to being laborious and time-

Next-Generation Sequencing in the Study of Infectious Diseases, Fig. 1 Metagenomics workflow

Next-Generation Sequencing in the Study of Infectious Diseases

consuming, characterization of cultured viruses may be hampered by nonnatural accumulation of mutations as a result of repeated passaging of the virus in cultures or the usage of non-appropriate cultures [34–36]. In addition, these methods focus on a single virus and cannot be utilized to explore all viruses in the sample, i.e., the virome. Capturing the virome is challenging, as viruses do not have a universally conserved marker like, e.g., the bacterial 16S ribosomal RNA [7]. Viruses vary not only across viral families, but mutation-driven variants are also generated in the same virus within the same host (quasispecies). In recent years, culture-free approaches, applied directly to clinical or environmental samples, are being increasingly used to sequence viruses. However, challenges arise from direct NGS due to the relatively small abundance and size of the viral nucleic acids in the clinical/environmental samples compared to those of other organisms such as bacteria or human. These background genomes affect the sensitivity of viral particle detection by NGS as the larger genomes are sampled more during sequencing and produce a greater amount of sequence reads compared to organisms with smaller genomes such as viruses. These challenges are slightly overcome with increased sequencing depth or length of the short sequence reads. Nevertheless, achieving higher proportions of viral nucleic acids increases the probability that viral sequences will be sufficiently represented in the data in order to, e.g., be discovered or for obtaining complete viral genomes. Viral-enrichment techniques, applied to samples prior to sequencing, enhance viral detection via NGS and in addition spare unnecessary expenditure and generation of data. Myriad of physical enrichment methods, e.g., ultracentrifugation, filtration, and nuclease treatments, which are the most commonly used, have been explored as single treatments or in combination and applied to enrich viruses from various sample types such as serum, feces, tissues, and even soil [37]. Ultracentrifugation separates the small viral particles from the rest of the mass existing in samples. Filtering also takes advantage of the small size of viruses to get rid of human and bacterial content in the

39

samples, with 0.45 and 0.22 mm syringe filters most commonly used, sometimes successively. Nucleases, e.g., OmniCleave, cleave free DNA and/or RNA while leaving capsid-protected viruses unharmed [37, 38]. Viral-enrichment methods have been demonstrated to greatly improve sensitivity to virus discovery via NGS and metagenomics approaches. A combination of filtration, nucleases, and random RNA and DNA amplification was optimized to reliably recover adeno, herpes, polio, and influenza viruses in plasma clinical samples, with correlation to the number of sequenced reads for each virus [38]; pre-treatment of cerebrospinal fluid (CSF) with DNA digestion alone aided in discovering viral etiological agents in pediatric patients with acute encephalitis and encephalopathy [39]; and numerous types of adenoviruses discovered in urban sewages following filtration and DNA digestion enrichments [40], to name a few examples. However, different types of samples greatly vary in their composition and complexity; therefore enrichment methods should be specifically designed according to the sample type and viruses in it [41]. In contrast to the abovementioned enrichment methods, which focus on subtraction of the host genome and other microbes prior to viral sequencing, a different group of enrichment approaches aim at capturing the virus or group of viruses from the sample prior to sequencing. These methods are more sensitive and specific and can be applied to samples with limited volumes; thus they are mostly utilized for clinical purposes such as diagnostics. Virus-capture approaches are versatile and can be designed to capture a large group of viruses, e.g., all human viruses; a family of viruses, e.g., all enteroviruses; and the complete genome of a single virus or part of a viral genome, e.g., one envelope gene. The latter approach is widely used for genotyping viruses in clinical laboratories; the viral gene is captured and amplified via PCR using specific primers and probes and sequenced via Sanger [42]. Two main virus-capture approaches are amplicon-based and hybridization-based. Amplicon-based sequencing is widely used and involves PCR amplification via specific primers and probes designed to capture the required virus/

40

Next-Generation Sequencing in the Study of Infectious Diseases

viruses [43]. This approach has been utilized to capture single viral genes, e.g., the envelope gene in HIV-1 [44], but more studies are moving toward whole genome capture, e.g., as demonstrated with coronavirus [45] and measles virus [46]. However, this approach includes several disadvantages, including technically challenging and timeconsuming normalization and optimization processes and longer PCR cycles which could introduce mutations [47, 48]. Hybridization-based capture relies on hybridization of specific probes to the virus/viruses of interest, followed by capture with magnetic beads, is able to capture the entire genome of multiple viruses with only one sequencing reaction [43], and was even demonstrated to discriminate between viral serotypes in clinical samples [48]. VirCapSeq-VERT (virome capture sequencing platform for vertebrate viruses) was the first method developed to capture, via specific probes, 207 different virus taxa that infect vertebrates including humans. The method increased targeted sequence reads by thousandfold, in addition to reducing host background and increasing viral genome coverage to above 90% [49]. Similarly, ViroCap [50] and ViroFind [51] are able to capture 337 and 535 selected DNA and RNA viruses infecting humans, respectively. Multitude of studies describe capture protocols, hybrid and/or amplicon capture-based, to enrich viruses in clinical samples in order to obtain their complete genomes via NGS [52].

Library Preparation and Sequencing NGS is nowadays an accessible technology that is widely applied to numerous fields in biological research. As such, various methods and pipelines have evolved to answer research needs depending on the objectives, accuracy, and available costs. The types of analyses made possible by NGS include not only deep sequencing which facilitates variant calling but also enables study of gene expression and regulation, exome interrogation, epigenetics, and more. In the field of virology, NGS may be utilized to (a) study the host response to viral infection via, for example, application of RNA sequencing followed by gene

expression analysis, and/or (b) sequence the virus itself, completely or partially, where the viral genome can be applied to study, e.g., virus mutation and evolution, perform epidemiological investigations of viral outbreaks, aid in vaccine design, or explore the virome. Our ability to sequence nucleic acids using high-throughput automatic machinery has undergone three major stages (or – generations) of development to date, with numerous platforms available for sequencing nowadays. The first sequencing technology developed was by Sanger [53, 54], termed now first-generation sequencing, which utilized chain-termination inhibitors. Originally, data acquisition following sequencing was done manually, and now it is automatic. Sanger sequencebased platforms provide low-throughput, highquality reads of up to 1 kb [55]. Although automated Sanger sequencing has led to numerous breakthrough discoveries since it has been invented, including sequencing the complete human genome [56], second-generation sequencing technologies have made it almost obsolete for some applications such as will be discussed in the following sections. However, currently in the field of virology, Sanger sequencing is still considered the gold standard to study or confirm variants with high accuracy [57] and standardized genotyping [15, 58], as its sequencing error rates are much lower compared to more advanced technologies with higher-throughput and shorter read lengths [59]. In addition, as many public health laboratories have not yet transformed to second-generation sequencing, simplified protocols for whole genome sequencing via Sanger are still being utilized [46, 60]. As opposed to Sanger sequencing that produces a single sequence, second-generation sequencing technologies, also known as next generation or high throughput, are able to perform massively parallel sequencing and produce large numbers of sequence reads with exceptionally high genomic coverages and in less time and lower costs [54]. Second-generation sequencing platforms include, the Roche 454 [61] – the first commercially available platform, Illumina, and Ion Torrent. Although different platforms use different chemistries, they all involve the step of library preparation prior to sequencing, in which amplified DNA

Next-Generation Sequencing in the Study of Infectious Diseases

sequences are sheared, fit with adapters, and are amplified via synthesis [32, 62]. These platforms currently dominate the DNA sequencing space; however, limitations in these technologies include short-read lengths due to signal deterioration [63]. Indeed, short-read data lack the accuracy to resolve complex genomic regions, do not always uniquely map to the genome thereby generating repetitive areas, and cannot access high GC content areas, to name a few examples [64]. In contrast, “third-generation” technologies such as Pacific Biosciences’ recent development – singlemolecule real-time sequencing (SMRT) – Oxford Nanopore, and Helicos do not require the timeconsuming and costly steps of library preparation and can produce much longer sequence reads [54, 65]. In addition to longer reads, some of these technologies are advantageous in their small size, portability and availability of data collection, and analysis in real time [66]. Oxford Nanopore’s MinION [67], for example, was recently used to sequence 142 Ebola virus samples collected in West Africa and aid with outbreak surveillance and application of control measures by identification of sub-strains and mutation rates within 24 hours of sample collection [68]. Moreover, recent studies report usage of this technology to generate complete genomes of viruses from seawater, which was not possible through short sequence read assembly [Beaulaurier et al., BioRxiv 2019] and influenza virus sequencing directly from respiratory samples [Lewandowski et al., BioRxiv 2019]. Thus, despite the still high error rates produced by these technologies compared to secondgeneration sequencing [69] and lack of evidence favoring these technologies over existing ones [64], third-generation sequencing platforms are constantly improving and may soon become the leading sequencing technology. Acquisition of an NGS platform commits institutions and/or research labs to long-term financial arrangements and to the different sets of advantages and disadvantages of the platform [70, 71]. Commitments include infrastructure, workforce development, and training, efficiency, and cost [71, 72]. Efforts may be needed to develop standard protocols, quality controls, and proficiency testing programs to satisfy ISO standards,

41

good laboratory practices, and regulatory requirements [72]. Given the above information, choice of NGS platform is not trivial. Considerations should include the application it will be used for, e.g., low-quality sequences may often be sufficient for known pathogens [73]; however, for de novo virome assembly, long accurate reads and/or high depth are preferred. In addition, budget, envisionment of workflow and bioinformatic requirements, quality (limits of detection, limits of quantitation, precision, analytic specificity, base call accuracy, etc.), maintenance, and support should be considered as well. In addition to choosing the right technology and platform for a specific study, there are many considerations and choices to be made in planning a sequencing experiment. These may influence the accuracy, coverage, and depth in which viral genomes will be sequenced and/or represented in the metagenomics dataset. Second-generation sequencing is currently the one mostly utilized in the context of viral sequencing; hence it will be the technology referenced to in the following sections. Already at the step of library preparation, there are numerous kits to choose from, designed to prepare either DNA or RNA genomic material for sequencing, depending on the nature of the virus to be sequenced. Specifically, in the context of viruses which possess small genomes and exist in small quantity in clinical samples, low-input library preparation kits that require very low initial RNA or DNA amounts are mostly utilized. These include, for example, Illumina’s Nextera XT DNA/cDNA library preparation [74] and TaKaRa Bio’s SMARTer Pico RNA Kit [75]. In the case of virome discovery via metagenomics analysis, both DNA and RNA library preparations may be applied to identify both DNA and RNA viruses [76, 77]. Following library preparation, sequencing kits also offer many options. Sequencing can be conducted in a single-end fashion, meaning DNA is sequenced from one end, while paired-end sequencing ensures sequencing both ends of each DNA fragment (also known as “read”) resulting in twice the number of reads for each fragment at the same time and effort of library preparation. Paired-end sequencing enables a more accurate downstream analysis,

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Next-Generation Sequencing in the Study of Infectious Diseases

for example, in detecting low-frequency viral variants [78], which is not possible via single-end sequencing. In addition to the number of copies of each read, there are different options for the length of the read sequenced as well, depending on the type of sample, downstream application and analyses, and coverage requirements. Generation of longer reads requires additional cycles and reagents from the sequencer; Illumina sequencing kits, for example, offer up to 300 base pairs per read. Following sequencing, the overlapping regions between the reads are used to assemble them together; thus longer reads are useful for de novo assembly of genomes (without a reference genome) and more accurate determination of repetitive areas, for example, while shorter reads are sufficient in case of a well-known reference genome and in addition are more economical. Finally, one has to choose the total number of reads to allocate for sequencing each sample in the study. Sequencing kits range in the amount of data they are able to generate, with Illumina MiSeq V3 kit, for example, offering up to 15GB and 25 million reads. The larger the output, the costlier it is, and it is therefore important to balance information gaining without generation of excess data. Efficient study designs are based on allocating the optimal amount of sequence reads per sample to accurately detect or characterize viruses in the samples, depending on the purpose of the study, downstream analyses, and, of course, available funds. Greater amounts of reads provide higher coverages and depths of sequencing. Correspondingly, these increase data accuracy and specificity to, for example, obtaining whole genomes, identifying new viruses in clinical samples, characterizing rare virus variants, and more. The number of reads to allocate per sample changes with the type of sample, sample processing, and virus(es) that exist in the sample. Studies utilize computational subsampling of the number of reads, where random sets of reads are extracted to simulate different sequencing depths to conclude the number of reads to allocate per sample. For example, using this method, one million reads were shown to provide sufficient genomic coverage to detect closterovirus in plants [79]; complete genomic

coverage of the measles virus was obtained with one million reads when the virus was in high quantities in patient urine samples and five million reads when it was in higher quantities [Bucris et al., unpublished data]. Remarkably, it was recently demonstrated that samples are so diverse, either by nature or by distortion of population composition by experimental procedures, that even high depth of sequencing cannot provide consistent results in case of short-read data and results should be validated via replicates [80].

Bioinformatics Analyses: Viroinformatics NGS technologies are rapidly evolving to generate immense amounts of data while spending substantially less funds and time. The major bottlenecks of NGS are in fact data analysis [71]. To decipher the heaps of information generated by sequencing and derive meaningful conclusions, various downstream bioinformatics methodologies, tools, and pipelines (a combination of programs run consecutively) are progressively being developed. However, despite the increasing need for research in the field of virology and the available technological advances that enable it, there had been a remarkably low bioinformatics focus on viruses, and even a call to promote communication between virologists and bioinformaticians recently came out [81]. Indeed, virus bioinformatics, or viroinformatics, is a relatively novel field of development in an era where most bioinformatics research efforts and tool development were thus far directed toward the bacterial portion of the microbiome. There are many challenges in developing tools for viroinformatics which do not exist for analyses of other cellular organisms. Viruses lack a universal marker, e.g., the 16S ribosomal RNA in bacteria, making it difficult to comprehensively capture them. They have small genomes and exist in low abundance in clinical or environmental samples and thus may be concealed by larger background genomes. Some RNA viruses have the ability to mutate at a high rate and are able to substantially diverge from their origin genome, which is often improperly used as a reference genome, and finally – viruses may display high sequence diversity, even within the same viral

Next-Generation Sequencing in the Study of Infectious Diseases

species, within and among their hosts. The type of bioinformatics analyses applied to NGS data largely depends on the study question and desired output. However, few frequently used workflows and available programs to execute them have become customary in the field. For example, virome discovery bioinformatics pipeline usually includes the sequential steps of subtraction of background genomes, de novo assembly, mapping to a virus database, and annotation. In the following sections, such popular approaches are reviewed, in addition to other challenges and available resources in the field of viroinformatics. The following section will describe fields of development in viroinformatics which are crucial for metagenomics viral investigation. In addition, tools and algorithms that execute these methods will be highlighted and are summarized in Table 1. Virus-Specific Databases Databases which may or may not be publically available exist, which store viral sequences and related clinical and/or epidemiological annotation data. The largest publically available sequence database for all species is currently GenBank, and it is maintained by the National Center for Biotechnology Information (NCBI), National Library of Medicine, and National Institutes of Health (Bethesda, MD). The nr/nt database, containing sequences from GenBank and additional databases such as the European Molecular Biology Laboratory-European Bioinformatics Institute Nucleotide Sequence Database (EMBLEBI) [82], RefSeq [83], and the Protein Data Bank (PDB) [84], is also widely used. However, virusspecific databases, which have just recently become available resources, are crucial for a more accurate and comprehensive analysis of viral sequences. Detecting viruses in an allspecies database is challenging due to lack of closely related sequences, missing or misannotations, or hits of background genomes that exist in the sample sequenced, such as human and bacterial sequences. Virus-specific databases include NCBI’s viral genomes, which to date includes 9228 complete or nearly complete validated viral RefSeq sequences [85], the curated reference viral database (RVDB) that includes

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any virus-related/like sequences, excluding bacteriophages, which was established specifically to facilitate detection of known and novel viruses via bioinformatics analyses [86], and ViralZone, which provides molecular and epidemiological information [87], to name a few examples. Databases for viral groups include the endogenous viral elements databases gEVE (genome-based endogenous viral element), which was curated using bioinformatics tools and is not yet fully validated [88], bacteriophage-specific databases such as phiSITE – which catalogs regulatory elements from bacteriophages [89] – and the interesting recent microbe-versus-phage (MVP) database which pairs phage-microbe interactions to aid in selecting phages that target microbes of interest [90]. Single virus-centered databases are available as well, most of which usually exist for well-studied viruses such as influenza virus (EpiFlu [91], e.g., is currently the most comprehensive collection of genetic sequence data) and HIV (Stanford HIV Drug Resistance Database [92], e.g., includes drug resistance mutations/predictions in HIV-1). Above are some specific examples; however, resources are becoming more and more available and easy to find on the Internet. Additional selected virus-specific database are summarized in [81, 93]. Given the vast number of sequences deposited in databases on a daily basis, different approaches for searching and identifying these sequences have evolved. With the development of sequencing, early methods for detecting similarities between sequences used dynamic programming algorithms. These algorithms, for example, Needleman-Wunsch and Smith-Waterman, compared each nucleotide of one sequence to each nucleotide of a second sequence and kept track of how well the sequences aligned at every step. These approaches required long processing times and utilized parallel computing or supercomputers [94]. Today, the most well-known methods for sequence searching in databases are by far the BLAST (basic local alignment search tool) program suite, with about 200,000 queries made every week via its free website application. In contrast to previous methods, BLAST uses short “word” segments instead of one letter at a time.

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Next-Generation Sequencing in the Study of Infectious Diseases

Next-Generation Sequencing in the Study of Infectious Diseases, Table 1 Summary of highlighted tools used in viroinformatics Category Database

Name NCBI viral genomes RVDB

Function Validated viral RefSeq sequences

ViralZone

Resource for genomic and proteomic viral sequences Bioinformatically curated, endogenous viral elements Phage regulatory elements

gEVE phiSITE

Search tool

Mapping and sequence manipulation

EpiFlu HIV drug resistance database BLAST

Influenza virus sequences Curated HIV drug resistance data

FASTA suite

Sequence similarity

PSI-BLAST HMMER ExPASy BWA

Position-specific Sequence analysis via hidden Markov models Bioinformatics resource portal Map sequences to a reference genome

Bowtie2

Map sequences to a reference genome

GSNAP

Map sequences utilizing probabilistic models and known splice sites Program suite for sequence manipulation Genomic sequence assembly, OLC-based De novo sequence assembly, OLC-based De novo sequence assembly, DBG-based De novo sequence assembly, DBG-based De novo sequence assembly, DBG-based De novo sequence assembly for viral genomes De novo sequence assembly for viral genomes Web-based viral genome assembly pipeline Web-based viral genome assembly pipeline Web-based viral genome assembly pipeline

SAMtools Sequence assembly

Curated reference viral database

Arachne Newbler METAVELVET ABySS SPAdes VICUNA IVA virusTAP VirAmp V-GAP

Local alignment

Link/reference www.ncbi.nlm.nih.gov/ genome/viruses [85] https://hive.biochemistry.gwu. edu/rvdb [86] https://viralzone.expasy.org [87] http://geve.med.u-tokai.ac.jp [88] http://www.phisite.org/main [89] https://www.gisaid.org [91] https://hivdb.stanford.edu [92]

https://blast.ncbi.nlm.nih.gov/ Blast.cgi [95] www.ebi.ac.uk/Tools/sss/fasta/ nucleotide.html [96] [97] http://hmmer.org [98] https://www.expasy.org [99] http://bio-bwa.sourceforge.net [100] http://bowtie-bio.sourceforge. net/bowtie2/index.shtml [101] https://omictools.com/gsnaptool [102] http://www.htslib.org [104] https://omictools.com/arachnetool [108] https://omictools.com/newblertool [109] http://metavelvet.dna.bio.keio. ac.jp [110] https://github.com/bcgsc/abyss [112] http://cab.spbu.ru/software/ spades [113] https://www.broadinstitute.org/ viral-genomics/vicuna [105] https://sanger-pathogens. github.io/iva [116] https://gph.niid.go.jp/cgi-bin/ virustap/index.cgi [118] https://galaxyproject.org/use/ viramp [119] https://repository.kaust.edu.sa/ handle/10754/581472 [121] (continued)

Next-Generation Sequencing in the Study of Infectious Diseases

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Next-Generation Sequencing in the Study of Infectious Diseases, Table 1 (continued) Category Haplotype construction

Name HaploClique Virus-VGas SAVAGE

Virome discovery pipelines

MEGAN4

VIP

Phylogenetic analysis and related tools

Function Reconstruction of viral haplotypes via maximal clique finding Reconstruction of viral haplotypes from pre-assembled cotigs Reference-free reconstruction of viral haplotypes Virome discovery pipeline from metagenomic data

Clustal omega

Computational pipeline for virus identification and discovery from metagenomic Multiple sequence alignment

FigTree

Phylogenetic tree visualization

R package ggTree jmodeltest2

Phylogenetic tree visualization

ModelTest-NG MEGA BEAST

Selection of best-fit nucleotide substitution model Selection of best-fit nucleotide substitution model, enhanced functions Phylogenetic tree construction and additional analyses Construction of phylogenetic trees using the Bayesian inference approach

Although this approach increases the speed of alignment, it slightly decreases the accuracy of the algorithm [95]. The BLAST suite is freely available online and includes nucleotidenucleotide searches as well as additional features, such as translated nucleotide to protein search. In addition to BLAST, other similar methods for comparing a single sequence to a database, for example the FASTA suite [96]. More sensitive search tools are available, which create profiles from multiple sequences to provide a score for each type of amino acid at each position. These include, for example, PSI-BLAST (positionspecific iterative basic local alignment search tool) [97] and HMMER, which implements probabilistic hidden Markov models [98]. A different approach for increasing sensitivity is using just the invariant (or conserved) positions in the alignment, stored and implemented in ExPASy [99] (a bioinformatics resource portal operated by the

Link/reference https://github.com/cbg-ethz/ haploclique [124] http://cefg.uestc.cn/vgas [125] https://bitbucket.org/jbaaijens/ savage/src/master [126] https://software-ab.informatik. uni-tuebingen.de/download/ [128] https://github.com/keylabivdc/ VIP [130] https://www.ebi.ac.uk/Tools/ msa/clustalo [155] http://tree.bio.ed.ac.uk/ software/figtree [138] https://guangchuangyu.github. io/software/ggtree [139] https://github.com/ddarriba/ jmodeltest2 [146] https://github.com/ddarriba/ modeltest [147] https://www.megasoftware.net/ index.html [156] https://beast.community [154]

SIB Swiss Institute of Bioinformatics), for example. Obtaining Viral Sequences The outputs generated by second-generation sequencers are FASTQ files, which contain millions of short sequences, also named “reads.” The length of these reads varies according to the sequencing kit chosen, with an average of ~150 base pairs – too short of a length for any species’ genome. Therefore, one of the first steps in any bioinformatics pipeline is to assemble the short reads to form longer sequences to be used in downstream analyses. Two main approaches for sequence assembly are (a) assembly via mapping/ alignment (these terms are used interchangeably), where the short reads are assembled based on their identity to known viral genome reference sequence(s), and (b) de novo assembly, where the short reads are assembled into longer

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contiguous stretches of nucleotide sequences (called “contigs”) without using a reference genome. Mapping or aligning sequence reads to known reference viral genomes is mostly utilized when a specific reference genome is known a priori, for example, in cases of sequencing a known virus from a tissue culture or from a sample known to be infected with a specific virus. There are numerous algorithms designed to perform mapping which are used to map short sequence reads to a myriad of genomes, from small viral genomes to the vast human genome. These include wellknown programs such as the Burrows-Wheeler Aligner (BWA) [100], Bowtie2 [101], and GSNAP [102], to name a few examples. These algorithms aim to match the short reads onto the reference and differ in the compromises they take into consideration in terms of the accuracy and speed of mapping [103]. In addition, parameters, some which may be user defined, can affect the accuracy of the mapping; for example, the number of differences allowed between the reference and query reads and the number of gaps to allow and how much to penalize for them. Following mapping, a Sequence/Alignment Map (SAM) tabdelimited text file that includes the position in the reference genome to which each read mapped and quality score of the mapping [104] is created; the binary equivalent and compressed version of SAM files are BAM files, which occupy less space. SAM and BAM files are used for any subsequent downstream bioinformatics analysis. SAMtools software package [104], for example, includes commands to manipulate such files and may be used to output a consensus sequence (via mpileup command). However, performance of such reference-based aligners is not optimized specifically for viruses. Viral genomes are very small, and in addition viral populations, especially RNA viruses, exhibit diversity within the infected host and often diverge greatly from existing reference genomes. Moreover, read depths (the number of repeats for each position along the genome) are highly variable across the genome, stemming from polymerase chain reaction (PCR) amplification biases prior to library preparation. These properties render standard aligners to map multiple reads to the same

region or leave areas of the genome unmapped. Also, even following successful assembly of viral consensus sequences, these may substantially vary between different patients with the same virus, thus posing difficulties in deciding on the “correct” reference genome to be used which will allow unbiased alignment [105, 106]. The de novo assembly approach aims at solving the problem of biases arising from using reference genomes as a basis for alignment of short reads. These algorithms do not assume any prior knowledge of the source sequence length, layout, or composition, and in general they try to combine short sequence reads into longer contigs. Currently there are many de novo assembly programs designed specifically to handle short-read NGS data of viruses, most based on two classes of algorithms – the overlap layout consensus graph (OLC) and de Bruijn graph (DBG) [107]. OLC algorithms create contigs based on overlaps between reads and utilize multiple alignment to create the consensus contig. OLC has been adapted by programs such as Arachne [108] and Newbler [109], to name a few examples. DBG algorithms start by breaking reads into shorter k-mers (sequences with k nucleotides) and create a graph in which each node is a k-mer, and nodes are connected if they appear consecutively in a read; contigs are inferred from paths in this graph. Popularly used programs which adopted DBG and are now used in the field of metagenomics are METAVELVET [110, 111], ABySS [112], and SPAdes (St. Petersburg genome assembler) [113], for example. However, these programs have been demonstrated to yield different outputs depending on the read length, depth, and number of repeats [114]. In addition, as these tools were initially designed to assemble single genomes with uniform sequencing depths (e.g., the human genome) rather than multiple genomes from a metagenomics dataset, they have difficulties in generating long contigs for data that includes repetitive elements (such as in viral UTR regions) or with uneven/low sequencing depth [115]. A few de novo assemblers have been developed which are specifically designed to handle viral metagenomics datasets. These include VICUNA, which is able to recover complete viral genomes

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and identify insertions and deletions in diverse viral populations via clustering of reads which should be in the same contig [105], and IVA (iterative virus assembler) [116], based on the PRICE [117] algorithm, in which sequences are iteratively extended into contigs. Finally, pipeline implementation tools are available for quick and easy assembly of viral genomes. These include virusTAP [118], which provides a free online service including subtraction of various host genomes, de novo assembly with an algorithm of choice, and Blast validation. VirAmp [119] combines existing assembly and annotation tools into a pipeline via the Galaxy interface (a web-based platform for data analysis [120]), and V-GAP [121] is a viral assembly pipeline that utilizes resampling to assemble the viral genomes. In addition to obtaining consensus viral sequences from samples, some studies are interested in capturing the whole viral quasispecies, that is – the collection of the different genetically related mutant strains which are generated during viral replication. Viral quasispecies undergoes continuous genetic variation, competition, and selection for the most fit variants in the environment in which they are generated [122]. These characteristics provide viruses, especially RNA viruses, the capacity to adapt and survive despite selective constraints such as the host immune response or antiviral drugs [123]. Thus, strategies for identification and/or prediction of mutants are highly desirable for clinical applications, for example, to assist with selecting the correct treatment. With the development of NGS and the ability to sequence viral populations with high depth to provide more detail, techniques for inferring all viral variants (also termed haplotypes) within the quasispecies from NGS data are becoming available. The challenges these methods aim to overcome include an unknown number of strains, minor differences – sometimes only one point mutation – across the strains, extremely low abundance rates that are sometimes as low as the sequencing error rates and are therefore hard to detect, and the lack of unbiased reference genome(s). De novo assembly approaches overcome some of these obstacles; however, most assemblers, even those that specifically handle

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viral sequence assembly, are designed to reconstruct only one single consensus sequence. Such existing algorithms include HaploClique [124], which reconstructs viral quasispecies from NGS data of bulk sequencing of mixed virus samples by utilizing clique graphs; however, it requires excessive computation resources when there is low coverage. Virus-VGas [125] is able to reconstruct haplotypes from pre-assembled contigs without a reference genome and SAVAGE (strain aware viral genome assembly) – [126] referencefree assembly method based on OLC that is able to exploit consensus sequences which were generated by other programs.

Virome Discovery Metagenomics and NGS have revolutionized the rate and nature of virus discovery, as described in detail in the introduction. Numerous studies are recently launching with the aim of revealing the virome in different settings, and these discoveries will most probably facilitate our understanding of viral communities in ecology, infectious disease diagnostics and surveillance, and advance new therapeutic approaches. The global virome project launched in 2018, for example, is aimed to identify viral threats worldwide and provide timely data for public health interventions against future pandemics [9]. Along with the great strides in sequencing platform and ambitious research motivations, a wealth of bioinformatics approaches and workflows to analyze and interpret the results streaming from NGS are being developed. As of 2018, ~50 such methods were evaluated to be published and are reviewed by Nooij et al. [127]. All workflows of viral metagenomics discovery include the basic step of matching the sequence data onto a viral sequence database, followed by annotation and summarization to reveal the identity of the viruses in the sample. Some workflows may include additional steps (summarized in Fig. 2): 1. Quality control and preprocessing of the raw reads. This step removes low-quality data and technical errors from subsequent analysis and

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Next-Generation Sequencing in the Study of Infectious Diseases, Fig. 2 Generic metagenomics workflow. (Adapted from Nooij et al. [127])

is critical in some cases for the success of the workflow, as it can decrease the false-positive classification of reads as viral. 2. Filtering of non-viral reads. In this step, reads mapping to non-viral genomes such as the host (e.g., human) or bacterial genomes are removed. In addition to reducing false-positive identification of viral reads, filtering speeds up subsequent analysis in reducing the number of reads and queries and prevents assembly of chimeric virus-host sequences. On the other hand, filtering may render loss of potentially important viral sequences. 3. Assembly of short reads into longer contigs. This step aids in reducing errors in identification from individual reads and reduces the amount of sequences processed in subsequent analysis. The assembly usually involves de novo approaches and generates consensus sequences by mapping the individual reads to these contigs. However, de novo assembly may generate erroneous contigs by either linking together reads from different viruses

or reads which should not have passed filtering such as adapter reads. Therefore, assembly into contigs should be preceded by preprocessing and even a step of grouping the sequences according to their respective taxa prior to assembly. 4. Matching sequences to viral reference databases. In most workflows, different methods for similarity search of nucleotide sequences such as BLAST or BWA are utilized to identify which read or contig matches which virus. All workflows utilize known reference databases which include viral sequences such as NCBI GenBank or BLAST nucleotide (nt) and nonredundant protein (nr) databases, and in some instances the user can input their own customized database according to their research needs. 5. Post-processing and validation. In cases where the database search outputs several taxonomic assignments, post-processing steps may aid in evaluating the most likely one, utilizing, for example, phylogenetic trees to place the sequence with homologous reference sequences and thereby best classifying it. Validation of the findings may be conducted to apply the method to clinical settings and include molecular validation by one or several groups. To mention several workflows, MEGAN4 [128], that implements the latter two steps in the workflow of searching and post-processing, is the most cited. This workflow, which includes assignment of reads to a taxonomic group prior to de novo assembly, was used to discover that the enteric virome is abnormal in Crohn’s disease and ulcerative colitis patients [129]. Another noteworthy workflow to mention is VIP (virus identification pipeline) [130], a “one-touch computational pipeline for virus identification and discovery from metagenomics NGS data,” as it implements all five steps of the workflow. VIP’s speedy outputs were utilized in obtaining a near-complete dengue virus genome from a clinical sample retrieved from an outbreak in only 10 minutes and confirmed by PCR [130].

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Phylogenetic Analysis Phylogenetics refers to the study of the evolutionary history and the relationships among organisms using phylogenetic trees and has become essential to understanding biodiversity, evolution, ecology, and genomes. Phylogenetic trees are diagrams which depict evolutionary relationships among, for example, a group of species represented by branches and nodes, where a node represents an emerging new lineage and a branch represents the persistence of a genetic lineage through time [131]. The study of phylogeny relationships and their inference is widely used in virology to describe viral mutation evolutionary events and in epidemiology to track the spread of pathogens and infer transmission chains, to name a few examples. With the available resources provided by the advances of sequencing and NGS, the field of molecular phylogenetics has gained momentum as a tool for classification of genomes according to similarities in their sequences. In epidemiology, for example, tracking of transmission chains is part of the work public health facilities perform as response to outbreaks and is critical in interrupting virus spread and reducing outbreak magnitude. Conventional epidemiological investigations are based on questionnaires, which are time-consuming and are limited by the availability and goodwill of the patients. This approach is especially challenging during largescale outbreaks, where large numbers of co-existing transmission chains may occur [31]. The usage of molecular phylogenetics are now boosting conventional epidemiological investigations in characterizing disease transmission patterns using partial and recently even complete viral genomes. Thus, a clearer picture of the viral outbreak is painted via evolutionary transmission patterns. Examples include characterization of the Zika virus (ZIKV) emergence and spread in the Americas utilizing over a hundred complete ZIKV genomes [132], investigation of the recombination events and potential transmission routes within five different host species of the Middle East respiratory syndrome (MERS) coronavirus [133], and identification of a subset of Ebola virus patients with slowed or acute-like substitution rates which possibly led to continued

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reemergence of the virus outside of the expected incubation period [134]. With the aid of NGS, studies are progressively advancing toward molecular phylogenetics using complete rather than partial genomes. Analyses based on complete genomes expand our ability to understand viral outbreaks, as the nucleotide-level resolution can discern between isolates of the same strain [135]. Complete genomes have been shown to enrich molecular epidemiological analyses, for example, with measles virus (MeV) outbreak investigations. MeV is highly contagious in humans but undergoes a low number of mutations upon replication, relative to other RNA viruses. Studies investigating measles transmission in Vancouver [136] and Wales [46] demonstrated that sequencing the complete MeV genome can enhance resolution of molecular epidemiologic analyses in that complete genomes contain more information. Phylogenetic tree reconstruction from sequence data involves several steps: (a) Perform multiple sequence alignment of all the sequences to identify mutated positions, gaps, or inserts across the sequences. Programs that perform multiple sequence alignment are freely available online, including the popular Clustal group of algorithms such as Clustal Omega with its increased scalability, allowing thousands of sequences to be aligned in only a few hours [137]. (b) Estimate the molecular evolution model to be applied. (c) Build the phylogenetic tree. There are many factors which go into choosing the correct models and approaches in (b) and (c), and these will be discussed briefly. (d) Create a figure of the tree for presentation and interpretation. Many programs that edit, manipulate, and produce nice tree figures are available, such as FigTree [138] and the R package ggtree [139]. Molecular evolutionary models describe the rates at which one nucleotide replaces another during evolution and are used in the process of phylogenetic tree construction to estimate the

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evolutionary distance between the sequences from the differences observed between them. Since the development of the first evolutionary model by Jukes and Cantor in 1969, JC69 [140], that assumes equal nucleotide frequencies and equal mutation rates, many more-detailed statistical models that estimate the number of substitutions in a position in time have been developed, and studies suggest there is still room for improvement [141]. Substitution models differ in the parameters they require and assumptions they make. In the Kimura 1980 model [142], for example, different rates are assigned to transitions (mutations between purine (A/G) to purine A/G and pyrimidines (C/T) to pyrimidine) and transversions (mutations between purines and pyrimidines); the HKY85 model distinguishes between the rate of transitions and transversion mutations and allows for unequal base frequencies [143]; and the generalized time-reversible model (GTR) [144] requires all six substitution rates and four equilibrium base frequency parameters, rendering it the most general, independent, finite-sites, timereversible model possible. In general, complex models fit the data better than simple models; however, complex models require estimation of numerous parameters, which can be computationally difficult and timely to compute and may incorporate errors. Statistical testing is used to fit the data to different evolutionary models and choose the best fit one through likelihood ratio tests or information criteria [145]. Tools for selecting the best fit nucleotide and amino acid substitution models exist, for example, jmodeltest2 [146] implements five different model selection strategies, including hierarchical and dynamical likelihood ratio tests, Akaike and Bayesian information criteria, and a decision theory method; in addition it provides estimates of uncertainty and parameter estimates. The recently published ModelTest-NG is faster and includes additional features such as bias correction [147]. The goal of phylogenetic tree inference is to assemble a phylogenetic tree that represents a hypothesis about the evolutionary processes underlying the data. Phylogenetic trees can be computationally constructed via several methods, including distance matrix, maximum parsimony,

maximum likelihood, and Bayesian inference. Distance-matrix methods rely on the genetic distance measures between the sequences, where distance is usually defined as the number of mismatches at aligned positions, with gaps either ignored or counted as mismatches [148]. Examples for methods within this group are UPGMA (unweighted pair group method with arithmetic mean), WPGMA (weighted pair group method with arithmetic mean), and neighbor joining, where the latter method applies cluster analysis techniques [149]. The maximum parsimony method identifies the tree that minimizes the total number of evolutionary steps required to explain the data [150], and the maximum likelihood method infers the tree that best maximizes the probability of observing the sequences and requires a substitution model to assess the probability that a particular mutation occurred [151]. Similarly, based on prior probabilities, Bayesian inference uses likelihood functions to create posterior probabilities of trees to produce the most likely tree for the data [152]. Numerous programs have been developed which apply these and additional methods to construct phylogenetic trees. MEGA (molecular evolutionary genetics analysis), for example, is an easy-to-use tool for exploring, discovering, and analyzing DNA and protein sequences from an evolutionary perspective [153]. BEAST (Bayesian evolutionary analysis sampling trees) is a platform for performing analysis of molecular sequences using the Bayesian inference approach, via Markov chain Monte Carlo (MCMC, a class of algorithms for sampling from probability distributions) [154].

Conclusions Viruses are the most rapidly mutating and abundant genetic elements in our world. They come in different shapes and structures, have different types of genomes, and infect a different range of hosts. Particularly because of their diverse nature, viruses lack conserved elements and thus pose challenges in identifying and characterizing their communities in different settings. Due to these challenges, virome studies have lagged behind those of

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bacteria and other species which were easier to study, and even microbial genomics experts term viral sequences “dark matter” when encountered in metagenomics studies [157]. Nevertheless, thanks to advances of NGS in shotgun sequencing and metagenomics that enable accurate sequencing of nucleic acid from any entity in the sample at hand, discovery of novel viruses, and, in general, research in the virome field, is greatly increased. Side by side with these advances, the development of bioinformatic algorithms and workflows is increasing, to handle the massive amounts of data generated by these studies, analyze, and interpret them. Our current understanding of the virome is still incomplete and changes rapidly with research and technological advances. We are now on the cusp of a new era, where the effects of the virome, as a communicable part of the metagenome, are increasingly revealed. “Studying viral sequences means working at the edge of human knowledge” [157], and indeed, only a small fraction of sequences out of the ~1031 viruses that are estimated to inhabit our planet can be found in databases, meaning that there are still numerous challenges to overcome within each step of a metagenomics study on the way to deciphering the virome. These challenges arise from the nature of viruses – their genome is orders of magnitude smaller compared to genomes of their hosts, bacteria, or other organisms in the sample investigated, making them hard to detect. They mutate rapidly, which often causes their genomes to deviate considerably from reference genomes available in databases that are used for their detection. The quasispecies formed in some viral infections may affect precise sequence reconstruction. There are no global viral conservative regions, such as the 16S gene in bacteria, and thus viral populations cannot simply be captured. Viral fractions may be active or silent, and viral-like particles may hinder successful viral isolation or nucleic acid extraction. Finally, the high-rate recombination events, horizontal gene transfer, and assortment of viral genomic segments perplex bioinformatic analyses approaches [127, 158, 159]. Additional challenges arise from lack of sensitivity of the sequencing platform, which may not detect all viruses in the sample.

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On the other hand, false-positive detections may arise from methods that misclassify viruses because of their homology to other viruses or species or falsely annotate due to crosscontamination. Indeed, existing databases do not fully represent viral diversity [159]. On top of these platform and methods of analysis-related obstacles, there is a growing need for standardization in viral metagenomics in order to bring these methods to the clinic for diagnostic purposes. Despite these challenges, the future viral metagenomics is promising. Innovative novel technologies and bioinformatics approaches are constantly developing with the aim of addressing emerging obstacles. The growing mass of publications and sequence data generated on a daily basis demonstrates the motivation to utilize this technology and introduce it to viral discovery, disease surveillance, and clinical diagnoses. In fact, many funded initiatives have been recently established for these purposes. For example, the global virome project [9] is aimed to identify and characterize currently unknown viruses, and VIROPLANT (virome NGS analysis of pests and pathogens for plant protection, www. viroplant.eu) spans 17 partners from 8 European partners and is aimed to apply NGS approaches to develop new environmentally friendly virusbased control strategies, with the hopes that these developments in the field will advance accelerated viral surveillance and discovery.

Future Directions NGS platforms and pipelines are being rapidly improved and will most likely increase the length of the reads generated along with reduction in error rates. Bioinformatics approaches developed alongside these improvements will be able to overcome, correct, or better understand biases introduced during sample preparation and sequencing. In the near future, our collective knowledge of the virome in humans and the environment will increase, as NGS technologies improve, and numerous previously unknown viruses will be characterized, annotated, and

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added to the databases. With the growth of information in such databases, reanalysis of publicly available NGS data using newly developed approaches will become possible and may identify previously masked information, as already illustrated by several studies [10, 160]. Finally, identification of novel pathogens, associated molecular patterns and antigens within the virome that stimulate innate and adaptive immunity, will contribute to our understanding of the virome for our own benefit, including development of therapies, vaccines, and preparedness for future outbreaks by yet undiscovered viruses.

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Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics Jeremy D. Driskell and Ralph A. Tripp Department of Infectious Diseases, College of Veterinary Medicine, Animal Health Research Center, University of Georgia, Athens, GA, USA

Article Outline Glossary Definition of the Subject and Its Importance Introduction Spectroscopy-Based Diagnostics Future Directions Bibliography

Glossary Chemometrics A term to describe the use of multivariate statistics used to extract chemical information. Fourier-transform infrared spectroscopy (FTIR) A specific technique for acquiring IR absorption spectra in which all wavelengths are simultaneously measured. Infrared spectroscopy An absorption-based vibrational spectroscopic technique which primarily probes non-polar bonds. Polymerase chain reaction (PCR) An enzymatic method for amplifying a specific nucleic acid sequence. Raman spectroscopy A scattering vibrational spectroscopic technique which primarily probes polar bonds. Surface-enhanced Raman spectroscopy (SERS) A technique used to amplify Raman scattered signal via adsorption to a nanometerscale metallic surface.

Vibrational (molecular) spectroscopy A general term for the use of light to probe vibrations in a sample as a means of determining chemical composition and structure.

Definition of the Subject and Its Importance Importance of Rapid Diagnosis of Infectious Diseases Infectious diseases are a major burden on human health with the World Health Organization (WHO) reporting that infectious diseases are responsible for one in ten deaths in the world’s richest nations. The impact of infectious diseases is even greater in poorer regions of the world where six of every ten deaths are caused by a spectrum of infectious diseases that include bacteria, viruses, parasites and fungi. These infectious agents can further be described as classical pathogens, e.g., tuberculosis and malaria, seasonal epidemics, e.g., influenza and rhinoviruses, emerging infectious disease, e.g., highly pathogenic avian influenza and hemorrhagic fevers, or global pandemics such as the most recent outbreak of novel H1N1 influenza virus. Central to the management of each of these diseases are diagnostics. Early and rapid detection of an infectious agent is not only imperative to prevent the spread of disease, but it is also an essential first step to identify appropriate therapeutics that target the disease, as well as to overcome inappropriate administration of ineffective drugs that may drastically lead to drug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). This is just a succinct example which highlights the importance of diagnostic testing; however, the sections that follow discuss the current status of diagnostics and introduce an emerging approach to diagnostics based on vibrational spectroscopy which has tremendous potential to significantly advance the field.

© Springer Science+Business Media, LLC 2012 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_532 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media LLC, 2012 https://doi.org/10.1007/978-1-4419-0851-3_532

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Introduction Classical Culture-Based Diagnostics Despite the importance of diagnostic tests for infectious diseases, relatively few technological advances have supplanted classical microbiological approaches, e.g., in vitro culture, as a diagnostic standard. Clinical laboratories routinely rely on selective and chromogenic growth media to identify bacterial agents. For example, an aß-chromogenic medium, which includes two substrates, has been developed to selectively isolate Salmonella spp. with 100% sensitivity and 90.5% specificity [1]. More recently chromogenic media have been developed to identify Staphylococcus aureus and distinguish methicillinresistant strains (MRSA) [2, 3]. Culture-based diagnostics provide a method for definitive identification of many bacteria, and the tests are relatively inexpensive; however, the identification process has generally low throughput and substantial time is required for diagnostics. Typically, culture requires 24–72 h to allow the bacteria to grow while slow-growing organisms such as mycobacteria require substantially longer (6–12 week) incubation times. Obviously, this is not ideal as the time frame can delay patient treatment. It should also be noted that not all pathogens can be cultured in a laboratory environment, thus the technique cannot be universally applied. There are several additional drawbacks of culture-based diagnostic methods including the requirement for species specific reagents, appropriate culture and storage environments, and labor intensive procedures. Antibody-Based Diagnostics Antigen detection and serology are common approaches used in clinical laboratories as alternative or complementary tools to culture-based detection. Common to both of these methods is the use of antibodies either to directly label and detect the antigen or to capture the host response, e.g., antibody responses to infection. Typically, an enzyme-linked immunosorbent assay (ELISA) is employed for antigen detection in diagnostic laboratories. As a first step, ELISA requires an unknown amount of antigen in a sample to be

specifically, via a capture antibody in a sandwich assay format, or nonspecifically, via adsorption, immobilized to a solid phase such as a microtiter plate. After removing excess antigen, a known amount of detection antibody specific to the pathogen is then introduced to bind any immobilized antigen. The detection antibody is either directly labeled with an enzyme, or in an additional step, detected with an enzyme-labeled secondary antibody. After removal of excess reagent, a substrate is introduced to react with the enzyme producing a quantifiable color change. A slight modification of this approach replaces the enzyme with a fluorophor for fluorescence-based readout, eliminating the final substrate incubation step. A similar approach is taken for ELISA-based serological assays in which a known amount of purified antigen is immobilized onto the solid phase and incubated with serum to detect the presence of antibodies. While the procedure requires multitudinous steps, reagents, and substantial labor, ELISA is considered rapid relative to culture-based diagnostics as in many cases the assay can be completed within several hours. ELISA-based assays continue to be an integral part of laboratory diagnostics, but in their original form they are limited to the laboratory. Lateral flow immunoassays, also called dipstick assays, immunochromatography, sol particle immunoassays, or rapid diagnostic tests, have been developed to overcome many limitations of ELISAs by eliminating the complex multi-step procedure, reducing labor, and allowing field or point-of-care testing. Lateral flow assays, like ELISAs, utilize pathogen-specific antibodies for the direct detection of antigen or detection of antibody response. However, for the case of lateral-flow assays the labeled detection antibody, capture antibody, and control reagents are dried on a prefabricated carrier strip. By design, these assays overcome diffusion-limited kinetics to exploit the rapid kinetics of antibody-antigen recognition [4, 5] to yield results in 10–20 min. Thus, because of the “reagentless” nature and rapid results, these assays are well suited for field use and resource-poor regions where reagent storage and test sites are severely limited. It should be noted, however, that these benefits are at the loss

Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics

of quantitative information and often a lower threshold of detection. Numerous lateral flow immunoassays have been developed commercially for clinical diagnostics. Several competing manufacturers offer rapid diagnostic tests for influenza virus in which a conserved antigen is detected in a lateral flow assay format. Some detect influenza A and influenza B without distinction of the subtypes, e.g., QuickVue Influenza Test (Quidel), others detect and differentiate A and B strains, e.g., QuickVue Influenza A þ B Test (Quidel) and 3 M™ Rapid Detection Flu A þ B Test, and some only identify A or B strains, e.g., SAS Influenza A Test, (SA Scientific). Similarly, commercial rapid diagnostic tests are available for detection of a conserved protein for rotavirus A, e.g., IVD Rotavirus A Testing Kit. Not all lateral flow assays are designed for antigen detection; a rapid diagnostic test developed for leptospirosis diagnosis target anti-Leptospira IgM antibodies [6]. Despite continued advancements in antibodybased diagnostics these platforms will always be limited by the need for species-specific reagents, i.e., antibodies where the assays can only perform with the sensitivity and specificity inherent to the antibody. For example, commercial lateral flow assays for influenza only provide 50–70% sensitivity and 90–95% specificity with respect to culture-based diagnosis [7]. While the lateral flow assays may be performed rapidly, their low sensitivity may preclude early diagnosis due to low levels or unsustained levels of antigen through disease available for detection. Moreover, serological-based assays developed to detect agent-specific antibodies require that the infection elicit a detectable sustained immune response before the assay can be performed, a feature which substantially delays diagnosis. Molecular Diagnostics Nucleic acid and sequence-based methods for diagnostics offer significant advantages over conventional culture- and antibody-based diagnostics with regard to sensitivity, specificity, speed, cost, and portability. Central to molecular diagnostics is the use of a complementary nucleic acid probe that hybridizes to a unique species-

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specific region of the infectious agents RNA or DNA. While several molecular platforms have been developed for infectious diseases diagnostics, e.g., fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) is the most commonly employed molecular method for diagnostics. PCR is a method of amplifying targeted segments of nucleic acid by several orders of magnitude to facilitate detection. In principal, complementary primer sequences are used to hybridize to a target nucleic acid sequence. A thermostable DNA polymerase, e.g., Taq polymerase, is then employed to extend the primer sequence. Thermal control facilitates extension, melting, and annealing, and via temperature-controlled cycling, the number of target sequences increases exponentially with each cycle. Amplification of the target sequence can be read out in an ethidium bromide-stained agarose gel or in real-time via cleavage of a fluorescent tag from the primer during the extension step. Appropriate selection of the primers provides extremely specific detection, while the amplification of the target nucleic acid provides excellent sensitivity. PCR has been demonstrated to be sensitive to single-copy numbers of DNA/RNA targets. In these most sensitive PCR assays, primers are chosen to fully complement a region of the target sequence. Perfect complement probes are also ideal for maximizing the assay specificity to a particular infectious agent. However, in practice, genetic mutations, particularly prevalent in RNA viruses such as influenza, can render a primer/ probe set ineffective for diagnosis. Thus, degenerate probes are sometimes chosen to encompass some genetic diversity at the expense of sensitivity. Multiplexed PCR utilizes multiple primer/ probe sets that target different pathogens. Multiplexed assays are implemented when the sample size is limited, preventing multiple individual singleplex PCR analyses, and the clinician is unable to determine the most likely causative agent based on early clinical presentation. Multiplexed PCR assays are not quantitative due to target competition for reagents, are typically less sensitive than singleplex assays, and because of

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increased reagents, are more expensive to perform than singleplex assays. Moreover, multiple PCR products cannot be simultaneously read out by fluorescence, thus microarray analysis or electrophoresis to identify PCR products of different lengths is required to detect multiple PCR products. However, breakthroughs in multiplexed detection and quantitation are forthcoming [8–10]. Thus, for detection and diagnosis of many diseases such as viruses, PCR offers many advantages over classical methods of diagnostics, and its role will continue to expand in clinical diagnostic laboratories. However, there are challenges associated with PCR. For pathogens in which culture and microscopy can be used, molecular diagnostics are not the most cost effective. For example, the cost of a commercial PCR assay for tuberculosis ranges from $40 to $80, whereas microscopy and culture can be implemented for $1 and $5, respectively. Another consideration is how to interpret PCR results. Due to the sensitivity afforded by PCR, extremely low levels of infectious agent can be detected which may be below clinically relevant thresholds for disease presentation. Thus, quantitative PCR rather than qualitative PCR is typically more informative when correlating to clinical diagnosis. PCR assays developed in the laboratory are not always translational to analysis of clinical samples. In general, PCR cannot be performed directly on biological fluids such as blood because compounds such as hemoglobin, lactoferrin, heme, and IgG inhibit amplification [11–13]. Therefore, DNA and RNA are extracted as a first step, prior to PCR, but inefficiency of extraction kits often lead to a decrease in analytical performance compared to laboratory cultures [14, 15]. Moreover, isolation of nucleic acids is time consuming and technically challenging unless automated, which requires expensive equipment and reagents. A final consideration is the need for temperature-sensitive reagents, thermocyclers, skilled workers, and a clean laboratory environment to prevent contamination leading to false-negative results. While tremendous efforts are focused on PCR automation, incorporation of microfluidics [16, 17], and isothermal amplification [18–21], e.g., loop-

mediated isothermal amplification (LAMP), to overcome these challenges, the current status of PCR precludes widespread use of PCR diagnostics in point-of-care settings and developing nations. Limitations of Classical and Conventional Diagnostics As discussed above, diagnostics are essential to healthcare and disease management. While there is merit in further advancing current diagnostic methods, there are shortcomings for each approach. As noted above, culture is sensitive, but the length of time prevents rapid and early diagnosis. Antibody-based techniques are often limited in sensitivity, and like molecular diagnostics, are expensive, and require species-specific detection reagents. It is likely that any improvements in these methods to address these challenges will be incremental; however there are several next generation diagnostics that offer potential paradigm shifting approaches to detection and diagnoses that are currently being investigated. One important area is the use of molecular or vibrational spectroscopy for “whole-organism” fingerprinting. This innovative approach to diagnostics promises to be rapid, specific, and truly reagentless.

Spectroscopy-Based Diagnostics Vibrational spectroscopy includes a number of nondestructive analytical techniques which provide molecular information about the chemical makeup, e.g., functional groups, of a sample. Subtle changes in the frequency of a particular functional group vibration, e.g., group frequency, provides additional details of chemical structure, local environment surrounding the bond, bond angle, length, geometry, and conformation. These attributes of vibrational spectroscopy have led to the development of vibrational spectroscopic approaches to generate whole-organisms fingerprints to serve as unique biochemical signatures for pathogen identification. Unique to this approach of infectious agent identification is rapid readout, and perhaps most importantly, there is no

Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics

need for species-specific reagents or other reagents of any kind. Three of the most developed vibrational spectroscopic techniques include infrared absorption spectroscopy, Raman scattering spectroscopy, and surface-enhanced Raman spectroscopy (SERS). These three methods, as well as their development for diagnosing infectious diseases are described in detail below. Infrared Spectroscopy Infrared spectroscopy is an absorption technique in which the sample is irradiated most commonly with mid-infrared light with wavelengths in the range of 2.5–50 mm. Photons of appropriate energy to bring about a transition from one vibrational state to an excited vibrational state are absorbed by the analyte. Selection rules govern which vibrations are allowed to absorb IR photons, providing chemical and structural information. These rules require a net change in the dipole moment of the molecule as a result of the vibration. Thus, IR absorption spectra are dominated by asymmetrical vibrations. With consideration to these selection rules, proteins and nucleic acids (building blocks of bacteria, viruses, etc.) are excellent absorbers of IR radiation making IR spectroscopy an ideal tool for characterization of infectious agents. The chemical complexity and sheer size of bacteria and viruses tend to produce complex spectra with broad and overlapping bands. Yet subtle changes in band shape, slight shifts in band peak positions, and variation in relative band intensities provide significant insight into chemical structure. Thus, careful evaluation of the full spectral fingerprint of whole-organisms, rather than analysis of single peaks common to small molecule studies, can lead to identification and classification of microorganisms. IR spectroscopy as a technique for wholeorganism fingerprinting dates back to 1952 [22]. In this early study, Stevenson and Boulduan showed that the IR spectra for Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and six other species of cultured bacteria are unique to each species. In addition to species identification, six strains of Bacterium tularense were spectroscopically differentiated. This initial work did not

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immediately translate to the diagnostic applications of IR spectroscopy. Two major breakthroughs that did not occur for several decades after the original findings were essential to further increase the utility of IR for whole-organism fingerprinting. First, prior to the 1980s when Fourier transform infrared spectroscopy (FTIR) was introduced, dispersive instruments were typically used. Dispersive instruments do not provide the speed or analytical performance required for IR-based diagnostics. FTIR instruments provide much better signal-to-noise spectra with improved spectral resolution, and are acquired in less time. These advantages provided by FTIR were essential to accurately analyze these complex biological spectra by distinguishing subtle changes in spectral band shape and to rapidly collect data. Additional developments in the methods of data analysis were also essential to move IR wholeorganism fingerprinting forward. The large number of variables, i.e., wavelengths, each containing relevant information, inherent to IR spectra and rather subtle changes in intensity prevented traditional single-peak analysis for bacterial identification. Moreover, visual inspection of the spectra by the analyst is too labor intensive, prone to operator error, and unrealistic for large numbers of spectra and/or organisms from which to identify. The introduction of chemometrics, i.e., multivariate statistics applied to spectroscopy, led to the continued interest and advancement of IR-based diagnostics. These methods, including principal component analysis [23, 24], discriminant analysis, multiple regression analysis [25], and artificial neural networks [26, 27], function to simplify the high dimensional dataset by identifying the most significant variables with the ultimate goal of sample identification or quantification. FTIR has received the greatest attention with respect to vibrational spectroscopic techniques over the last 2 decades and has been developed to the point that it can be considered an established method for the identification of both bacterial species and strains [28–33]. Researchers continue to investigate laboratory cultures of bacteria in an effort to standardize sampling protocols so that spectral databases can be shared among laboratories and to standardize the methods of

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analysis including spectral preprocessing, feature selection, and classification algorithms. As the method has matured, and while it continues to be tweaked and standardized, FTIR is now being applied to the analysis of clinical specimens. For example, FTIR has been successfully used to analyze clinical sputum samples collected from cystic fibrosis patients [26]. This FTIR capability is important as historically, lung infection caused by Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae were the major causes of morbidity and mortality in cystic fibrosis patients; however, the number of emerging nonfermenting species is on the rise [34], and many of these species are closely related and not appropriately identified using typical clinical diagnostics and microbiological approaches [35]. Using a FTIR spectral library and an artificial neural network built for pathogen identification, the results from the FTIR method were compared to conventional microbiology detection methods. A two-tiered ANN classification scheme was built in which the top-level network identified P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and Burkholderia cepacia complex (BCC) bacteria. The second-level network differentiated among four species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis. Ultimately, this method resulted in identification success rates of 98.1% and 93.8% for the two ANN levels, respectively. However, before this optimized method was established, the research highlighted three important considerations. First, not all bacterial isolates produce poly-ß-hydroxybutyric acid (PHB) which contributes to the IR spectra and confounds classification. To overcome this, each isolate was cultured on TSA medium and harvested after 5 h of growth, prior to the expression of PHB. This step enriches the bacteria for analysis and eliminates interference from PHB. Second, flagella or pilus fibers were determined to contribute to spectral heterogeneity. Vigorous vortexing and subsequent centrifugation removes the fibers to significantly improve spectral reproducibility and classification results (Fig. 1). Third, the classification algorithm significantly affects the classification results. The authors show that hierarchical clustering

algorithms (HCA) discriminate between reference and clinical strains rather than based on bacterial identity. Advanced methods, such as ANN, that determine spectral variables that vary only as a result of the bacteria was necessary to correctly classify according to strain. This example work demonstrates the power of IR-based diagnostics, but suggests that these methods may require problem-specific standardization of experimental protocols and data analysis. These groundbreaking efforts to develop IR for bacterial analysis have led to the realization that spectroscopic methods have advantages for exploring detection of other pathogens. For example, FTIR has been employed for the distinction of yeast and fungi with success [36, 37]. More recently IR has been investigated as a method to detect viral infections, although current experiments are limited to viral infection of cells in culture [38–41]. While mock-infected and herpes simplex virus type 1-infected Vero cells are readily distinguished via IR, infection-induced spectral changes are inconsistent [39, 40]. Thus, substantially more research effort is necessary to standardize protocols and correlate the spectral response to the biochemical response upon infection. Reports continue to support the utility of FTIRbased diagnostics in the clinical laboratory, but there are certain limitations to consider. First, water is a particularly strong absorber of IR light. Thus, care must be taken to completely dehydrate the sample prior to data acquisition. This obviously does not prevent IR-based diagnostics, it is merely an inconvenience. Second, IR absorption spectroscopy is not an inherently sensitive method and trace levels of a pathogen are not readily apparent. Hence, clinical samples will likely require a culture step to generate sufficient biomass for IR analysis. As noted above, this sample enrichment can be as short as 5 h, and with IR data acquisition on the order of minutes, the total analysis time is still more rapid, less labor intensive, and more informative in many cases than conventional diagnostic methods and does not require species-specific reagents. Raman Spectroscopy Raman spectroscopy is a scattering technique, in which the sample is irradiated with a

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clinical isolates (isolates 57 and 69) in the 1500–800 cm1 range. (a) The heterogeneity of 15 replicate measurements for each strain in the spectral ranges of 1200–900 cm1 and

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monochromatic light source, almost always a laser. The majority of the scattered photons are elastically scattered and maintain the same frequency as the excitation source; however, a small fraction of the photons are shifted in frequency relative to the excitation source. The difference in the energy between the excitation and inelastically, i.e., Raman, scattered photons correspond to the energy necessary to bring about a transition from one vibrational state to an excited vibrational state. Thus, much like IR spectroscopy, Raman spectra provide insight into the chemical structure, local environment, geometry, and conformation of the sample and can serve as a whole-organism fingerprinting method. Selection rules also govern which vibrations are Raman active. These rules require a change in the polarizability during the vibration to be Raman active. Thus, Raman spectra are dominated by symmetrical vibrations and the technique is often seen as a complementary rather than competing technique with IR spectroscopy. However, for application to the analysis of biological materials and wholeorganism fingerprinting methods, Raman offers many inherent advantages over IR spectroscopy. Because of the selection rules, the main chain and aromatic side chains of peptides rather than aliphatic side chains are probed via Raman scattering in contrast to IR. Raman bands of nucleic acids are limited to heterocyclic bases or phosphodiester groups making up the backbone. Raman bands are narrower and less likely to overlap, thus the spectra are much less complicated compared to IR spectra because of the many more nonsymmetric vibrations that are possible. Another major advantage of Raman is that water does not interfere since its vibrations do not fit the selection rules criteria. This is an extremely important consideration when analyzing biological samples which are endemic to aqueous

environments. Other advantages of Raman include the flexibility to analyze samples in any state, e.g., gas, liquid, or solid, and the ability to analyze small sample volumes and masses because of the tight focus of incident laser light (square microns) compared to the incident IR beams (square centimeters). Viruses were the first infectious agent analyzed by Raman spectroscopy, although not in a diagnostic capacity [42]. In this first work, Raman spectroscopy was used to probe the RNA and protein structure upon viral packaging. In the 1970s, Raman spectroscopy suffered from poor sensitivity due to instrument limitations. The first evaluation of Raman spectroscopy for pathogen detection was not until 1987 when spectra were collected for five species of bacteria including E. coli, P. fluorescens, S. epidermidis, B. subtilis, and E. cloacae [43]. To overcome the limited sensitivity of the instruments at the time, an ultraviolet laser was used for excitation to enhance spectral features of RNA, DNA, tyrosine, and tryptophan via resonance Raman. Unique spectra were observed for each bacterium, although analysis relied on visual interpretation since chemometrics had not been implemented for spectral analysis yet. UV Raman instruments, while producing the requisite sensitivity for pathogen analysis, is quite expensive and nonresonant vibrations are not observed which results in a significant loss in information that is valuable for differentiation. Despite the recognized benefits of Ramanbased diagnostics, particularly when compared to conventional and IR-based diagnostics, instrumentation has limited the maturation of Ramanbased diagnostics. After development of UV Raman for pathogen detection [43–46], Fourier transform Raman (FT-Raman) instruments were introduced for microbiological studies which

ä Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics, Fig. 1 (continued) 1500–1300 cm1 and the corresponding micrographs obtained by TEM are shown. (b) Vector-normalized firstderivative spectra measured after vortexing of similar cells at the maximum intensity for 15 min and subsequent

centrifugation at 8000  g for 5 min to separate the cells from free pilus appendages in the supernatants. Micrographs of the cells obtained by TEM after they were vortexed without centrifugation show the small fragments of pili or fibers suspended in the supernatants. (From [26])

Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics

increased instrument sensitivity [47, 48]. Raman instruments have now evolved to include NIR lasers to reduce fluorescence from biological and NIR-sensitive CCD detectors. These modern instruments have only been developed in this decade to fully explore the potential of Raman as a diagnostic technique [49–55]. Thus Ramanbased whole-organism fingerprinting is less developed than IR-based methods and examples are generally limited to the analysis of laboratory cultures. In an early study, Maquelin et al. [54] utilized Raman spectroscopy to directly analyze five bacterial strains, including three strains of Staphylococcus spp., E. coli, and E. faecium, on solid culture medium. The flexibility in sample type afforded by Raman spectroscopy allowed direct measurement on the culture plate that would not be possible using IR spectroscopy. The background Raman spectrum resulting from the culture medium was subtracted from those spectra collected from the bacterial microcolonies. Hierarchical cluster analysis yielded two major groupings, one consisting of the three Staphylococcus strains and one consisting of the E. coli and E. faecium. The E. coli and E. faecium spectra clearly grouped according to species within the latter subcluster while spectra in the Staphylococcus subcluster grouped according to strain. While chemometric analysis of these spectra collected from same-day cultures yielded a successful classification rate of 100% for external validation samples, combined data collected from 3 days dropped the accuracy to 83% for classification of two S. aureus strains (ATCC 29213 and UHR 28624). However, these two strains are extremely similar and in general the results demonstrate the utility of Raman-based diagnostics. The most rigorous evaluation of Raman spectroscopy for reagentless detection and identification of pathogens was performed in collaboration with a US government laboratory. In this work, a comprehensive library of Raman spectra has been established for over 1,000 species, including 281 CDC category A and B biothreats, 146 chemical threats, 310 environmental interferents, and numerous others [52]. Spectral signatures were collected using Raman chemical imaging

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spectroscopy (RCIS) [56]. RCIS technology combines digital imaging and Raman spectroscopy. Digital imaging automatically discriminates against background particulates and identifies regions of interest on a sample platform that are then targeted for Raman analysis. Sample analysis is faster and completely automated using this approach. Two commercially available instruments were tested, one in the laboratory (ChemImage Corp., Falcon) and the other in the field (ChemImage Corp., Eagle). To test the robustness of the Raman spectral library and classification scheme, blinded samples containing one of four Bacillus strains were analyzed and identified. The predictive performance ranged from 89.4% to 93.1% for these closely related bacteria. It was concluded that key to the success of this diagnostic approach is the extensiveness of the spectral library. There are many more bacterial phenotypes than genotypes, and it has been found that Raman fingerprints correlate with cell phenotype, thus an all-inclusive library must contain spectra for each bacterial strain grown under different conditions and at different stages of development. In a subsequent study untrained personnel at the Armed Forces Institute of Pathology evaluated 14 bacteria to generate a spectral library and sent 20 blinded samples to ChemImage for external validation in which all 20 samples were correctly identified. This comprehensive study is the first to establish the true utility of automated Raman-based diagnostics carried out off-site by untrained personnel. However, these samples were prepared in water, cell culture media, or spiked nasal swabs, none of which are truly clinical samples. An early study to evaluate clinical samples for Acinetobacter by Raman spectroscopy and compare the results with an established diagnostic method were among the first showing the power and speed of Raman-based detection [55]. In this study, 25 Acinetobacter isolates from five hospitals in three countries were analyzed using selective amplification of restriction fragments (AFLP), an established molecular technique for typing bacteria strains. Dendograms resulting from the hierarchical cluster analysis of Raman and AFLP fingerprints for the isolates were

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generated and compared (Fig. 2). Both dendograms resulted in five clusters that separate the strains according to the five outbreaks, with the exception of one Basildon isolate RUH 3242 which clustered with isolates from Venlo in the Raman-based dendogram. Overall results from Raman fingerprinting of these clinical isolates were very similar to those obtained for established methods, but with the advantage of faster analysis and less complicated procedures. Despite the advancement of Raman spectroscopy instrumentation and methods for pathogen fingerprinting, Raman is still often limited by poor sensitivity. Only ~1 in 106–108 photons are inelastically scattered as the vast majority are elastically scattered. This means that high quality spectra with the requisite signal-to-noise can take minutes to acquire. While this may not be a limitation in laboratory experiments, or developmental stages in research, it prohibits its usefulness in clinical diagnostic laboratories which analyze hundreds to thousands of samples per day. Thus, there is great interest in enhancing the Raman signal. One such method is to excite the sample with a frequency that resonates with an electronic transition, so called resonance Raman spectroscopy. For biological samples, this requires UV lasers for excitation, and as noted above, is cost prohibitive to widespread adoption of this method. Moreover, chemical information is lost when performing resonance Raman which would likely reduce classification accuracy of closely related pathogens. An alternative method to amplify Raman scattering is surface-enhanced Raman spectroscopy (SERS). SERS has received a great deal of attention, particularly with respect to whole-organism fingerprinting and is the subject of the next section. SERS Surface-enhanced Raman spectroscopy is a technique in which the Raman signal of a sample is significantly amplified via adsorption onto a metallic nanostructured surface. A laser excitation frequency is selected such that it is in resonance with the collective oscillation of the conduction electrons in the nanostructures, i.e., surface plasmon resonance. When resonance conditions

are met, the local electromagnetic field experienced by molecules in close proximity to the surface is significantly increased to yield rather large enhancements in the Raman scattering. While the signal enhancement is substrate and sample dependent, typical enhancements are on the order of 104–1014 with respect to normal Raman intensities, with several studies reporting the detection of single molecules using this technique [57, 58]. SERS offers the benefits of normal Raman compared to IR spectroscopy while providing a markedly improved sensitivity. Recent advances in nanofabrication methods and SERS theory has led to significant improvements in SERS substrates in the last several years and has driven increased efforts to develop SERS for whole-organism fingerprinting [59–78]. The major focus of whole-organism fingerprinting via SERS has been on bacteria identification [51, 64–74, 77, 78]. Most of these studies report differentiation among bacteria species, with many demonstrating discrimination of different strains of the same species. However, there are several inconsistencies that have been noted by researchers, particularly in the earlier studies. For example, Grow et al. found SERS spectra for strains that belong to the same species were sometimes less similar than spectra collected from different species [65], and Jarvis and Goodacre observed similar spectra for the same bacteria using different preparations of silver nanoparticles, but noted subtle changes in signal intensities among nanoparticle batches [68]. These discrepancies evident in these early studies highlight the primary challenge of SERS-based diagnostics, i.e., the enhancing substrate. The SERS signal is highly dependent on the enhancing substrate, thus a reliable means of fabricating nanostructured materials is vital to the success of SERS-based diagnostics. Several research laboratories have analyzed and published SERS spectra for both Bacillus subtilis and E. coli; however, each reported incongruent spectral fingerprints [67, 68, 71, 72]. The experimental protocols, however, varied among each study. For example, in two different reports Jarvis et al. used two different chemical synthesis preparations to generate colloidal silver, citrate

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Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics, Fig. 2 Dendrograms resulting from the hierarchical cluster analysis of (left) AFLP analysis and (right) Raman analysis of the isolates. The asterisk marks the strain RUH 3242 misclassified via Raman fingerprinting. (From [55])

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reduction [67] and borohydride reduction [51], to serve as the SERS substrate. The SERS spectra were drastically different in each study. It is well known that spectra are dependent on the enhancing nanostructure, e.g., material, size, shape, interparticle spacing, etc., but given the same final nanostructure similar spectra were expected. The authors attributed the differences to the effect of diverse chemistries used to prepare each silver colloid [79]. However, it should be noted that different excitation sources, 532 nm and 785 nm, were employed in the two studies. For normal Raman, the Raman shifts should be independent of the excitation source, thus spectral fingerprinting should not be affected by the choice of the laser. SERS spectra, however, can be influenced by the excitation source because of the requisite pairing of the excitation frequency and plasmon resonance of the substrate. Therefore, it is perhaps more probable that spectral differences observed by Jarvis et al. are due to greater signal enhancement for the 532 nm excitation source rather than due to differences in chemical preparation of the colloidal silver. This interpretation is supported by a study in which a third variation in experimental parameters was implemented utilizing citratereduced silver colloid but acquired spectra with a 647 nm laser [71]. Results from this study closely resembled the results for B. subtilis obtained by Jarvis et al. employing borohydride reduced silver nanoparticles and 532 nm excitation. Collectively, these studies also demonstrate the need for procedural consistency. In a pivotal study, scientists at a US Army research laboratory evaluated the SERS signatures for many bacteria using a standardized sampling protocol and instrumentation. To date, three SERS substrates were directly compared using the standardized protocol: silver nanoparticles, silver film over nanospheres (FONS), and commercially available Klarite. Interaction between substrate and bacteria vary significantly as visualized with electron microscopy which likely results in different spectral fingerprints. Moreover the signal intensities varied significantly among the substrates reflecting differences in enhancing quality. Details of these experiments are approved for public release as a technical report (ARL-TR-4957).

In another key study, SERS and Raman fingerprints were directly compared to assess the advantages of SERS analysis [72]. Raman and SERS spectra were collected for several bacteria, including four strains of Bacillus, S. typhimurium, and E. coli. As noted above, the substrate is a critical factor in SERS analysis, and in this study aggregated gold nanoparticle films were grown in-house and established as a reliable means of substrate preparation for acquisition of repeatable spectra. As anticipated, SERS yielded much greater signal-to-noise spectra compared to normal Raman. The study also identified two unexpected benefits of SERS. Normal Raman signal for Bacillus species was overwhelmed by native fluorescence of the sample; however, in the SERS analysis, the metal substrate functioned to quench the fluorescence component in addition to enhancing the Raman signal. It was also observed that normal Raman spectra are more complex than SERS spectra. This is explained by the fact that bulk Raman interrogates all components throughout the entire bacterium equally, while the distance-dependence of SERS enhancement preferentially probes the region of the bacterium closest to the metal substrate and bands for the internal components are not detected. Fortunately, most chemical variation among bacterial strains and species are expressed on the cell surface, thus greater spectral differences are observed among SERS spectra of different samples than compared to bulk Raman spectra. This added advantage is exemplified by greater discrimination of bacteria when utilizing SERS spectra as compared to Raman spectra [72]. A number of novel nanofabrication methods have recently emerged for producing SERS substrates with the potential for addressing the issues noted above due to substrate heterogeneity. These include electron beam lithography [80, 81], nanosphere lithography [82–84], a template method [85–88], oblique angle vapor deposition (OAD) [89–91], and a proprietary wet-etching technology used to produce commercially available Klarite (D3 technologies). It should be noted, however, that with the exception of OAD and Klarite, these fabrication methods are not adaptable to large-scale production due to the

Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics

complexity of the fabrication procedure. Not only is it likely that these substrates will lead to significant advances in SERS-diagnostics of bacteria, the use of OAD and Klarite substrates has already lead to successful application to virus identification [59, 60, 62, 63, 75, 76]. In the most recent investigation of SERS-based viral fingerprinting, eight strains of rotavirus were analyzed [63]. These isolates were recovered from clinical fecal samples and propagated in MA104 cells and represent the 5 G and 3 P genotypes responsible for the most severe infections. Unique SERS fingerprints were acquired for each strain when adsorbed onto OAD-fabricated silver nanorods. Representative spectra for each strain and negative control, as well as the difference spectra which subtract out the background cell lysate signal are displayed in Fig. 3. Classification algorithms based on partial least squares discriminant analysis were constructed to identify the samples according to (1) rotavirus positive or negative, (2) P4, P6, or P8 genotype, (3) G1, G2, G3, G4, or G9 genotype, or (4) strain. Respectively, these

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four classification models resulted in 100%, 98–100%, 96–100%, and 100% sensitivity and 100%, 100%, 99–100%, and 99–100% specificity. Compilation and critical analysis of reports to date demonstrate the potential of Raman-based diagnostics and its advantages over IR, normal Raman spectroscopy, and convention diagnostic methods, but also highlight the need for standardization. The challenge in the future is standardization of substrates and sampling protocols since background can “quench” signal from the analyte. For example, blood analysis requires sample processing to remove some competing elements [92], yet SERS spectra highly dependent on the sample pretreatment procedure as remaining chemical species will also contribute signal and degrade the performance of matching in spectral library databases. The outlook of SERS is not a question of spectral quality and reproducibility in a controlled environment, the question is how to control the environment across laboratories.

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Future Directions The future of spectroscopic-based diagnostics is bright as demonstrated by the many studies cited and discussed above. In addition to the success found in these studies, areas of improvement have also been identified. An important area of potential development is the methods used for statistical analysis. Well-established algorithms such as PCA, HCA, and discriminant analysis continue to provide high predictive accuracy, but recent examples have shown that more creative and novel approaches such as artificial neural networks, “bar-coding” [70], or innovative uses of PLS [59] can further improve the predictive value. A revolution in instrumentation is also occurring. Vibrational spectroscopy has recently filled niches in quality control of pharmaceuticals and raw materials as well as identification of chemical threats. The nature of these applications and explosion in interest have driven the instrumentation industry to invest in the development and production of high-quality yet affordable handheld instruments for mobile, on-site analysis. This market-driven commercialization, in effect, is paving the way for point-of-care, mobile, and cost-effective spectroscopy-based diagnostics. The most important factor for widespread realization of spectroscopic diagnosis will be the emergence of a universal protocol for sampling, and for the case of SERS, a standard substrate. The accepted protocol must then be used to build a spectral database covering a variety of phenotypes and developmental stages as illustrated above. Implementing a standard practice is crucial for the success of the technique, but once developed this technology has the potential to become the first and immediate response to clinical cases in which infection is suspected.

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Infectious Diseases, Vibrational Spectroscopic Approaches to Rapid Diagnostics 73. Premasiri WR, Moir DT, Lawrence DZ (2005) Vibrational fingerprinting of bacterial pathogens by surface enhanced Raman scattering (SERS). Proc SPIE 5795: 19–29 74. Sengupta A, Laucks ML, Davis EJ (2005) Surfaceenhanced Raman spectroscopy of bacteria and pollen. Appl Spectrosc 59:1016–1023 75. Shanmukh S, Jones L, Driskell J, Zhao Y, Dluhy R et al (2006) Rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array SERS substrate. Nano Lett 6:2630–2636 76. Shanmukh S, Jones L, Zhao Y-P, Driskell JD, Tripp RA et al (2008) Identification and classification of respiratory syncytial virus (RSV) strains by surfaceenhanced Raman spectroscopy and multivariate statistical techniques. Anal Bioanal Chem 390:1551–1555 77. Yan F, Vo-Dinh T (2007) Surface-enhanced Raman scattering detection of chemical and biological agents using a portable Raman integrated tunable sensor. Sens Actuator B Chem 121:61–66 78. Zeiri L, Bronk BV, Shabtai Y, Czege J, Efrima S (2002) Silver metal induced surface enhanced Raman of bacteria. Colloids Surf A Physicochem Eng Asp 208:357–362 79. Jarvis RM, Goodacre R (2008) Characterisation and identification of bacteria using SERS. Chem Soc Rev 37:931–936 80. DeJesus MA, Giesfeldt KS, Oran JM, Abu-Hatab NA, Lavrik NV et al (2005) Nanofabrication of densely packed metal-polymer arrays for surface-enhanced Raman spectrometry. Appl Spectrosc 59:1501–1508 81. Kahl M, Voges E, Kostrewa S, Viets C, Hill W (1998) Periodically structured metallic substrates for SERS. Sens Actuator B Chem 51:285–291 82. Haynes CL, Van Duyne RP (2001) Nanosphere lithography: a versatile nanofabrication tool for studies of size-dependent nanoparticle optics. J Phys Chem B 105:5599–5611 83. Hulteen JC, Treichel DA, Smith MT, Duval ML, Jensen TR et al (1999) Nanosphere lithography: sizetunable silver nanoparticle and surface cluster arrays. J Phys Chem B 103:3854–3863 84. Jensen TR, Malinsky MD, Haynes CL, Van Duyne RP (2000) Nanosphere lithography: tunable localized surface plasmon resonance spectra of silver nanoparticles. J Phys Chem B 104:10549–10556 85. Broglin BL, Andreu A, Dhussa N, Heath JA, Gerst J et al (2007) Investigation of the effects of the local environment on the surface-enhanced Raman spectra of striped gold/silver nanorod arrays. Langmuir 23: 4563–4568 86. Lombardi I, Cavallotti PL, Carraro C, Maboudian R (2007) Template assisted deposition of Ag nanoparticle arrays for surface-enhanced Raman scattering applications. Sens Actuator B Chem 125:353–356 87. Ruan CM, Eres G, Wang W, Zhang ZY, Gu BH (2007) Controlled fabrication of nanopillar arrays as active substrates for surface-enhanced Raman spectroscopy. Langmuir 23:5757–5760

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Syndromic Surveillance of Infectious Diseases Aharona Glatman-Freedman1,2 and Zalman Kaufman1 1 Israel Center for Disease Control, Israel Ministry of Health, Ramat Gan, Israel 2 Department of Epidemiology and Preventive Medicine, School of Public Health, Tel Aviv University Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Article Outline Glossary Terms Definition of the Subject Introduction Data Retrieval and Analysis in Syndromic Surveillance Applications of Syndromic Surveillance Web-Based Developments Conclusions Future Directions Bibliography

Glossary Terms Syndromic surveillance: A surveillance approach that uses medical data from different sources to monitor disease trends in real-time and to detect disease outbreaks.

Definition of the Subject Infectious diseases continue to present major challenges throughout the world. Since the spread of infectious diseases occurs irrespective of geographic boundaries, they place at risk both individuals and populations [1]. Optimal surveillance is paramount for the detection and the combat

against infectious diseases. Several types of surveillance can be applied to follow infectious diseases. They include the traditional form of surveillance which rely on the report of notifiable diseases by clinicians and clinical laboratories and syndromic surveillance [2]. Syndromic surveillance is an exploratory method in which disease indicators can be retrieved through automated data acquisition, analysis, and the generation of statistical signals [2]. These signals are monitored in real-time or near real-time in order to detect disease outbreaks earlier and more comprehensively than with traditional public health approaches [2]. Syndromic surveillance follows patterns of disease and can detect changes in illness data [3].

Introduction Syndromic surveillance was first applied in the 1990s, with the purpose of detecting bioterrorism events [3]. Syndromic surveillance systems use medical data from different sources [4, 5]. Unlike traditional surveillance systems, which rely on specific clinical and laboratory results, syndromic surveillance relies on nonspecific clinical data, as well as administrative data [4, 6]. These sources include general practitioners and emergency departments visits but can also utilize data regarding pharmacy sales, medical telephone help lines, as well as web queries [4]. Although syndromic surveillance systems rely on nonspecific data, they have the advantages of sensitivity and timeliness. In addition, since the data used for syndromic surveillance is collected automatically, no specific additional efforts is required for data collection [6].

Data Retrieval and Analysis in Syndromic Surveillance Syndromic surveillance utilizes nonspecific clinical data, administrative data, and web-based data

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_1088 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2021 https://doi.org/10.1007/978-1-4939-2493-6_1088-1

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Data sources Clinic visits

Emergency department visits

Medicaons prescripons

Medicaons purchase

Selecon of syndromes/ Diagnoses/ Key terms

Retrieval of data by syndromes/ Diagnoses/ Key terms

Idenfying data aberraon

Signal verificaon

Medical telephone help-lines

Absence reports

Use by public health authories

Web queries

Syndromic Surveillance of Infectious Diseases, Fig. 1 Key components in the process of syndromic surveillance

(Fig. 1). In order to generate relevant data, syndromes, diagnoses, or key terms are selected (Fig. 1). For example, influenza-like illness (ILI) constitutes a syndrome for the surveillance of influenza. Several diagnostic terms can be used for ILI: flu, fever and cough, fever and sore throat, or fever with cough and sore throat [7]. Specific clinical diagnoses such as measles, rubella, mumps, and others can also be selected [8]. Medication names can be used as a key term to search for prescriptions that were dispensed or for overthe-counter medications that were purchased at pharmacies [9, 10]. Data is then retrieved through the use of the selected syndromes, diagnoses, or key terms. The data is then screened for the purpose of identifying aberrations (Fig. 1). This screening is frequently done through the use of algorithms [11]. The data that constitute the aberration are then reviewed to determine if the signal identified is verified to be of concern (Fig. 1).

Following signal verification, the information can be used by public health authorities (Fig. 1).

Applications of Syndromic Surveillance Syndromic surveillance systems have been implemented worldwide [5, 12–14]. Usually, they are integrated within surveillance systems for monitoring influenza and acute gastrointestinal morbidity [3]. In addition to these routine applications, syndromic surveillance has been recommended for detecting and managing public health events of high concern, such as severe acute respiratory syndrome (SARS), avian influenza (H5N1), or pandemic influenza [3]. Syndromic surveillance has been recently used in COVID-19 surveillance [15–17]. Various methodologies can be applied to analyze the data used in syndromic surveillance.

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These include full automatization with the use of algorithms, on the one hand, and manual collection and analysis of data, on the other [3].Since syndromic surveillance relies on clinical syndromes, it can be used in places with little or no availability of timely laboratory services, such as low-income countries [3, 13, 18]. Syndromic surveillance has demonstrated the ability to adapt to different public health needs [3]. The following sections describe the application of syndromic surveillance in response to such needs. Continuous Disease Surveillance The most commonly used syndromic surveillance tool for monitoring influenza consists of physicians’ visits for influenza-like illness (ILI), which is used by multiple countries around the globe. A study from the USA demonstrated that ILI physicians’ visits and ILI medical claims used to generate regional time series graphs that were strongly correlated with influenza laboratory surveillance data, during seasonal and pandemic influenza periods [19, 20]. Syndromic surveillance of acute gastrointestinal morbidity is routinely performed in the USA [21], France [22], the United Kingdom [23], and Israel [24]. Syndromic surveillance has also been applied for a variety of illnesses. While for certain illnesses clinical syndromes such as upper respiratory illness and lower respiratory illness are used, in other cases, specific diagnoses are used. In this regard, Public Health England (PHE) collects, analyzes, and distributes ongoing data regarding the following diseases: pharyngitis, Asthma, pneumonia, scarlet fever, mumps, measles, rubella, chicken pox, impetigo, and cellulitis [8]. Syndromic surveillance has been applied also for diseases such as malaria, dengue fever, and sexually transmitted illness [13]. Active Case Finding Using Syndromic Surveillance Syndromic surveillance systems can be used when responding to an emerging or reemerging infectious agent. In the beginning of 2016, the Florida Department of Health used its Electronic Surveillance System for Early Notification of Communitybased Epidemics (ESSENCE-FL) to run Zika

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queries. During a period of 7 weeks, five cases of imported Zika virus disease were identified using ESSENCE-FL [25]. Syndromic surveillance was employed in two healthcare centers in Gabon to detect measles cases [25] and in Australia to detect cases of leptospirosis during an outbreak that occurred among raspberry workers [26]. The New York City Department of Health used an emergency department syndromic surveillance system to identify patients with Ebola-like illness among travelers who returned from West Africa [27]. A mobile telephone-based syndromic surveillance system established in Southern Sierra Leone allowed the detection of Ebola hemorrhagic Fever [28]. Situational Awareness Syndromic surveillance can be used as a tool for following the course and progress of an outbreak [29]. The use of syndromic surveillance was evaluated during the 2009 influenza H1N1 pandemic. During the first phase of the of sustained humanto-human transmission of the pandemic influenza virus, laboratory-confirmed cases were used to track the outbreak. However, once the number of cases increased substantially, the use of laboratory confirmation for each suspected case became nonpractical. At such point, syndromic surveillance can become the main modality for evaluating community disease burden [30]. Detection of Infectious Diseases Among Specific Populations Mass Gatherings

Mass gatherings present a particular risk for the transmission of infectious diseases. For the 2012 London Olympic Games, syndromic surveillance in the UK was enhanced by the use of daily reporting of primary physicians’ out of hours contacts and emergency department visits to sentinel sites [31]. Syndromic surveillance was also applied to the 2015 special Olympic Games in Los Angeles [32]. A syndromic surveillance system was established in Iran to determine the trends of common illness among Iranian Hajj pilgrims. The system evaluated trends during five Hajj events. The infectious diseases syndromes included in that evaluation were: influenza-like illness,

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common cold, pneumonia, sinusitis, gastroenteritis, and conjunctivitis [33]. The most common infectious diseases detected were common cold, influenza-like illness, and sinusitis [33]. In addition, three gastroenteritis outbreaks were identified during the evaluation period [33]. A syndromic surveillance consisting of several infectious diseases syndromes was established during the Arbaeenia, a major religious mass gathering in Iraq, in 2014. The data was collected from 40 mobile clinics and was found useful in demonstrating the most prevalent infectious entities in that gathering [34]. Migrant Populations

Several studies described the application of syndromic surveillance to specific populations. Massive migration can predispose migrant populations and host populations to a variety of infectious diseases due to crowding and less than optimal health services. Italy established a syndromic surveillance system using 13 syndromes, to provide early warning of outbreaks among migrants from North Africa [35]. During a 6-month surveillance period which followed the 2011 civil unrest in North Africa, four alarm signals were issued by the system. The alarms which were triggered by cases of watery diarrhea, respiratory tract disease, and parasite skin infection subsided spontaneously within 1–3 days, indicating to health authorities that there was no need to initiate an outbreak response [35]. A recent syndromic surveillance system, using 14 syndromes, was established in migrant accommodations in Berlin, Germany, to detect infectious diseases among asylum seekers [36]. The most common infectious diseases syndromes, reported during the evaluation period which lasted 1.5 years, were respiratory infections, cutaneous parasites, and gastrointestinal infections. A total of 204 signals were generated by the syndromic surveillance system. However, no significant outbreak was detected during the evaluation period [36].

Web-Based Developments The recent availability of new and diverse digital data sources has led to the development of a variety

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of nontraditional methods for monitoring epidemics [37]. These new data methods attempted to capture relevant surveillance signals that can complement more traditional surveillance data, such as those arising from primary physicians practices [38]. Google Flu Trends is one example. It was a Google-operated web service, first launched in 2008, which attempted to provide estimates of influenza activity for 29 countries [39]. By aggregating search queries that were related to influenza-like illness, it sought to provide faster estimates about influenza activity [40]. A study that examined the correlation between Google Flu Trends incidence estimates and ILI consultation rates based on sentinel networks in several European countries found a strong correlation between the two systems during the 2009 influenza pandemic [41]. Google flu trends data also correlated well with emergency department ILI data from Manitoba, Canada, and demonstrated that data from both sources peaked 1–2 weeks prior to laboratory-confirmed influenza cases during the 2009 influenza pandemic [42]. Furthermore, good correlation was also found between Google Flu Trends data and prescription sales data for the years 2007–2011 [43]. Google trends data was recently evaluated for dengue fever using specific search terms. A study from Indonesia found high correlation between data retrieved from google trends using three search key words and the official national surveillance reports of dengue fever [44]. Evaluation of Google Dengue trends in Mexico found that it is most accurate in areas of high transmission, especially where the climate is most favorable for transmission [45]. Google trends data was also evaluated for other infectious diseases such as tick-borne encephalitis, [46], pertussis [47], malaria, and chikungunya [48] demonstrating significant correlation of various levels with reported cases of these diseases. However, the data indicates that Google trends data cannot be used as a stand-alone method, but rather as a supplementary method to traditional syndromic surveillance methods. Several attempts have been made to evaluate Twitter messages for syndromic surveillance.

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Twitter massages containing words synonymous to those of influenza case definition were identified by an algorithm. High correlation was found between relevant tweets and ILI trends generated through US traditional surveillance platforms [49]. Tweeter messages were found to have a high positive correlation with dengue fever cases reported to the Brazilian Ministry of Health [50]. Twitter data was also evaluated for other infectious diseases such as Zika virus [51] and Ebola [52]. The weekly Zika virus tweets and case reports in the USA followed similar temporal and special patterns [51]. The frequency of Ebola virus-related tweets increased during the days that preceded the distribution of Ebola-virus official news in Nigeria [52], indicating that the use of relevant tweets could be valuable in the places that do not have optimal surveillance systems [52]. Another innovative approach consists of involving the public directly in disease surveillance. An example of a participatory disease surveillance is the Influenzanet project, which operates in ten European countries [53]. It is a web-based surveillance system, established in 2011, for monitoring seasonal influenza epidemics. It relies on volunteers from the general population who monitor and report their symptoms weekly, through internetbased surveys [53]. This methodology can be regarded as a near-real-time-flexible surveillance structure, which can capture data of several groups of syndromes, not restricted to influenza alone [53]. Overall, the ILI incidence that was determined through influenza net data was found to correlate well with the ILI incidence that was reported by the European Center for Disease Prevention and Control Control (ECDC) [54] and by individual countries’ traditional surveillance systems.

Conclusions Syndromic surveillance of infectious diseases has become an important addition to the traditional surveillance methods. The main advantages of syndromic surveillance are the ability to be used adaptively to

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meet various needs, and the availability of data which is collected, in most cases, for administrative or other purposes. In addition, new sources of data can be added to syndromic surveillance over time, as they become available. The integration of syndromic surveillance with other forms of surveillance can provide a comprehensive picture that can assist public health professionals to make decisions based on multiple data sources. Furthermore, since syndromic surveillance relies on detecting changes in patterns of the disease frequency in target populations, it has the potential to provide early warning for emerging and reemerging diseases.

Future Directions Despite the development and evolution of syndromic surveillance over the past 30 years, several challenges require additional research and attention. The greatest challenge for syndromic surveillance is improving the capacity for early warning of evolving outbreaks. In this regard, finding reliable syndrome-specific algorithms that will provide sensitive yet specific alarm signals is paramount [55]. Localizing evolving outbreak signals in realtime is an equally important challenge that requires additional work. Optimally, localization of outbreaks around the globe should be done by a single algorithm. However, disease baselines vary geographically due to differences in the utilization of healthcare services in different geographical areas [55]. Furthermore, localization data vary among healthcare systems and countries [55]. The use of web-based data for syndromic surveillance shows promise for improving early warning of public health threats. However, despite the progress made, additional research is still required to overcome certain challenges such as “noise” generated by irrelevant data, privacy concerns, demographic bias, and language interpretation [56]. Last, additional efforts are required to identify optimal methods of syndromic surveillance in regions with weak healthcare services.

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82 51. Masri S, Jia J, Li C, Zhou G, Lee MC, Yan G et al (2019) Use of Twitter data to improve Zika virus surveillance in the United States during the 2016 epidemic. BMC Public Health 19(1):761 52. Odlum M, Yoon S (2015) What can we learn about the Ebola outbreak from tweets? Am J Infect Control 43(6):563–571 53. Kalimeri K, Delfino M, Cattuto C, Perrotta D, Colizza V, Guerrisi C et al (2019) Unsupervised extraction of epidemic syndromes from participatory influenza surveillance self-reported symptoms. PLoS Comput Biol 15(4):e1006173

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Antibiotics for Emerging Pathogens Vinayak Agarwal1 and Satish K. Nair2 1 Center for Biophysics and Computational Biology, University of Illinois at UrbanaChampaign, Urbana, IL, USA 2 Department of Biochemistry and Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Tuberculosis A highly contagious bacterial disease of humans and animals caused by various strains of Mycobacterium, which normally affects the lungs but can also spread to other organs of the body. Malaria A highly contagious protozoal disease caused by various strains of mosquito-borne Plasmodium. Peptide Short chains of amino acids connected by peptide bonds. Polyketide Compounds characterized by more than two carbonyl groups connected by single intervening carbon atoms.

Article Outline

Definition of the Subject Glossary Definition of the Subject Introduction Emerging Targets of Antibiotic Molecules Emerging Chemical Classes of Antibiotic Molecules Future Directions Bibliography

Glossary Microorganism A microscopic or submicroscopic organism, too small to be seen by unaided human eye, comprising of bacteria, virus, yeast, protozoa and fungi. Pathogen A disease-causing microorganism which may or may not be infectious. Antibiotic A chemical substance, usually organic in nature, capable of destroying or inhibiting the growth of pathogenic microorganisms. Antibiotic resistance The developed or acquired ability of antibiotic susceptible pathogenic microorganisms to grow and survive despite the inhibitory action of the antibiotic molecules. Plasmid Small, linear or circular genetic elements which can replicate independently of the chromosomal DNA inside a cell.

Antibiotics are organic small molecules, many of which are from natural sources, which are used to treat human infections caused by pathogenic microorganisms. While most validated antibiotics are initially very useful clinically, the pathogenic microorganisms that these compounds target are able to evade the action of the antibiotic by development of resistance mechanisms, which eventually render these antibiotics ineffective. Moreover, these resistance mechanisms can be passed on among different types of bacteria in a very simplistic manner that further compromises the usefulness of antibiotics. As a consequence, many diseases that were thought to have been eradicated by antibiotics (such as tuberculosis) have reemerged within these antibiotic-resistant strains. Hence, there is a constant need for the development of new and better antibiotic molecules that can be used to target these drug-resistant microbial populations. Traditionally, the discovery and development of new antibiotics and other drug molecules has relied on two parallel and complimentary approaches: the discovery of new molecules of clinical relevance from natural sources, and the use of synthetic chemistry methods to derive compounds, based on the scaffold of naturally occurring molecules, with enhanced favorable

© Springer Science+Business Media, LLC 2012 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_523 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media LLC, 2012 https://doi.org/10.1007/978-1-4419-0851-3_523

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antimicrobial properties. More recent development includes the identification of molecules without any natural precedents, based on the use of high-throughput screening methods of potential antibiotic targets against a large number of synthetic compound libraries to identify new classes of antibiotic molecules. Continued developments in all of these approaches, by an amalgam of both academic and industrial efforts, are essential for the development of compounds aimed at treating the evolution of drug-resistant pathogenic microorganisms.

Introduction The interaction of prokaryotic microorganisms with human beings is widespread and significant, and the number of bacterial cells within the human body far exceeds the number of the human eukaryotic cells. This interaction between humans and bacteria is designed to be mutually beneficial to both [1, 2]. However, a small number of these bacterial microorganisms are known to cause disease in human beings and are hence called pathogens. The mechanisms by which these pathogenic organisms cause disease are extremely diverse and affect nearly all types of tissues and organs. The advancement of medical science has resulted in the development of two frontline defense mechanisms to fight infections and diseases caused by microbes. The first line of defense is the vaccines that specifically elicit the human host’s adaptive immune response to recognize and eliminate pathogenic and harmful microbes, while selectively preserving the beneficial microbe population. Vaccines are generally biological macromolecules, derived from the pathogenic microbes themselves, which provide the adaptive immune response with the information necessary to elicit an antibody or cellular immune response against future invasions by the pathogenic microbes. Vaccines have been developed against several pathogens and have led to very significant reductions of deadly diseases such as tuberculosis and tetanus. However, vaccinations are not currently available against all pathogens, and the natural evolution of the pathogens themselves may render these

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vaccinations ineffective against future infections, as exemplified by the inherent difficulties in vaccine development against viral infections such as influenza and HIV/AIDS. The second frontline defense against pathogenic infections is the discovery and development of small molecule antibiotics that are designed to target and kill the harmful microorganisms with minimal side effects against the human host. Antibiotics are naturally occurring molecules produced by a number of different bacterial species to give them a selective competitive advantage for nutrients or other growth necessities over other microorganisms with which they share their surroundings. Serendipitous discovery and exploitation of these molecules dates back to ancient history [3–5] with numerous early reports, such as the use of the bark from cinchona tree, which contains the antibiotic quinine, used to treat malaria. Antibiotics generally have well-defined mechanisms of action against microbes and target specific biological processes to either retard growth (bacteriostatic antibiotics) or kill the microbe (bacteriocidal antibiotics). The first report of an isolated antibiotic molecule used to effectively treat infections was the development and use of penicillin from the fungus Penicillium notatum by Alexander Fleming in 1928. Penicillin was used to treat diseases such as syphilis and other infections caused by staphylococci and streptococci. The term “antibiotic” was coined by the microbiologist Selman Waksman in 1942 [6] and is derived from the Greek antibiosis (“against life”). Hailed as a wonder drug, the discovery of penicillin marked the beginning of the antibiotic age, during which time antibiotics were developed to treat almost all major human infectious diseases. During this era, thousands of antibiotic molecules were discovered and these compounds were applied to fight bacterial, fungal, protozoan, and yeast infections. Concurrently, synthetic organic chemistry was also applied for the production of derivatives of naturally occurring antibiotics, and these synthetic compounds exhibited more desirable properties, such as decreased incidences of allergic reactions in humans. The discovery and development of these molecules marked a decreased

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incidence of outbreaks of the most common infectious diseases and contributed to an increase in life expectancy around the world. However, repeated challenge of the pathogenic microorganisms with these antibiotics, in combination with the natural evolution of their genetic information, has resulted in the development of antibiotic resistance in these pathogens [7–9]. This process has led to the development of microbial resistance against nearly all available antibiotic molecules and has precipitated to an immense medical and financial challenge. The arsenal available to fight infections is becoming increasingly limited, and the rate of development of new molecules has not kept pace with the emergence of antibiotic resistance. Factors Contributing to Increased Antibiotic Resistance 1. Widespread and indiscriminate overuse of antibiotics in the clinical environment, which exposes pathogenic microorganisms to an ever increasing concentration of different antibiotic molecules, which in turn increases the selection pressure to develop resistance against these antibiotic molecules [10, 11]. 2. Use of antibiotics in agriculture and livestock rearing also leads to increased exposure of pathogenic bacteria to antibiotic molecules. Many of the antibiotics in use in agriculture and livestock feeds are also used for treating humans, and hence, development of resistant bacterial strains can be transferred from one setting to the other very easily [12–14]. 3. Genetic recombination and transfer of genes from an antibiotic-resistant microbe to a nonresistant microbe, which leads to spread of resistance among microbial species. Antibiotic resistance genes are usually borne on highly mobile genetic vehicles called plasmids, which can easily be transmitted among microbes [15]. Microorganisms are able to sequester multiple plasmids, resulting in the evolution of plasmids that contain more than one resistance determinant. For example, the Shigella epidemic that caused nearly a quarter million deaths in Guatemala in 1968 was caused by a pathogen that contained a plasmid with resistance against

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four of the most commonly used antibiotics [16–18]. Mechanisms by Which Microorganisms Develop Resistance to Antibiotics 1. Efflux pumps in the microbial cell walls and membranes which actively transport the antibiotic molecule out of the cell and hence decrease its concentration within the cell. Consequently, increasing doses of antibiotics are required to target such microbes [19–21]. This mechanism is typified by the resistance to the broad-spectrum antibiotic tetracycline, which is mediated by the genetically mobile tet genes that encode for active efflux pumps for tetracycline and its derivatives [22–24]. 2. Chemical modification of the antibiotic molecule by specific enzymes within the target pathogenic microbe, which render the antibiotic ineffective [25]. This mechanism is of particular concern against β-lactam antibiotics, such as penicillin and its derivatives [26, 27]. 3. Modulation of the antibiotic target within the target cell so that the antibiotic is no longer able to bind and engage the target. Random mutations within the antibiotic target genes may lead to the emergence and subsequent selection of microbe species with a desensitized antimicrobial target. This mechanism of resistance is operative for development of resistance against the macrolide antibiotic erythromycin, and resistance results from the modification of its target, the bacterial ribosome, such that the antibiotic is no longer able to find the target [28, 29]. Another example is resistance to the “drug of last resort” vancomycin, which results as a change in its target of the D-Ala- D-Ala dipeptide moiety of the bacterial cell wall to D-Ala- D-Lac or to D-Ala- D-Ser [30–32]. 4. Alteration of the metabolic pathways that are targeted by the antibiotic molecule so that inhibition of these metabolic pathways is not inhibitory or lethal to the microbe. A primary example of this class of resistance is the alterations in the sterol biosynthesis pathway in fungi which confer resistance to azole antibiotics [33, 34].

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In light of the above factors, there is an evergrowing need for academic laboratories as well as for the pharmaceutical industries to search for antibiotics that have new and as yet unexplored mode of actions. New chemical classes of drug molecules are needed to mitigate the effects of resistance against the current drug chemical entities.

Emerging Targets of Antibiotic Molecules Several new classes of antimicrobial targets have been identified, and drug development based on these findings, though still in its infancy, have taken major leaps forward. Microbial Fatty Acid Biosynthesis Inhibition The microbial fatty acid biosynthesis pathway presents numerous targets for drug development, particularly against the causative agent of tuberculosis – Mycobacterium tuberculosis. The metabolic pathways and chemical mechanisms for the synthesis of fatty acids are shared between a majority of prokaryotes and eukaryotes, including humans. However, as a result of differences in protein sequence and different arrangement of the active sites, compounds that target the bacterial fatty acid synthesis pathway are not crossreactive or toxic against humans. The most wellcharacterized class of fatty acid biosynthesis inhibitor molecules is the antituberculosis molecule isoniazid, which inhibits the biosynthesis of mycolic acid, an essential component of the mycobacterial cell wall. Triclosan, an extensively used molecule in consumer products, such as toothpastes and mouthwashes, is also a fatty acid biosynthesis inhibitor. An example of an emerging molecule in this class of antimicrobials is platensimycin (Fig. 1), produced by the organism Streptomyces platensis [35], which inhibits the growth of several pathogenic Gram-positive bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus pneumonia [36]. Platensimycin inhibits the 3-ketoacyl-ACP synthase (KAS) enzyme FabF. Continued high-throughput

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screening and host genetic engineering efforts led to the identification of a platensimycin analog, platencin [37–39], which, in addition to FabF, inhibits another KAS enzyme called FabH and displays enhanced pharmacodynamic properties as compared to platensimycin. Chemical structures of microbial fatty acid biosynthesis inhibitor molecules discussed in this section are illustrated in Fig. 1. Other KAS inhibitor molecules include cerulenin and thiolactomycin [40, 41]. Cerulenin is a broad-spectrum antifungal antibiotic produced by fungus Caephalosporium caerulens and preferentially inhibits the KAS enzymes FabB and FabF. Cerulenin forms a stable covalent adduct with the active site cysteine residue of these enzymes, and thus inhibits malonyl-group incorporation in the growing fatty acid chains [40, 42]. Thiolactomycin and related compounds have been isolated from various Streptomycetes and show potent inhibitory activity against fatty acid biosynthesis and mycolic acid biosynthesis in mycobacteria as well as against protozoan parasites such as malaria-causing Plasmodium falciparum. Thiolactomycin mimics the binding of malonyl coenzyme A molecule in the active site of KAS enzymes FabB, FabF, and FabH [41, 43]. Attempts for the preparation of semisynthetic derivatives of thiolactomycin are currently underway, and some of these derivatives are expected to improve upon its antimicrobial properties of thiolactomycin [44]. Molecules such as triclosan, isoniazid, diazaborines, and ethionamide target the enoylACP reductase enzyme FabI of the fatty acid biosynthesis pathway. Each of these molecules binds in the active site of the enzyme and forms a tight complex with the nicotinamide adenine dinucleotide (NAD+) cofactor required for catalysis by FabI. However, pseudomonads and S. pneumoniae contain an additional enoyl-ACP reductase enzyme, FabK, which is not susceptible to inhibition by these compounds, which allows these pathogens to overcome the antibacterial activities of these compounds. The fatty acid biosynthesis pathways also present several other enzymes that can be targeted for drug development. The isomerase activity of the

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Antibiotics for Emerging Pathogens, Fig. 1 Chemical structures of representative microbial fatty acid inhibitor molecules

enzyme FabA can be inhibited by the drug candidate compound 3-decynoyl-N-acetylcysteamine. Metabolic interaction of the acetyl coenzyme A carboxylase enzyme with the fatty acid biosynthetic pathway also poses a potential target for inhibitor screening. Finally, compounds such as phenethyl alcohol, which inhibit the enzyme PlsB, responsible for the link between fatty acid biosynthesis and phospholipid biosynthesis, are also antimicrobial agents. Isoprenoid Biosynthesis Inhibition Isoprenoids are naturally occurring diverse organic compounds derived from the condensation of two or more isoprene monomeric units. They represent the largest class of small molecules on Earth. The isoprenoid pathways between

prokaryotes and eukaryotes are very divergent: Humans synthesize isoprenoids by the mevalonate pathway, while microbes synthesize isoprenoids via the nonmevalonate pathways. Both pathways present numerous potential drug targets. One of the most widely used cholesterollowering drug, Lipitor, targets the mevalonate pathway in humans which is responsible for cholesterol biosynthesis. Antimicrobials such as terbinafine target the nonmevalonate pathways in fungi and yeast. Several promising lead compounds that target the nonmevalonate pathway in the malaria parasite Plasmodium falciparum have been characterized. Principal among these are the phosphonate molecules fosmidomycin, first isolated from Streptomyces lavendulae, and its derivative

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FR900098, isolated from Streptomyces rubellomurinus [45, 46]. Fosmidomycin inhibits the enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) of the malaria parasite and has progressed to the stage of human phase II clinical trials for use in combination therapy with lincosamide antibiotic clindamycin [47–49]. However, the bioavailability of this molecule is low in bacterial models, with tuberculosis-causing bacterial pathogen essentially resistant to this molecule due to lack of uptake [50]. FR900098 is a structural analog of fosmidomycin and has been shown to demonstrate better efficacy in vivo than the parent compound. Synthetic and biosynthetic routes for the production and manipulation of this compound are currently being pursued [51, 52], as is the synthesis of several other structural analogs of fosmidomycin [53]. Fosmidomycin and FR900098 belong to the phosphonate class of antimicrobials, which are characterized by the presence of a stable carbon– phosphorous covalent linkage [54]. In addition to the DXR enzyme, a second potential drug target is the isoprenoid biosynthesis enzyme IspH, which is present in the vast majority of pathogenic bacteria and the malaria parasite but not in humans [55]. IspH is an attractive target for the development of inhibitors which could be potential drug and herbicide candidates. A series of diphosphate and bisphosphonate compounds have been synthesized, some of which inhibited the IspH enzyme at nanomolar concentrations [56, 57]. Other isoprenoid biosynthetic drug targets include the enzyme dehydrosqualene synthase CrtM, from the human pathogen Staphylococcus aureus, which can be inhibited by phosphonosulfonates and phosphonoacetamide compounds [58]. These compounds lead to inhibition of production of the S. aureus virulence factor staphyloxanthin, which makes these otherwise recalcitrant pathogenic cells susceptible to reactive oxygen species and subsequent clearance by the human immune system [59, 60]. Finally, the antiarrythmia drug amiodarone, which blocks various metal ion channels in the cell membrane [61], in conjunction with itraconazole, a fungal cytochrome P450 inhibitor [62], is also being

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reevaluated for the treatment of Chagas disease and cutaneous leismaniasis [63]. Cell Wall Biosynthesis Inhibition Inhibition of cell wall biosynthesis has been one of the earliest validated targets for antibiotic development and still remains one of the most fruitful areas for the development of new drugs. However, drug development against cell wall biosynthesis is particularly challenging due to the presence of an additional lipopolysaccharide layer in Gram-negative bacteria and the fungal chitin cell wall in fungi, both of which are extremely rigid and impermeable to most small molecule drugs. The following section focuses on some new nucleosidic antibiotic molecules that inhibit microbial cell wall biosynthesis and hold promise for future development. Chemical structures of microbial cell wall biosynthesis inhibitor molecules discussed in this section are illustrated in Fig. 2. Nucleoside antifungal polyoxin molecules, such as nikkomycins, were isolated from several strains of Streptomyces [64] and have been shown to inhibit the biosynthesis of the fungal chitin cell wall. Nikkomycins display a wide spectrum of bioactivity while being nontoxic to animals and plants and have traditionally been used for controlling plant fungal diseases in agriculture. The parent nikkomycin compounds show relatively weak activity against opportunistic human pathogenic fungi, and semisynthetic derivatives are now being prepared which may display better pharmacological properties [65]. Capuramycin was first isolated from Streptomyces griseus in the 1980s and shows potent antibacterial properties against the human pathogens Streptococcus pneumoniae and Mycobacterium smegmatis [66]. Structure activity relationships have been explored by the synthesis of capuramycin derivatives [67, 68]. In in vivo studies against Mycobacterium tuberculosis, capuramycin demonstrated unique phenotypic changes in the pathogen which are not shown by any other currently available antibiotic and which may signify a mode of action distinct from those of known antibiotics [69].

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Antibiotics for Emerging Pathogens, Fig. 2 Chemical structures of representative microbial cell wall biosynthesis inhibitor molecules

Structurally related uridylpeptide molecules such as mureidomycins, pacidamycins, napsamycins, and sansanmycins have demonstrated activity against infections of the opportunistic human pathogen Pseudomonas aeruginosa in rodent models. Such infections are difficult to treat in immune-compromised patients as this pathogen can form a protective biofilm layer resulting in chronic infections [70–72]. These molecules do not show any cross-reactivity with mammalian cells.

Emerging Chemical Classes of Antibiotic Molecules Peptidic Antibiotics Peptidic antimicrobials are typically synthesized by the ribosome and consist of short polymers of the 20 naturally occurring amino acids linked together by amide bonds. A second class of peptide antimicrobials are generated by a molecular assembly line, called nonribosomal peptide synthases (NRPS), which can incorporate

nonnatural amino acids. The use of peptides to target pathogenic microbes is exemplified by the production of short cationic peptides by the human and plant immune systems, which either recruit the immune system for pathogen clearance or disrupt the outer cell and organelle cell membranes of microbes. A common conserved feature among all peptidic antimicrobials is posttranslational modifications conferred upon the peptide backbone, which are essential for their biological activities [73–75]. A well-known example of one class of ribosomally produced antimicrobial peptides is lantibiotics. Although these molecules are synthesized on the ribosome, they undergo posttranslational processing, resulting in the formation of several ring-like structures that add rigidity [76]. These compounds can be further modified on the amino and carboxy termini of these peptides as well, as shown in the structures of the lantibiotics epilancin [77] and microbisporicin [78]. Lantibiotics demonstrated multiple modes of action and exert their biological activities by binding to the lipid II cell wall moiety in bacteria [79],

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Antibiotics for Emerging Pathogens, Fig. 3 Chemical structures of lantibiotic nisin. Some amino acids are abbreviated by their three-letter codes

Antibiotics for Emerging Pathogens, Fig. 4 Chemical structures of lipopeptide antibiotic daptomycin

which leads to both inhibition of peptidoglycan biosynthesis as well as cell membrane permeabilization. The most extensively used and wellcharacterized lantibiotic to date is Nisin (Fig. 3), initially identified as a by-product of milk fermentation, which contains seven lantibiotic rings, and has been used in the livestock industry for the treatment of bovine mastitis [80, 81]. Nisin, produced by the bacterium Lactococcus lactis, shows no adverse effects in humans and is being evaluated in human trials for the treatment of staphylococcal mastitis in lactating women [82].

Among the NRPS peptides, the most promising drug candidates are the lipopeptide antibiotic daptomycin and glycopeptides vancomycin and teicoplanin. Lipopeptide antibiotic such as daptomycin (Fig. 4) contains a long lipid chain attached to the peptide, which helps in targeting the bacterial cell wall. Daptomycin is produced by Streptomyces roseosporus, and genetic manipulations of the producer organism have led to the production of a combinatorial library of daptomycin derivatives [83–86]. Daptomycin shows potent antimicrobial activity against methicillin-resistant Staphylococcus aureus

Antibiotics for Emerging Pathogens

(MRSA) [87], infections by which are a rising global concern, and is already registered for the treatment of skin and soft tissue infections [88], bacteremia, and bacterial endocarditis [89]. The glycopeptide antibiotics are produced as cyclic aglycones (without sugars) and are then decorated by a number of different sugar molecules that are covalently attached by dedicated glycosyltransferase enzymes [90–92]. Among these, vancomycin (Fig. 5) [93], which prevents cell wall biosynthesis in Gram-positive bacteria, is clinically approved as the “drug of last resort” against MRSA infections [94]. Oritavancin (Fig. 5), a semisynthetic derivative of vancomycin, is potent against vancomycin-resistant enterococci (VRE) and is currently under human trials for the treatment of soft tissue infections by Grampositive bacteria [95]. Also of note are the cyclic lactone linkage–containing depsipeptides katanosins/plusbacins [96] and glycolipodepsipeptide ramoplanin [97], which are also active against MRSA. Polyketide Antibiotics Polyketide antibiotics are similar to nonribosomal peptide antibiotics as they are synthesized by multimodular polyketide synthases. These enzymes are analogous to the nonribosomal peptide synthases but typically catalyze the condensation of malonyl coenzyme A–derived monomeric units into cyclic lactone rings, onto

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which further modifications such as hydroxylations, glycosylations, and methylations are added. The most well-known polyketide antibiotic is erythromycin (Fig. 6), produced by Streptomyces erythreus, which has been in clinical use for more than 50 years. Several derivatives of erythromycin, such as clarithromycin and azithromycin (Fig. 6), have been developed for clinical use [98]. The mode of action of these compounds is inhibition of protein synthesis through binding to the various subunits of the bacterial ribosome [99]. Advancements in genetic technologies have opened up avenues for the manipulation and reprogramming of polyketide synthases for the production of new and novel molecules, which may yield drug candidates with enhanced pharmacological properties [100–102]. Of particular note are the ansamycin group of macrolide molecules, which differ from the molecules previously discussed in that the monomeric unit is 3-amino5-hydroxybenzoic acid rather than a malonyl coenzyme A derivative. Various ansamycin molecules, such as rifamycin, geldanamycin, herbimycin, and ansamitocins, have been isolated, and their biosynthetic routes have been characterized. Of these, rifamycin, which inhibits bacterial RNA polymerase [103], is widely used in the treatment of tuberculosis. Geldanamycin, biosynthesized by Streptomyces hygroscopicus, and its analogs [104] inhibit heat shock protein

Antibiotics for Emerging Pathogens, Fig. 5 Chemical structures of representative glycopeptides and lipoglycopeptide antibiotics vancomycin and oritavancin

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Antibiotics for Emerging Pathogens

Antibiotics for Emerging Pathogens, Fig. 6 Chemical structures of representative polyketide antibiotics

90 (Hsp90) [105] and are being evaluated in human clinical trials as antitumor agents [106]. Trojan Horse Antibiotics A limiting factor for many antimicrobials and antibiotics is the lack of permeability across the microbial cell membranes. The outer membrane of the Gram-negative bacteria presents a formidable obstacle for drug entry [107]. Such Gramnegative bacteria employ a variety of active importer channels and pores to internalize requisite nutrient material. Nature has ingeniously designed several antimicrobial compounds that utilize a “Trojan horse” strategy to overcome permeability barriers; specifically, these drug molecules are covalently attached to a carrier that facilitates recognition and import through the active transporters. Once inside the target cell, the carrier is removed to release the active drug, which then exerts its antimicrobial activities. This strategy for drug delivery has several advantages.

First, active transport of the molecule inside susceptible cells alleviates the need for very high doses for the drug. Second, targeting of the molecule is highly specific only for those microbial cells that have the required transporter to internalize the molecules. Consequently, these “Trojan horse” are not taken up in humans and can be effective microbials even at very low doses. Notable among this class of compounds are the sideromycins [108, 109], which are characterized by the attachment of the antibiotic molecule to ferric ion–binding chemical components called siderophores. The siderophore–iron complexes are specifically recognized and internalized by dedicated transport machinery in bacteria. Sideromycins consists of structurally diverse molecules including danomycins, salmycins, albomycins, ferrimycins, and microcins. Bioactivities for all these molecules have been established against several human pathogens [110]. Interestingly, as rapidly dividing cancerous cells also

Antibiotics for Emerging Pathogens

display siderophore uptake receptors on their cell surfaces, these molecules are also being evaluated as potential anticancer compounds [109]. Several synthetic conjugates of antibiotic molecules with siderophores have also been generated for evaluation of antimicrobial properties [111]. A different class of “Trojan horse” molecules consists of drugs that are attached to peptide carrier molecules. An example of one such drug is the compound (Z)-l-2-amino-5-phosphono-3pentenoic acid (APPA), which is a potent inhibitor of the metabolic enzyme threonine synthase, and the “Trojan horse” version consists of APPA attached to a dipeptide. Depending on the amino acids in the dipeptide, the resultant conjugate can either be an antifungal (rhizocticins) or antibacterial (plumbemycin) [112]. Thus, a variety of activities can be encoded in the identical molecule by simply altering the nature of the conjugated peptide. A similar strategy for peptide attachment is utilized in the protein synthesis inhibitor microcin C7 [113].

Future Directions Maintenance of global health in light of the reemergence of life-threatening infectious diseases requires a continuous influx of new and potent antibiotic molecules that are efficacious and safe for human and animal clinical use. Pathogenic organisms have displayed a remarkable resilience in both developing and propagating mechanisms for overcoming the activities of these antibiotic molecules. The development of new antibiotic molecules relies on two principal paradigms: the characterization of new microbial targets against which new drug molecules can be developed, and the identification of new chemical entities which can be used as antibiotics. Both routes have resulted in the production of very successful drug molecules, and future prospects for antibiotic development are reliant on further progress in these directions. Recent biotechnological advances in the area of microbial genome sequencing have led to a rapid increase in exploration of the microbial metabolome for both novel pathways to target in pathogens and new candidates for use as antibiotics. Semisynthetic

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chemical derivatization of naturally occurring antibiotics has further expanded upon the repertoire of candidate drugs, and combinatorial synthetic approaches have generated vast chemical libraries which may be screened for compounds with desired biological properties. Future antibiotic development will require an amalgamation of various academic and industrial approaches such as (1) systems biology to explore metabolic connections and effects of antibiotic molecules on the human host and targeted pathogenic microbes, (2) synthetic biochemistry to generate and diversify arrays of compounds, (3) genomics and bioinformatics to identify and analyze antibiotic biosynthetic gene clusters, and (4) microbial ecological evaluations to better understand the role of naturally occurring antibiotic scaffolds in their native microbial niches. Another promising developing area is combinatorial therapy, in which multiple antibiotic molecules are administered together in order to overcome microbial resistance mechanisms and achieve better efficacy and pathogen clearance. This approach has proven to be very successful in antiviral and oncological applications, and combination regimens have been used for the treatment of tuberculosis for several decades now. Another area of rapid progress in the future promises to be the development of narrowspectrum antibiotics and better diagnostic tools which would aid in curtailing the indiscriminate use of broad-spectrum antibiotics, and thus reduce the incidence of microbial resistance against antibiotics. Acknowledgments Research in the Nair lab is supported by the NIGMS. We thank Neha Garg, Yue Hao, and Zhi Li for stimulating discussions.

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Climate Change Effects on Infectious Diseases Matthew Baylis and Claire Risley LUCINDA Group, Institute of Infection and Global Health, University of Liverpool, Neston, UK

Article Outline Glossary Definition of the Subject and Its Importance Introduction Weather, Climate, and Disease Climate Change and Disease Other Drivers of Disease Climate Change and Disease in Wildlife Evidence of Climate Change’s Impact on Disease Future Directions Bibliography

Glossary Climate The weather averaged over a long time or, succinctly, climate is what you expect, weather is what you get! El Niño Southern Oscillation (ENSO) A climate phenomenon whereby, following reversal of trade winds approximately every 4–7 years, a vast body of warm water moves slowly west to east across the Pacific, resulting in “an El Niño” event in the Americas and leading to a detectable change to climate (mostly disruption of normal rainfall patterns) across 70% of the earth’s surface. Emerging disease An infection or disease that has recently increased in incidence (the number of cases), severity (how bad the disease is), or distribution (where it occurs). Endemic stability The counter-intuitive situation where the amount of disease rises as the amount of infection falls, such that controlling infection can exacerbate the problem.

Infection The body of a host having been invaded by microorganisms (mostly viruses, bacteria, fungi, protozoa, and parasites). Infectious disease A pathology or disease that results from infection. Note that many diseases are not infectious and not all infections result in disease. Intermediate host A host in which a parasite undergoes an essential part of its lifecycle before passing to a second host, and where this passing is passive, that is, not by direct introduction into the next host (see vector). Vector Usually, an arthropod that spreads an infectious pathogen by directly introducing it into a host. For diseases of humans and animals, the most important vectors are flies (like mosquitoes, midges, sandflies, tsetse flies), fleas, lice, and ticks. Aphids are important vectors of diseases in plants. In some instances, other means of carriage of pathogens, such as human hands, car wheels, etc., are referred to as vectors. Vector competence The proportion of an arthropod vector population that can be infected with a pathogen. Zoonosis An infection of animals that can spread to, and cause disease in, humans (plural, zoonoses).

Definition of the Subject and Its Importance Infectious diseases of humans continue to present a significant burden to our health, disproportionately so in the developing world. Infectious diseases of livestock affect their health and welfare, are themselves important causes of human disease and, exceptionally, can threaten our food security. Wildlife infections again present a zoonotic risk to humans, but additionally, such diseases may threaten vulnerable populations and be a cause of extinction and biodiversity loss. Wild populations are inherently more susceptible to environmental change, largely lacking any

© Springer Science+Business Media, LLC 2012 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_524 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media LLC, 2012 https://doi.org/10.1007/978-1-4419-0851-3_524

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human protective influence that domesticated species and human populations may benefit from. Many infectious diseases of humans and farmed or wild animals are influenced by weather or climate, affecting where or when disease occurs, or how severe outbreaks are, and it is therefore likely that future climate change, whether human caused or natural, will have an impact on future disease burdens. Understanding the processes involved may enable prediction of how disease burdens will change in the future and, therefore, allow mitigative or adaptive measures to be put in place. While climate change will likely be an important cause of change in some infectious diseases in the future, there are other disease drivers which will also change over similar time scales and which may exacerbate or counteract any effects of climate change. Assessment of the future importance of climate change as an influence over future disease burdens must therefore be considered alongside other causes of change.

Introduction The impact of infectious diseases of humans and animals seems as great now as it was a century ago. While many disease threats have disappeared or dwindled, at least in the developed world, others have arisen to take their place. Important infectious diseases of humans that have emerged in the last 30 years, for a range of reasons, include Acquired immune deficiency syndrome (AIDS), variant Creutzfeldt-Jakob disease (vCJD), multidrug resistant tuberculosis, severe acute respiratory syndrome (SARS), E. coli O157, avian influenza, swine flu, West Nile fever, and Chikungunya [1, 2]. The same applies to diseases of animals: Indeed, all but one of the aforementioned human diseases have animal origins – they are zoonoses – and hence the two subjects of human and animal disease, usually studied separately by medical or veterinary scientists, are intimately entwined. What will be the global impact of infectious diseases at the end of the twenty-first century? Any single disease is likely to be affected by

Climate Change Effects on Infectious Diseases

many factors that cannot be predicted with confidence, including changes to human demography and behavior, new scientific or technological advances including cures and vaccines, pathogen evolution, livestock management practices and developments in animal genetics, and changes to the physical environment. A further, arguably more predictable, influence is climate change. Owing to anthropogenic activities, there is widespread scientific agreement that the world’s climate is warming at a faster rate than ever before [3], with concomitant changes in precipitation, flooding, winds, and the frequency of extreme events such as El Niño. Innumerable studies have demonstrated links between infectious diseases and climate, and it is unthinkable that a significant change in climate during this century will not impact on at least some of them. How should one react to predicted changes in diseases ascribed to climate change? The answer depends on the animal populations and human communities affected, whether the disease changes in severity, incidence, or spatiotemporal distribution and, of course, on the direction of change: Some diseases may spread but others may retreat in distribution. It also depends on the relative importance of the disease. If climate change is predicted to affect mostly diseases of relatively minor impact on human society or global biodiversity/ecosystem function, while the more important diseases are refractory to climate change’s influence, then our concerns should be tempered. To understand climate change’s effects on infectious diseases in the future it is necessary to first understand how climate affects diseases today. This entry begins by first presenting examples of climate’s effects on diseases of humans and livestock today and, from the understanding gained, then describes the processes by which climate change might affect such diseases in the future. Diseases of wildlife are important, to some extent, for different reasons to those of humans and livestock, and are therefore considered separately. The relative importance of climate change as a disease driver, compared to other forces, is considered, with examples provided of where climate change both is, and is not,

Climate Change Effects on Infectious Diseases

the major force. Finally, the future prospects and the uncertainties surrounding them are considered.

Weather, Climate, and Disease Many diseases are affected directly or indirectly by weather and climate. Remarkably, no systematic surveys of links between diseases and weather/climate seem to exist and, therefore, it is not possible to indicate whether these diseases represent a minority or majority. The associations between diseases and weather/climate fall broadly into three categories. The associations may be spatial, with climate affecting the distribution of a disease; temporal with weather or climate affecting the timing of outbreaks; or they may relate to the intensity of an outbreak. Temporal associations can be further broken into at least two subcategories: seasonal, with weather or climate affecting the seasonal occurrence of a disease, and interannual, with weather or climate affecting the timing, or frequency of years in which outbreaks occur. Here a selection of these associations is presented, which is by no means exhaustive but is, rather, intended to demonstrate the diversity of effects. Furthermore, the assignment of diseases into the different categories should not be considered hard-and-fast as many diseases could come under more than one heading. Spatial • Schistosomosis is an important cause of human mortality and morbidity in Africa and, to a lesser extent, in Asia. The disease is caused by species of Schistosoma trematode parasite, for which water-living snails are intermediate hosts. The distribution of suitable water bodies is therefore important for its distribution. However, there must also be suitable temperature: In China, Oncomelania hupensis snail intermediate hosts cannot live north of the January 0  C isotherm (the “freezing line”) while Schistosoma japonicum only develops within the snail at temperatures above 15.4  C. Shistosomosis risk in China is therefore

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restricted to the warmer southeastern part of the country [4]. • Diseases transmitted by tsetse flies (sleeping sickness, animal trypanosomosis) and ticks (such as anaplasmosis, babesiosis, East Coast fever, heartwater) impose a tremendous burden on African people and their livestock. Many aspects of the vectors’ life cycles are sensitive to climate, to the extent that their spatial distributions can be predicted accurately using satellite-derived proxies for climate variables [5]. • Mosquitoes (principally Culex and Aedes) transmit several viruses of birds that can also cause mortality in humans and horses. Examples are West Nile fever (WNF) and the viral encephalitides such as Venezuelan, western, and eastern equine encephalitis (VEE, WEE, and EEE, respectively) [6]. The spatial distributions of the mosquito vectors are highly sensitive to climate variables. Temporal-Seasonal All of the previous examples of spatial associations between diseases and climate can also be classified as temporal-seasonal, as the effects of climate on the seasonal cycle of the intermediate hosts (snails, tsetse flies, and mosquitoes, respectively) also determines in part the seasonal cycle of disease. There are other diseases where the associations can be described as seasonaltemporal. • Salmonellosis is a serious food-borne disease caused by Salmonella bacteria, most often obtained from eggs, poultry, and pork. Salmonellosis notification rates in several European countries have been shown to increase by about 5–10% for each 1  C increase in ambient temperature [7]. Salmonellosis notification is particularly associated with high temperatures during the week prior to consumption of infected produce, implicating a mechanistic effect via poor food handling. • Foot-and-mouth disease (FMD) is a highly contagious, viral infection of cloven-footed animals, including cattle, sheep, and pigs. Most transmission is by contact between

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infected and susceptible animals, or by contact with contaminated animal products. However, FMD can also spread on the wind. The survival of the virus is low at relative humidity (RH) below 60% [8], and wind-borne spread is favored by the humid, cold weather common to temperate regions. In warmer drier regions, such as Africa, wind-borne spread of FMD is considered unimportant [9]. • Peste des petits ruminants (PPR) is an acute, contagious, viral disease of small ruminants, especially goats, which is of great economic importance in parts of Africa and the Near East. It is transmitted mostly by aerosol droplets between animals in close contact. However, the appearance of clinical PPR is often associated with the onset of the rainy season or dry cold periods [10], a pattern that may be related to viral survival. The closely related rinderpest virus survives best at low or high relative humidity, and least at 50–60% [11]. • Several directly transmitted human respiratory infections, including those caused by rhinoviruses (common colds) and seasonal influenza viruses (flu) have, in temperate countries, seasonal patterns linked to the annual temperature cycle. There may be direct influences of climate, such as the effect of humidity on survival of the virus in aerosol [12], or indirect influences via, for example, seasonal changes in the strength of the human immune system or more indoor crowding during cold weather [13]. Temporal-Interannual The previous examples of spatial associations between diseases and climate, which were further categorized as temporal-seasonal, can also be classified as temporal-interannual, as the effects of climate on the intermediate hosts (snails, tsetse flies, and mosquitoes, respectively) will determine in part the risk or scale of a disease outbreak in a given year. There are other diseases where the associations can be described as seasonalinterannual. • Anthrax is an acute infectious disease of most warm-blooded animals, including humans, with worldwide distribution. The causative

Climate Change Effects on Infectious Diseases









bacterium, Bacillus anthracis forms spores able to remain infective for 10–20 years in pasture. Temperature, relative humidity, and soil moisture all affect the successful germination of anthrax spores, while heavy rainfall may stir up dormant spores. Outbreaks are often associated with alternating heavy rainfall and drought, and high temperatures [14]. Cholera, a diarrheal disease which has killed tens of millions of people worldwide, is caused by the bacterium Vibrio cholerae, which lives amongst sea plankton [15]. High temperatures causing an increase in algal populations often precede cholera outbreaks. Disruption to normal rainfall helps cholera to spread further, either by flooding, leading to the contamination of water sources, such as wells, or drought which can make the use of such water sources unavoidable. Contaminated water sources then become an important source of infection in people. Plague is a flea-borne disease caused by the bacterium Yersinia pestis; the fleas’ rodent hosts bring them into proximity with humans. In Central Asia, large scale fluctuations in climate synchronize the rodent population dynamics over large areas [16], allowing population density to rise over the critical threshold required for plague outbreaks to commence [17]. African horse sickness (AHS), a lethal infectious disease of horses, is caused by a virus transmitted by Culicoides biting midges. Large outbreaks of AHS in the Republic of South Africa over the last 200 years are associated with the combination of drought and heavy rainfall brought by the warm phase of the El Niño Southern Oscillation (ENSO) [18]. Rift Valley Fever (RVF), an important zoonotic viral disease of sheep and cattle, is transmitted by Aedes and Culex mosquitoes. Epizootics of RVF are associated with periods of heavy rainfall and flooding [19–21] or, in East Africa, with the combination of heavy rainfall following drought associated with ENSO [20, 22]. ENSO-related floods in 1998, following drought in 1997, led to an epidemic of RVF (and some other diseases) in the Kenya/

Climate Change Effects on Infectious Diseases

Somalia border area and the deaths of more than 2000 people and two-thirds of all small ruminant livestock [23]. Outbreaks of several other human infections, including malaria and dengue fever have, in some parts of the world, been linked to ENSO events. Intensity In addition to associations between climate and the spatial and temporal distributions of disease outbreaks, there are some examples of associations between climate and the intensity or severity of the disease that results from infection. It is theoretically possible, for example, that climateinduced immunosuppression of hosts may favor the multiplication of some microparasites (viruses, bacteria, rickettsia, fungi, protozoa), thereby increasing disease severity or, alternatively, reduce the disease-associated immune response to infection, thereby reducing disease severity. However, the clearest examples pertain to macroparasites (helminth worms) which, with the notable exception of Strongyloides spp., do not multiply within the host. Disease severity is therefore directly correlated with the number of parasites acquired at the point of infection or subsequently, and in turn this is frequently associated with climate, which affects both parasite survival and seasonal occurrence. • Fasciolosis, caused by the Fasciola trematode fluke, is of economic importance to livestock producers in many parts of the world and also causes disease in humans. In sheep, severe pathology, including sudden death, results from acute fasciolosis which occurs after ingestion of more than 2,000 metacercariae (larval flukes) of Fasciola hepatica at pasture, while milder pathology associated with subacute and chronic fasciolosis occurs after ingestion of 200–1,000 metacercariae [24]. Acute fasciolosis is therefore most common in places or in years when rainfall and temperature favor the survival of large numbers of metacercariae. • Nematodirus battus is a nematode parasite of the intestine of lambs. Eggs deposited in the

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feces of one season’s lambs do not hatch straightaway; instead, they build up on the pasture during summer and remain as eggs over winter, not hatching until temperatures the following spring exceed 10  C [25]. Once the mean daily temperature exceeds this threshold the eggs hatch rapidly, leading to a sharp peak in the number of infective larvae on the pasture. If this coincides with the new season’s lambs grazing on the pasture, there is likely to be a large uptake of larvae and severe disease, called nematodiriasis. If, however, there is a warm spell early in the year, the peak in larvae on pasture may occur while lambs are still suckling rather than grazing, such that fewer larvae are ingested and the severity of nematodiriasis is reduced.

Climate Change and Disease There is a substantial scientific literature on the effects of climate change on health and disease, but with strong focus on human health and vectorborne disease [5, 26–42]. By contrast, the effects of climate change on diseases spread by other means, or animal diseases in general, have received comparatively little attention [43–48]. Given the global burden of diseases that are not vector-borne, and the contribution made by animal diseases to poverty in the developing world [49], attention to these areas is overdue. The previous section demonstrates the range of climate influences upon infectious disease. Such influences are not the sole preserve of vector-borne diseases: Food-borne, waterborne, and aerosol-transmitted diseases are also affected. A common feature of non-vector-borne diseases affected by climate is that the pathogen or parasite spends a period of time outside of the host, subject to environmental influence. Examples include the infective spores of anthrax; FMD viruses in temperate regions; the Salmonella bacteria that contaminate food products; the choleracausing vibrio bacteria in water; and the moisture- and temperature-dependent survival of the parasites causing schistosomosis and fasciolosis.

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By contrast, most diseases transmitted directly between humans (for example, human childhood viruses, sexually transmitted diseases (STDs), tuberculosis) have few or no reported associations with climate. This is also the case for animal infections such as avian influenza, bovine tuberculosis, brucellosis, Newcastle’s disease of poultry, and rabies. Clear exceptions are the viruses that cause colds and seasonal flu in humans, and PPR in small ruminants; these viruses are spread by aerosol between individuals in close contact but are nevertheless sensitive to the effects of ambient humidity and possibly temperature. The influence of climate on diseases that are not vector-borne appears to be most frequently associated with the timing (intra- or interannual) of their occurrence rather than their spatial distribution. There are examples of such diseases that occur only in certain parts of the world (for example, PPR) but most occur worldwide. By contrast, the associations of vector-borne diseases with climate are equally apparent in time and space, with very few vector-borne diseases being considered a risk worldwide. This is a reflection of the strong influence of climate on both the spatial and temporal distributions of intermediate vectors. If there are exceptions to this rule, the vectors are likely to be lice or fleas, with lives so intimately associated with humans or animals that they are relatively protected from climate’s influences. In the scientific literature, many processes have been proposed by which climate change might affect infectious diseases. These processes range from the clear and quantifiable to the imprecise and hypothetical. They may affect pathogens directly or indirectly, the hosts, the vectors (if there is an intermediate host), epidemiological dynamics, or the natural environment. A framework for how climate change can affect the transmission of pathogens between hosts is shown in Fig. 1. Only some of the processes can be expected to apply to any single infectious disease. Effects on Pathogens Higher temperatures resulting from climate change may increase the rate of development of certain pathogens or parasites that have one or

Climate Change Effects on Infectious Diseases

more lifecycle stages outside their human or animal host. This may shorten generation times and, possibly, increase the total number of generations per year, leading to higher pathogen population sizes [48]. Conversely, some pathogens are sensitive to high temperature and their survival may decrease with climate warming. Phenological evidence indicates that spring is arriving earlier in temperate regions [50]. Lengthening of the warm season may increase or decrease the number of cycles of infection possible within 1 year for warm or cold-associated diseases respectively. Arthropod vectors tend to require warm weather so the infection season of arthropod-borne diseases may extend. Some pathogens and many vectors experience significant mortality during cold winter conditions; warmer winters may increase the likelihood of successful overwintering [41, 48]. Pathogens that are sensitive to moist or dry conditions may be affected by changes to precipitation, soil moisture, and the frequency of floods. Changes to winds could affect the spread of certain pathogens and vectors. Effects on Hosts A proposed explanation for the tendency for human influenza to occur in winter is that the human immune system is less competent during that time; attributable to the effects of reduced exposure to light on melatonin [51] or vitamin D production [52]. The seasonal light/dark cycle will not change with climate change, but one might hypothesize that changing levels of cloud cover could affect exposure in future. A second explanation, the tendency for people to congregate indoors during wintertime, leads to a more credible role for a future influence of climate change. Mammalian cellular immunity can be suppressed following heightened exposure to ultraviolet B (UV-B) radiation – an expected outcome of stratospheric ozone depletion [53, 54]. In particular, there is depression of the number of T helper 1 lymphocytes, cells which stimulate macrophages to attack pathogen-infected cells and, therefore, the immune response to intracellular pathogens may be particularly affected.

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Indirect effects of climate change on disease

Direct effects of climate change on host immunity

Climate change Temperature, rainfall, humidity, floods, wind

Indirect effects of climate change on host genetic susceptibility to infection

1 Survival, growth rate, seasonal cycle, dispersal

Pathogen in air, soil, water, food, vector Vectors & intermediate hosts

Host 1

Survival, growth rate, seasonal cycle, dispersal

Host 2

Contact rates between infected and susceptible hosts 2

3

Climate sensitive drivers of disease emergence

Climate independent drivers of disease emergence

Environmental change

Microbial, demograpic, societal & technical change

Habitats, landscapes, communities, ecosystems, land use, agriculture

Microbial resistance, population increase, travel, war, technical advance

Climate Change Effects on Infectious Diseases, Fig. 1 A schematic framework of the effects of climate change on the transmission of diseases of humans and animals. Climate change can act directly on pathogens in a range of external substrates, or their vectors and intermediate hosts, thereby affecting the processes of survival, growth, seasonality, and dispersal. It can also directly affect hosts themselves or the contact rates between infected and susceptible individuals. Climate change can

have indirect effects on disease transmission via its effects on the natural or anthropogenic environment, and via the genetics of exposed populations. Environmental, demographic, social, and technical change will also happen independently of climate change and have as great, or much greater, influence on disease transmission than climate change itself. The significance of climate change as a driver of disease will depend on the scale of arrow 1, and on the relative scales of arrows 2 and 3

Examples of such intracellular pathogens include many viruses, rickettsia (such as Cowdria and Anaplasma, the causative agents of heartwater and anaplasmosis), Brucella, Listeria monocytogenes and Mycobacterium tuberculosis, the bacterial agents of brucellosis, listeriosis, and tuberculosis, respectively, and the protozoan parasites Toxoplasma gondii and Leishmania which cause toxoplasmosis and visceral leishmanosis (kala-azar), respectively, in humans [55]. A third host-related effect worthy of consideration is genetic resistance to disease. Some human populations and many animal species have evolved a level of genetic resistance to some of the diseases to which they are commonly exposed.

Malaria presents a classic example for humans, with a degree of resistance to infection in African populations obtained from heterozygosity for the sickle-cell genetic trait. Considering animals, wild mammals in Africa may be infected with trypanosomes, but rarely show signs of disease; local Zebu cattle breeds, which have been in the continent for millennia, show some degree of trypanotolerance (resistance to disease caused by trypanosome infection); by contrast, recently introduced European cattle breeds are highly susceptible to trypanosomosis. In stark contrast, African mammals proved highly susceptible to rinderpest which swept through the continent in the late nineteenth century, and which they had

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not previously encountered. It seems unlikely that climate change will directly affect genetic or immunologic resistance to disease in humans or animals. However, significant shifts in disease distributions driven by climate change pose a greater threat than simply the exposure of new populations. Naïve populations may, in some cases, be particularly susceptible to the new diseases facing them. Certain diseases show a phenomenon called endemic stability. This occurs when the severity of disease is less in younger than older individuals, when the infection is common or endemic and when there is life-long immunity after infection. Under these conditions most infected individuals are young, and experience relatively mild disease. Counter-intuitively, as endemically stable infections become rarer, a higher proportion of cases are in older individuals (it takes longer, on average, to acquire infection) and the number of cases of severe disease rises. Certain tick-borne diseases of livestock in Africa, such as anaplasmosis, babesiosis, and cowdriosis, show a degree of endemic stability [56], and it has been proposed to occur for some human diseases like malaria [57]. If climate change drives such diseases to new areas, nonimmune individuals of all ages in these regions will be newly exposed, and outbreaks of severe disease could follow. Effects on Vectors Much has already been written about the effects of climate change on invertebrate disease vectors. Indeed, this issue, especially the effects on mosquito vectors, has dominated the debate so far. It is interesting to bear in mind, however, that mosquitoes are less significant as vectors of animal disease than they are of human disease (Table 1). Mosquitoes primarily, and secondarily lice, fleas, and ticks, transmit between them a significant proportion of important human infections. By contrast, biting midges, brachyceran flies (e.g., tabanids, muscids, myiasis flies, hippoboscids), ticks, and mosquitoes (and, in Africa, tsetse) all dominate as vectors of livestock disease. Therefore, a balanced debate on the effects of climate

Climate Change Effects on Infectious Diseases

change on human and animal disease must consider a broad range of vectors. There are several processes by which climate change might affect disease vectors. First, temperature and moisture frequently impose limits on their distribution. Often, low temperatures are limiting because of high winter mortality, or high temperatures because they involve excessive moisture loss. Therefore, cooler regions which were previously too cold for certain vectors may begin to allow them to flourish with climate change. Warmer regions could become even warmer and yet remain permissive for vectors if there is also increased precipitation or humidity. Conversely, these regions may become less conducive to vectors if moisture levels remain unchanged or decrease, with concomitant increase in moisture stress. For any specific vector, however, the true outcome of climate change will be significantly more complex than that outlined above. Even with a decrease in future moisture levels, some vectors, such as certain species of mosquito, could become more abundant, at least in the vicinity of people and livestock, if the response to warming is more water storage and, thereby, the creation of new breeding sites. One of the most important vectors of the emerging Chikungunya virus (and to a lesser extent dengue virus) is the Asian tiger mosquito (Aedes albopictus) which is a container breeder and therefore thrives where humans store water. Equally, some vectors may be relatively insensitive to direct effects of climate change, such as muscids which breed in organic matter or debris, and myiasis flies which breed in hosts’ skin. Changes to temperature and moisture will also lead to increases or decreases in the abundance of many disease vectors. This may also result from a change in the frequency of extreme weather events such as ENSO. Outbreaks of several biting midge and mosquito-borne diseases, for example, have been linked to the occurrence of ENSO [18, 22, 59–62] and mediated, at least in part, by increase in the vector population size in response to heavy rainfall, or rainfall succeeding drought,

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Climate Change Effects on Infectious Diseases, Table 1 The major diseases transmitted by arthropod vectors to humans and livestock (Adapted from [58]) Vector Phthiraptera (Lice)

Reduvidae (Assassin bugs) Siphonaptera (Fleas)

Psychodidae (Sand flies) Culicidae (Mosquitoes)

Simulidae (Black flies)

Diseases of humans Epidemic typhus Trench fever Louse-borne relapsing fever Chagas’ disease Plague Murine typhus Q fever Tularaemia Leishmanosis Sand fly fever Malaria Dengue Yellow fever West Nile Filiariasis Encephalitides (WEE, EEE, VEE, Japanese encephalitis, Saint Louis encephalitis) Rift Valley fever Onchocercosis

Ceratopogonidae (Biting midges)

Glossinidae (Tsetse flies) Tabanidae (Horse flies)

Trypanosomosis Loiasis

Muscidae (Muscid flies)

Shigella E. coli

Muscoidae, Oestroidae (Myiasis-causing flies)

Bot flies

Hippoboscoidae (Louse flies, keds) Acari (Mites)

Ixodidae (Hard ticks) Argasidae (Soft ticks)

Chiggers Scrub typhus (tsutsugamushi) Scabies Human babesiosis Tick-borne encephalitis Tick fevers Ehrlichiosis Q fever Lyme disease Tick-borne relapsing fever Tularaemia

Diseases of livestock

Myxomatosis

Canine leishmanosis Vesicular stomatitis West Nile fever Encephalitides Rift Valley fever Equine infectious anemia

Leucocytozoon (birds) Vesicular stomatitis Bluetongue African horse sickness Akabane Bovine ephemeral fever Trypanosomosis Surra Equine infectious anemia Trypanosoma vivax Mastitis Summer mastitis Pink-eye (IBK) Screwworm Blow fly strike Fleece rot Numerous protozoa Mange Scab Scrapie? Babesiosis East coast fever (Theileriosis) Louping ill African Swine Fever Ehrlichiosis Q fever Heartwater Anaplasmosis Borreliosis Tularaemia

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that ENSO sometimes brings [18, 22]. Greater intra- or interannual variation in rainfall, linked or unlinked to ENSO, may lead to an increase in the frequency or scale of outbreaks of such diseases. The ability of some insect vectors to become or remain infected with pathogens (their vector competence) varies with temperature [63, 64]. In addition to this effect on vector competence, an increase in temperature may alter the balance between the lifespan of an infected vector, its frequency of feeding, and the time necessary for the maturation of the pathogen within it. This balance is critical, as a key component of the risk of transmission of a vector-borne disease is the number of blood meals taken by a vector between the time it becomes infectious and its death [65]. Accordingly, rising ambient temperature can increase the risk of pathogen transmission by shortening the time until infectiousness in the vector and increasing its feeding frequency at a faster rate than it shortens the vector’s lifespan, such that the number of feeds taken by an infectious vector increases. However, at even higher temperatures this can reverse [66] such that the number of infectious feeds then decreases relative to that possible at lower temperatures. This point is extremely important, as it means that the risk of transmission of vectorborne pathogens does not uniformly increase with rising temperature, but that it can become too hot and transmission rates decrease. This effect will be most important for short-lived vectors such as biting midges and mosquitoes [30]. Lastly, there may be important effects of climate change on vector dispersal, particularly if there is a change in wind patterns. Wind movements have been associated with the spread of epidemics of many Culicoides- and mosquitoborne diseases [67–72]. Effects on Epidemiological Dynamics Climate change may alter transmission rates between hosts by affecting the survival of the pathogen or the intermediate vector, but also by other, indirect, forces that may be hard to predict with accuracy. Climate change may influence human demography, housing, or movement or

Climate Change Effects on Infectious Diseases

be one of the forces that leads to changes in future patterns of international trade, local animal transportation, and farm size. All of these can alter the chances of an infected human or animal coming into contact with a susceptible one. For example, a series of droughts in East Africa between 1993 and 1997 resulted in pastoral communities moving their cattle to graze in areas normally reserved for wildlife. This resulted in cattle infected with a mild lineage of rinderpest transmitting disease both to other cattle and to susceptible wildlife, causing severe disease, for example, in buffalo, lesser kudu, and impala, and devastating certain populations [73]. Indirect Effects No disease or vector distribution can be fully understood in terms of climate only. The supply of suitable hosts, the effects of co-infection or immunological cross-protection, the presence of other insects competing for the same food sources or breeding sites as vectors [74], and parasites and predators of vectors themselves, could have important effects [48]. Climate change may affect the abundance or distribution of hosts or the competitors/predators/parasites of vectors and influence patterns of disease in ways that cannot be predicted from the direct effects of climate change alone. Equally, it has been argued that climate change-related disturbances of ecological relationships, driven perhaps by agricultural changes, deforestation, the construction of dams, and losses of biodiversity, could give rise to new mixtures of different species, thereby exposing hosts to novel pathogens and vectors and causing the emergence of new diseases [40]. A possible “example in progress” is the reemergence in the UK of bovine tuberculosis, for which the badger (Meles meles) is believed to be a carrier of the causative agent, Mycobacterium bovis. Farm landscape, such as the density of linear features like hedgerows, is a risk factor for the disease, affecting the rate of contact between cattle and badger [75]. Climate change will be a force for modifying future landscapes and habitats, with indirect and largely unpredictable effects on diseases.

Climate Change Effects on Infectious Diseases

Other Drivers of Disease The future disease burden of humans and animals will depend not only on climate change and its direct and indirect effects on disease, but also on how other drivers of disease change over time. Even for diseases with established climate links, it may be the case that in most instances these other drivers will prove to be more important than climate. A survey of 335 events of human disease emergence between 1940 and 2004 classified the underlying causes into 12 categories [2]. One of these, “climate and weather,” was only listed as the cause of ten emergence events. Six of these were non-cholera Vibrio bacteria which cause poisoning via shellfish or exposure to contaminated seawater; the remaining four were a fungal infection and three mosquito-borne viruses. The other 11 categories included, however, “land use changes” and “agricultural industry changes,” with 36 and 31 disease emergence events, respectively, and both may be affected by climate change. The causes of the remaining 77% of disease emergence events – “antimicrobial agent use,” “international travel and commerce,” “human demography and behavior,” “human susceptibility to infection,” “medical industry change,” “war and famine,” “food industry changes,” “breakdown of public health,” and “bushmeat” – would be expected to be either less or not influenced by climate change. Hence, climate change’s indirect effects on human or animal disease may exceed its direct effects, while drivers unsusceptible to climate change may be more important still at determining our disease future.

Climate Change and Disease in Wildlife Wildlife disease is important for different reasons to those which make disease in humans and domestic animals important. It has the potential for endangerment of wildlife and can be a source of zoonoses and livestock disease.

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Wildlife Disease as a Cause of Endangerment Disease in wild populations may limit or cause extreme fluctuations in population size [76] and reduce the chances of survival of endangered or threatened species [77]. Indeed, disease can be the primary cause of extinction in animals or be a significant contributory factor toward it. For example: • The Christmas Island rat, Rattus macleari, is believed to have been extinct by 1904. There is molecular evidence that this was caused by introduction of murine trypanosomes apparently brought to the island by black rats introduced shortly before 1904 [78]. • Similarly, the last known Po’o-uli bird (Melamprosops phaeosoma) in Hawaii died from avian malaria brought by introduced mosquitoes [79]. • Canine Distemper in the Ethiopian Wolf (Canis simensis) has brought about its decline [80]. • Devil facial tumor disease, an aggressive nonviral transmissible parasitic cancer, continues to threaten Tasmanian Devil (Sarcophilus harrisii) populations [81]. • The white nose fungus Geomyces destructans is decimating bat populations in Northeastern US states and is currently spreading in Europe [82]. This is perhaps the most recent emergence of a disease of concern to wildlife endangerment. Although disease can cause endangerment and extinction, its importance relative to other causes is uncertain. A review of the causes of endangerment and extinction in the International Union for Conservation of Nature (IUCN) red list of plant and animal species found that disease was implicated in a total of 254 cases, some 7% of the total examined [83]. Although the other factors may be more important overall, disease remains an important cause of endangerment and extinction for certain animal groups. A contender for the single issue of greatest current conservation concern is the epidemic of the chytrid fungus Batrachochytrium dendrobatidis in amphibians. With a

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broad host range and high mortality, this pathogen is likely to be wholly or partly responsible for all recent amphibian extinction events, which, remarkably, comprise 29% of all extinctions attributable to disease since the year 1500 [77]. Wildlife Disease as a Source of Infections for Humans and Livestock Many diseases of wildlife frequently cross the species barrier to infect humans or domestic animals [84, 85]. Closely related organisms often share diseases. A particular risk to humans is presented by diseases of primates: The human immunodeficiency viruses (HIV) that cause AIDS originated as simian immunodeficiency viruses in African monkeys and apes. Humans acquire or have acquired many other pathogens from mammals other than primates, especially those that humans choose to live close to (livestock, dogs, cats), or that choose to live close to us (rodents, bats) [86]. In addition, there are examples of human infections shared with birds (e.g., avian influenza), and reptiles and fishes (e.g., Salmonella spp.). Insects are frequent carriers of pathogens between vertebrate hosts, and there may even be a pathogen transmissible from plants to humans [87]. Wildlife populations are the primary source of emerging infectious diseases in humans. A search of the scientific literature published between 1940 and 2004 attempted to quantify the causes of disease introductions into human populations and found that about 72% were introduced from wildlife [2]. How Climate Change Can Influence Wildlife Disease The effects of climate change on wildlife disease are important when the changes produced lead to increased risk of endangerment or extinction of the wildlife, or increased transmission risk to humans or domestic animals. Climate change can increase the threat of endangerment or extinction, via reduction in population size of the wildlife host (by altering habitats, for example), or increase in pathogen range or virulence, such that the persistence of a host population is at risk, and climate change can

Climate Change Effects on Infectious Diseases

increase the risk of disease emergence and spread to humans or livestock via change to the distribution of wildlife hosts, such that encroachment on human or livestock populations is favored. Changes in species’ distribution may arise directly under climate change as a result of an organism’s requirement for particular climatic conditions or indirectly via ecological interactions with other species which are themselves affected. Climate change can cause the appearance of new assemblages of species and the disappearance of old communities [88], which can create new disease transmission opportunities or end existing ones. Climatic factors potentially affecting wildlife disease transmission more directly include the growth rate of the pathogen in the environment or in a cold-blooded (ectothermic) wildlife host (e.g., fish, amphibian, reptile). Therefore, effects which are more marked in wildlife disease in comparison to human or livestock disease include the occurrence of ectothermic hosts, and also the vast range of potential vectors that may transmit disease. It is therefore more difficult to generalize about the effects of climate change on wildlife. In colder climates, the parasite that causes the most severe form of human malaria, Plasmodium falciparum does not develop rapidly enough in its mosquito vectors for there to be sufficient transmission to maintain the parasite. Avian malaria (Plasmodium relictum) exhibits an elevational gradient due mainly to temperature and is subject to similar constraints [89]. An example of a wildlife pathogen constrained due to its dependence on environmental transmission is anthrax. Infective spores of anthrax bacilli can lie dormant in soils for decades, becoming dangerous when climatic conditions, particularly precipitation, favor it. It is well established that when a host population size is reduced, the pool of susceptible individuals may be too small for pathogen survival. This effect is particularly acute for host-specific diseases, such as the transmissible cancer of Tasmanian devils, Sarcophilus harrisii, DFTD or Devil facial tumor disease (discussed in [81]); at low host population sizes, DFTD may become extinct. By contrast, diseases with a broad host range may threaten individual species down

Climate Change Effects on Infectious Diseases

to the last individual. As anthrax has both a broad host range and can lie dormant in the environment, it is a particular threat for species with very low numbers, and is currently a conservation consideration for many species. For example the Javan rhinoceros population is down to fewer than 60 individuals and identified as a priority for conservation (an “EDGE” species) because of its uniqueness and scarcity [90]. Climate change may have particular impact on marine animals, because of the preponderance of ectothermic animals in the sea, the multiple ways in which climate change is predicted to affect the marine environment, and the multiple stresses that marine organisms and ecosystems are already experiencing due to anthropogenic influence. Disease is an important part of this impact. For example, warming of the Pacific in the range of the oyster Crassostrea virginica caused range expansion of the protozoan parasite Perkinsus marinus probably due to a combination of increased overwinter survival, greater summer proliferation, and increased host density [91]. Coral reefs are also sensitive to at least 12 potential factors associated with climate change: [92] CO2 concentration, sea surface temperature, sea level, storm intensity, storm frequency, storm track, wave climate, run off, seasonality, overland rainfall, and ocean and air circulation [92]. Although these factors might not all increase levels of disease, the synergism between disease, climate, and other stressors might lead to accelerating degradation of the coral reef habitat. From a geographic perspective, there is evidence that the greatest change in ecosystems attributable to climate change is likely to be in the tropics; the second being the arctic [88]. The impacts of this change on wildlife disease and its consequences may be particularly great in these two regions, and there is evidence that it is already occurring. The tropics have the most species in imminent danger of extinction [93] while tropical coral reefs comprise much of the biodiversity of the oceans. In addition to extinction risks, tropical forests may also pose a zoonosis risk. An increase in animal-human interaction is likely in tropical forests, which have a diverse fauna subject to increasing human encroachment.

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With regard to the Arctic, a model of the effect of global warming on a protostrongylid lungdwelling nematode Umingmakstrongylus pallikuukensis, in Canadian Arctic muskoxen Ovibos moschatus, found that warming was likely to significantly influence infection, making the muskoxen more susceptible to predation [94]. Muskoxen were also subject to climate-influenced outbreaks of fatal pneumonia [95]. Indeed, the combination of climate change’s effects on pathogen survival and transmission, and the stress to host species from changing environmental conditions, may have serious impact on arctic populations of fish, muskoxen, sheep, and others [96]. Dependency of Disease on Climate: The Example of Chytridiomycosis in Amphibians In wildlife epidemiology, the host may be of equal importance to the pathogen and vector when considering the impact of climate, as wildlife may be impacted by climate in more diverse ways than humans or domestic animals, and are subject to much reduced human mitigation of those impacts. The importance of climate in Batrachochytrium dendrobatidis epidemiology, the cause of chytridiomycosis, and numerous amphibian extinctions is fiercely debated. Although the pathogen B. dendrobatidis is neither vector-borne nor has a prolonged environmental phase, it is affected by temperature because the environment of its ectothermic hosts is not kept constant when external temperature varies. It belongs to a basal group within the fungi and has a brief motile zoospore stage for dispersal, followed by the penetration of the outer layers of amphibian skin and asexual intracellular multiplication [97]. Its growth is limited by warmer temperatures, perhaps because amphibians shed their outer layers of skin more frequently in warmer temperatures [97]. Pounds et al. [98] were the first to make a connection between climate and B. dendrobatidis-mediated amphibian extinctions, reporting spatiotemporal associations between warming and last year of detection of frog species. The development rate of the B. dendrobatidis fungus depends on summer temperature [99], and its survival is dependent on

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winter freezing [100]. The fungus appears to cause more mortality in mountainous regions [101], yet may be limited at the upper extremes of altitude. Climate may also affect the impact of the disease due to host factors. The habitat of the golden toad Bufo periglenes in 1987, the last year of its existence, was much reduced due to an especially dry summer. This may have caused crowded conditions in the remaining ponds, facilitating the spread of chytridiomycosis [102]. In addition, climate may affect mortality associated with the disease. The mortality of frogs exposed to B. dendrobatidis spores as adults has been shown to be dependent on the condition of the frogs [103]. In a changing climate where amphibians are shifting their ranges into suboptimal areas, hosts are likely to be more susceptible to the damaging effects of B. dendrobatidis infection. On the other hand, it has been argued that climate is not important in B. dendrobatidis outbreaks [104]. The authors contrast the climatelinked epidemic hypothesis with the hypothesis that disease outbreaks occur largely due to introduction into unexposed, naive populations, and describe spatiotemporal “waves” of declines across Central America as evidence that it is disease introduction (and not climatic variables) causing declines.

Evidence of Climate Change’s Impact on Disease Climate warming has already occurred in recent decades. If diseases are sensitive to such warming, then one might expect a number of diseases to have responded by changing their distribution, frequency, or intensity. A major difficulty, however, is the attribution of any observed changes in disease occurrence to climate change because, as shown above, other disease drivers also change over time. It has been argued that the minimum standard for attribution to climate change is that there must be known biological sensitivity of a disease or vector to climate, and that the change in disease or vector (change in seasonal cycle, latitudinal or altitudinal shifts) should be statistically

Climate Change Effects on Infectious Diseases

associated with observed change in climate [28]. This has been rephrased as the need for there to be change in both disease/vector and climate at the same time, in the same place, and in the “right” direction [105]. Given these criteria, few diseases make the standard: Indeed, only a decade ago one group concluded that the literature lacks strong evidence for an impact of climate change on even a single vector-borne disease, let alone other diseases. This situation has changed. One disease in particular, bluetongue, has emerged dramatically in Europe over the last decade and this emergence can be attributed to recent climate change in the region. It satisfies the right time, right place, right direction criterion [64], but in fact reaches a far higher standard: A model for the disease, with variability in time driven only by variation in climate, produces quantitative estimates of risk which fit closely with the disease’s recent emergence in both space and time. Bluetongue Bluetongue is a viral disease of sheep and cattle. It originated in Africa, where wild ruminants act as natural hosts for the virus, and where a species of biting midge, Culicoides imicola, is the major vector [106]. During the twentieth century bluetongue spread out of Africa into other, warm parts of the world, becoming endemic in the Americas, southern Asia, and Australasia; in most of these places, indigenous Culicoides became the vectors. Bluetongue also occurred very infrequently in the far extremes of southern Europe: once in the southwest (southern Spain and Portugal, 1955–1960), and every 20–30 years in the southeast (Cyprus, 1924, 1943–46); Cyprus and Greek islands close to Turkey (1977–1978); the presence of C. imicola was confirmed in both areas and this species was believed to be the responsible vector. Twenty years after this last 1977–1978 outbreak, in 1998 bluetongue once again reappeared in southeastern Europe [107]. Subsequent events, however, are unprecedented. Between 1998 and 2008 bluetongue accounted for the deaths of more than one million sheep in Europe – by far the longest and largest outbreak on record. Bluetongue has

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occurred in many countries or regions that have never previously reported this disease or its close relatives. There have been at least two key developments. First, C. imicola has spread dramatically, now occurring over much of southern Europe and even mainland France. Second, indigenous European Culicoides species have transmitted the virus. This was first detected in the Balkans where bluetongue occurred but no C. imicola could be found [108]. In 2006, however, bluetongue was detected in northern Europe (The Netherlands) from where it spread to neighboring countries, the UK and even Scandinavia. The scale of this outbreak has been huge, yet the affected countries are far to the north of any known C. imicola territory [109]. Recently, the outputs of new, observation based, high spatial resolution (25 km) European climate data, from 1960 to 2006 have been integrated within a model for the risk of bluetongue transmission, defined by the basic reproduction ratio R0 [110]. In this model, temporal variation in transmission risk is derived from the influence of climate (mainly temperature and rainfall) on the abundance of the vector species, and from the influence of temperature alone on the ability of the vectors to transmit the causative virus. As described earlier, this arises from the balance between vector longevity, vector feeding frequency, and the time required for the vector to

become infectious. Spatial variation in transmission risk is derived from these same climatedriven influences and, additionally, differing densities of sheep and cattle. This integrated model successfully reproduces many aspects of bluetongue’s distribution and occurrence, both past and present, in Western Europe, including its emergence in northwest Europe in 2006. The model gives this specific year the highest positive anomaly (relative to the long-term average) for the risk of bluetongue transmission since at least 1960, but suggests that other years were also at much higher-than-average risk. The model suggests that the risk of bluetongue transmission increased rapidly in southern Europe in the 1980s and in northern Europe in the 1990s and 2000s. These results indicate that climate variability in space and time are sufficient to explain many aspects of bluetongue’s recent past in Europe and provide the strongest evidence to date that this disease’s emergence is, indeed, attributable to changes in climate. What then of the future? The same model was driven forward to 2050 using simulated climate data from regional climate models. The risk of bluetongue transmission in northwestern Europe is projected to continue increasing up to at least 2050 (Fig. 2). Given the continued presence of susceptible ruminant host populations, the models suggest that by 2050,

R0 changes (%)

80 40 0 –40

Sim A1B mean

–80 1960

OBS 1980

2000

2020

2040

2060

Year

Climate Change Effects on Infectious Diseases, Fig. 2 Projections of the effect of climate change on the future risk of transmission of bluetongue in northern Europe. The y-axis shows relative anomalies (%) with respect to the 1961–1999 time period for the risk of bluetongue transmission, during August–October in northwest

Europe, as defined by the basic reproductive ratio, R0. R0 was estimated from climate observations (OBS – thick black line), and an ensemble of 11 future climate projections (SimA1B), for which the dashed line presents the mean and the grey envelope the spread (Adapted from [110])

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transmission risk will have increased by 30% in northwest Europe relative to the 1961–1999 mean. The risk is also projected to increase in southwest Europe, but in this case only by 10% relative to the 1961–1999 mean. The matching of observed change in bluetongue with quantitative predictions of a climate-driven disease model provides evidence for the influence of climate change far stronger than the “same place, same time, right direction” criterion described earlier. Indeed, it probably makes bluetongue the most convincing example of a disease that is responding to climate change. In this respect, bluetongue differs remarkably from another vector-borne disease, malaria. Malaria Some 3.2 billion people live with the risk of malaria transmission, between 350 and 500 million clinical episodes of malaria occur each year and the disease kills at least one million people annually [111]. Of these, each year about 12 million cases and 155,000–310,000 deaths are in epidemic areas [112]. Interannual climate variability primarily drives the timing of these epidemics. Malaria is caused by Plasmodium spp. parasites. Part of the parasite’s life cycle takes place within anopheline mosquitoes while the remainder of the life cycle occurs within the human host. The parasite and mosquito life cycles are affected by weather and climate (mainly rain, temperature, and humidity), allowing models of the risk of malaria transmission to be driven by seasonal forecasts from ensemble prediction systems [113], thereby permitting forecasts of potential malaria outbreaks with lead times of up to 4–6 months [114, 115]. Among scientists there are contrasting views about the overall importance of climate on the transmission of malaria, and therefore on the importance of future climate change. Some argue that climate variability or change is the primary actor in any changing transmission pattern of malaria, while others suggest that any changing patterns today or in the foreseeable future are due to non-climate factors [35, 116, 117].

Climate Change Effects on Infectious Diseases

A key insight is that while global temperatures have risen, there has been a net reduction in malaria in the tropics over the last 100 years and temperature or rainfall change observed so far cannot explain this reduction [118]. Malaria has moved from being climate sensitive (an increasing relationship between ambient temperature and the extent of malaria transmission) in the days before disease interventions were widely available to a situation today where regions with malaria transmission are warmer than those without, but within the malaria-affected region, warmer temperatures no longer mean more disease transmission. Instead, other variables affecting malaria, such as good housing, the running of malaria control schemes, or ready access to affordable prophylaxis, now play a greater role than temperature in determining whether there are higher or lower amounts of transmission. This would suggest that the importance of climate change in discussions of future patterns of malaria transmission is likely to have been significantly overplayed. What is clearly recognized, by all sides in the malaria and climate debate, is that mosquitoes need water to lay their eggs in, and for larval development, and that adult mosquitoes need to live long enough in an environment with high humidity and with sufficiently high temperature for transmission to be possible to the human host. Hence, while the spatial distribution of higher versus lower degrees of malaria transmission appears to have become, in a sense, divorced from ambient temperature, it seems likely that the weather plays as important a role as ever in determining when seasonal transmission will start and end. Climate change may therefore still have a role to play in malaria: not so much affecting where it occurs but, via changing rainfall patterns and mosquito numbers, when or for how long people are most at risk. Malaria has only recently become confined to the developing world and tropics. It is less than 40 years since malaria was eradicated in Europe and the United States; and the 15  C July isotherm was the northern limit until the mid nineteenth century [119]. Changes in land use and increased living standards, in particular, acted to reduce exposure to the mosquito vector in these

Climate Change Effects on Infectious Diseases

temperate zones, leading ultimately to the final removal of the disease. In the UK, a proportion of the reduction has been attributed to increasing cattle numbers and the removal of marshland [120]. In Finland, changes in family size, improvements in housing, changes in farming practices, and the movement of farmsteads out of villages lead to the disappearance of malaria [121], where it had formerly been transmitted indoors in winter. While future increases in temperature may, theoretically, lead to an increased risk of malaria transmission in colder climes than at present [120, 122], the much-altered physical and natural environment may preclude this risk increasing to a level that merits concern. Once again, a more important future driver of malaria risk, in the UK at least, may be the pressure to return some of our landscape to its former state, such as the reflooding of previously drained marshland.

Future Directions Climate change is widely considered to be a major threat to human and animal health, and the viability of certain endangered species, via its effects on infectious diseases. How realistic is this threat? Will most diseases respond to climate change, or just a few? Will there be a net increase in disease burden or might as many diseases decline in impact as increase? The answers to these questions are important, as they could provide opportunities to mitigate against new disease threats, or may provide the knowledge-base required for policy makers to take necessary action to combat climate change itself. However, both the methodology to accurately predict climate change’s effects on diseases and, in most cases, the data to apply the methodology to a sufficiently wide-range of pathogens is currently lacking. The majority of pathogens, particularly those not reliant on intermediate hosts or arthropod vectors for transmission, either do occur, or have the potential to occur, in most parts of the world already. Climate change has the capacity to affect the frequency or scale of outbreaks of these

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diseases: Good examples would be the frequency of food poisoning events from the consumption of meat (such as salmonellosis) or shellfish (caused by Vibrio bacteria). Vector-borne diseases are usually constrained in space by the climatic needs of their vectors, and such diseases are therefore the prime examples of where climate change might be expected to cause distributional shifts. Warmer temperatures usually favor the spread of vectors to previously colder environments, thereby exposing possibly naïve populations to new diseases. However, altered rainfall distributions have an important role to play. Many pathogens or parasites, such as those of anthrax, haemonchosis, and numerous vector-borne diseases, may in some regions be subject to opposing forces of higher temperatures promoting pathogen or vector development, and increased summer dryness leading to more pathogen or vector mortality. Theoretically, increased dryness could lead to a declining risk of certain diseases. A good example is fasciolosis, where the lymnaeid snail hosts of the Fasciola trematode are particularly dependent on moisture. Less summer rainfall and reduced soil moisture may reduce the permissiveness of some parts of the UK for this disease. The snail and the freeliving fluke stages are, nevertheless, also favored by warmer temperatures and, in practice, current evidence is that fasciolosis is spreading in the UK [123]. One way to predict the future for disease in a specific country is to learn from countries that, today, are projected to have that country’s future climate [37, 39]. At least some of the complexity behind the multivariate nature of disease distributions should have precipitated out into the panel of diseases that these countries currently face. For example, in broad terms, the UK’s climate is predicted to get warmer, with drier summers and wetter winters, becoming therefore increasingly “mediterranean.” It would seem reasonable, therefore, to predict that the UK of the future might experience diseases currently present in, or that occur periodically in, southern Europe. For humans, the best example would be leishmanosis (cutaneous and visceral) [124], while for animals, examples include West Nile

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fever [125], Culicoides imicola–transmitted bluetongue and African horse sickness [41], and canine leishmanosis [126]. The phlebotomid sandfly vectors of the latter do not currently occur in the UK, but they are found widely in southern continental Europe, including France, with recent reports of their detection in Belgium [127]. The spread of the Asian tiger mosquito into Europe and the recent transmission in Europe of both dengue fever [128] and Chikungunya [129] by this vector are further cause for alarm. However, the contrasting examples of bluetongue and malaria – one spreading because of climate change and one retreating despite it – show that considerations which focus entirely on climate may well turn out to be false. Why are these two diseases, both vector-borne and subject to the similar epidemiological processes and temperature dependencies, so different with respect to climate change? The answer lies in the relative importance of other disease drivers. For bluetongue, it is difficult to envisage epidemiologically relevant drivers of disease transmission, other than climate, that have changed significantly over the time period of the disease’s emergence [64]. Life on the farm for the midges that spread bluetongue is probably not dramatically different today from the life they enjoyed 30 years ago. Admittedly, changes in the trade of animals or other goods may have been important drivers of the increased risk of introduction of the causative viruses into Europe, but after introduction, climate change may be the most important driver of increased risk of spread. For malaria, change in drivers other than climate, such as land use and housing, the availability of prophylaxis, insecticides and, nowadays, insecticide-treated bed nets, have played far more dominant roles in reducing malaria occurrence than climate change may have played in increasing it. Two key reasons, then, for the difference between the two diseases are, first, that life for the human hosts of malaria has changed more rapidly than that of the ruminant hosts of bluetongue, and second, the human cost of malaria was so great that interventions were developed;

Climate Change Effects on Infectious Diseases

while the (previously small) economic burden of bluetongue did not warrant such effort and our ability to combat the disease 5 years ago was not very different from that of 50 years before. The very recent advent of novel inactivated vaccines for bluetongue may now be changing this situation. This entry began by asking whether climate change will affect most diseases or just a few. The examples of malaria and bluetongue demonstrate that a better question may be as follows: Of those diseases that are sensitive to climate change, how many are relatively free from the effects of other disease drivers such that the pressures brought by a changing climate can be turned into outcomes?

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Climate Change Effects on Infectious Diseases 70. Sellers RF, Pedgley DE, Tucker MR (1977) Possible spread of African horse sickness on the wind. J Hyg Camb 79:279–298 71. Sellers RF, Pedgley DE, Tucker MR (1978) Possible windborne spread of bluetongue to Portugal, JuneJuly 1956. J Hyg Camb 81:189–196 72. Sellers RF, Pedgley DE, Tucker MR (1982) Rift Valley fever, Egypt 1977: disease spread by windborne insect vectors? Vet Rec 110:73–77 73. Kock RA, Wambua JM, Mwanzia J, Wamwayi H, Ndungu EK, Barrett T, Kock ND, Rossiter PB (1999) Rinderpest epidemic in wild ruminants in Kenya 1993–97. Vet Rec 145:275–283 74. Davis AJ, Jenkinson LS, Lawton JH, Shorrocks B, Wood S (1998) Making mistakes when predicting shifts in species range in response to global warming. Nature 391:783–786 75. White PCL, Brown JA, Harris S (1993) Badgers (Meles meles), cattle and bovine tuberculosis (Mycobacterium bovis) – a hypothesis to explain the influence of habitat on the risk of disease transmission in southwest England. Proc R Soc Lond B 253:277–284 76. Tompkins DM, Dobson AP, Arneberg P, Begon ME, Cattadori IM, Greenman JV, Heesterbeek JAP, Hudson PJ, Newborn D, Pugliese A, Rizzoli AP, Rosa R, Rosso F, Wilson K (2001) Parasites and host population dynamics. In: Hudson PJ, Dobson AP (eds) Ecology of wildlife diseases. Oxford University Press, Oxford, pp 45–62 77. Smith KF, Sax DF, Lafferty KD (2006) Evidence for the role of infectious disease in species extinction and endangerment. Conserv Biol 20:1349–1357 78. Wyatt KB, Campos PF, Gilbert MTP, Kolokotronis SO, Hynes WH, DeSalle R, Daszak P, MacPhee RDE, Greenwood AD (2008) Historical mammal extinction on christmas island (Indian ocean) correlates with introduced infectious disease. PLoS One 3(11):e3602 79. Freed LA, Cann RL, Goff ML, Kuntz WA, Bodner GR (2005) Increase in avian malaria at upper elevation in Hawai’i. Condor 107:753–764 80. Haydon DT, Laurenson MK, Sillero-Zubiri C (2002) Integrating epidemiology into population viability analysis: managing the risk posed by rabies and canine distemper to the Ethiopian wolf. Conserv Biol 16:1372–1385 81. McCallum H, Jones M (2006) To lose both would look like carelessness: Tasmanian devil facial tumour disease. PLoS Biol 4:1671–1674 82. Frick WF, Pollock JF, Hicks AC, Langwig KE, Reynolds DS, Turner GG, Butchkoski CM, Kunz TH (2010) An emerging disease causes regional population collapse of a common North American bat species. Science 329:679–682 83. Smith KF, Acevedo-Whitehouse K, Pedersen AB (2009) The role of infectious diseases in biological conservation. Anim Conserv 12:1–12

119 84. Daszak P, Cunningham AA, Hyatt AD (2000) Wildlife ecology – emerging infectious diseases of wildlife – threats to biodiversity and human health. Science 287:443–449 85. Gortazar C, Ferroglio E, Hofle U, Frolich K, Vicente J (2007) Diseases shared between wildlife and livestock: a European perspective. Eur J Wildl Res 53: 241–256 86. Wolfe ND, Dunavan CP, Diamond J (2007) Origins of major human infectious diseases. Nature 447: 279–283 87. van der Riet FD (1997) Diseases of plants transmissible between plants and man (phytonoses) exist. Med Hypotheses 49:359–361 88. Williams JW, Jackson ST, Kutzbacht JE (2007) Projected distributions of novel and disappearing climates by 2100 AD. Proc Natl Acad Sci U S A 104: 5738–5742 89. Benning TL, LaPointe D, Atkinson CT, Vitousek PM (2002) Interactions of climate change with biological invasions and land use in the Hawaiian islands: modeling the fate of endemic birds using a geographic information system. Proc Natl Acad Sci U S A 99:14246–14249 90. Isaac NJB, Turvey ST, Collen B, Waterman C, Baillie JEM (2007) Mammals on the EDGE: conservation priorities based on threat and phylogeny. PLoS One 2(3):e296 91. Ford SE, Smolowitz R (2007) Infection dynamics of an oyster parasite in its newly expanded range. Mar Biol 151:119–133 92. Sokolow S (2009) Effects of a changing climate on the dynamics of coral infectious disease: a review of the evidence. Dis Aquat Org 87:5–18 93. Ricketts TH, Dinerstein E, Boucher T, Brooks TM, Butchart SHM, Hoffmann M, Lamoreux JF, Morrison J, Parr M, Pilgrim JD, Rodrigues ASL, Sechrest W, Wallace GE, Berlin K, Bielby J, Burgess ND, Church DR, Cox N, Knox D, Loucks C, Luck GW, Master LL, Moore R, Naidoo R, Ridgely R, Schatz GE, Shire G, Strand H, Wettengel W, Wikramanayake E (2005) Pinpointing and preventing imminent extinctions. Proc Natl Acad Sci U S A 102:18497–18501 94. Kutz SJ, Hoberg EP, Polley L, Jenkins EJ (2005) Global warming is changing the dynamics of Arctic host-parasite systems. Proc R Soc Lond B 272: 2571–2576 95. Ytrehus B, Bretten T, Bergsjo B, Isaksen K (2008) Fatal pneumonia epizootic in musk ox (Ovibos moschatus) in a period of extraordinary weather conditions. EcoHealth 5:213–223 96. Bradley M, Kutz SJ, Jenkins E, O’Hara TM (2005) The potential impact of climate change on infectious diseases of Arctic fauna. Int J Circumpolar Health 64:468–477 97. Berger L, Hyatt AD, Speare R, Longcore JE (2005) Life cycle stages of the amphibian chytrid

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Climate Change Effects on Infectious Diseases Batrachochytrium dendrobatidis. Dis Aquat Org 68: 51–63 Pounds JA, Bustamante MR, Coloma LA, Consuegra JA, Fogden MPL, Foster PN, La Marca E, Masters KL, Merino-Viteri A, Puschendorf R, Ron SR, Sanchez-Azofeifa GA, Still CJ, Young BE (2006) Widespread amphibian extinctions from epidemic disease driven by global warming. Nature 439: 161–167 Ribas L, Li MS, Doddington BJ, Robert J, Seidel JA, Kroll JS, Zimmerman LB, Grassly NC, Garner TWJ, Fisher MC (2009) Expression profiling the temperature-dependent amphibian response to infection by Batrachochytrium dendrobatidis. PLoS One 4(12):e8408 Gleason FH, Letcher PM, McGee PA (2008) Freeze tolerance of soil chytrids from temperate climates in Australia. Mycol Res 112:976–982 Fisher MC, Garner TWJ, Walker SF (2009) Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. Annu Rev Microbiol 63:291–310 Pounds JA, Crump ML (1994) Amphibian declines and climate disturbance – the case of the golden toad and the harlequin frog. Conserv Biol 8:72–85 Garner TWJ, Walker S, Bosch J, Leech S, Rowcliffe JM, Cunningham AA, Fisher MC (2009) Life history tradeoffs influence mortality associated with the amphibian pathogen Batrachochytrium dendrobatidis. Oikos 118:783–791 Lips KR, Diffendorfer J, Mendelson JR, Sears MW (2008) Riding the wave: reconciling the roles of disease and climate change in amphibian declines. PLoS Biol 6:441–454 Rogers DJ, Randolph SE (2003) Studying the global distribution of infectious diseases using GIS and RS. Nat Rev Microbiol 1:231–237 Mellor PS, Boorman J, Baylis M (2000) Culicoides biting midges: their role as arbovirus vectors. Annu Rev Entomol 45:307–340 Mellor PS, Wittmann EJ (2002) Bluetongue virus in the Mediterranean basin 1998–2001. Vet J 164: 20–37 Purse BV, Nedelchev N, Georgiev G, Veleva E, Boorman J, Denison E, Veronesi E, Carpenter S, Baylis M, Mellor PS (2006) Spatial and temporal distribution of bluetongue and its Culicoides vectors in Bulgaria. Med Vet Entomol 20:335–344 Mellor PS, Carpenter S, Harrup L, Baylis M, Mertens PPC (2008) Bluetongue in Europe and the Mediterranean basin: history of occurrence prior to 2006. Prev Vet Med 87:4–20 Guis H, Caminade C, Calvete C, Morse AP, Tran A, Baylis M (2011) Modelling the effects of past and future climate on the risk of bluetongue emergence in Europe. J Roy Soc Interface. (in press) WHO (2005) World Malaria report, rollback Malaria programme. World Health Organisation, Geneva

112. Worrall E, Rietveld A, Delacollette C (2004) The burden of malaria epidemics and cost-effectiveness of interventions in epidemic situations in Africa. Am J Trop Med 71:136–140 113. Palmer TN, Alessandri A, Andersen U, Cantelaube P, Davey M, Delecluse P, Deque M, Diez E, DoblasReyes FJ, Feddersen H, Graham R, Gualdi S, Gueremy JF, Hagedorn R, Hoshen M, Keenlyside N, Latif M, Lazar A, Maisonnave E, Marletto V, Morse AP, Orfila B, Rogel P, Terres JM, Thomson MC (2004) Development of a European multimodel ensemble system for seasonal-tointerannual prediction (DEMETER). Bull Am Meteorol Soc 85:853–872 114. Morse AP, Doblas-Reyes FJ, Hoshen MB, Hagedorn R, Palmer TN (2005) A forecast quality assessment of an end-to-end probabilistic multimodel seasonal forecast system using a malaria model. Tellus Ser A 57:464–475 115. Jones AE, Morse AP (2010) Application and validation of a seasonal ensemble prediction system using a dynamic malaria model. J Clim 23:4202–4215 116. Lafferty KD (2009) The ecology of climate change and infectious diseases. Ecology 90:888–900 117. Epstein P (2010) The ecology of climate change and infectious diseases: comment. Ecology 91:925–928 118. Gething PW, Smith DL, Patil AP, Tatem AJ, Snow RW, Hay SI (2010) Climate change and the global malaria recession. Nature 465:342–344 119. Reiter P (2008) Global warming and malaria: knowing the horse before hitching the cart. Malar J 7:S3 120. Kuhn KG, Campbell-Lendrum DH, Armstrong B, Davies CR (2003) Malaria in Britain: past, present, and future. Proc Natl Acad Sci U S A 100: 9997–10001 121. Hulden L (2009) The decline of malaria in Finland – the impact of the vector and social variables. Malar J 8:94 122. Lindsay SW, Hole DG, Hutchinson RA, Richards SA, Willis SG (2010) Assessing the future threat from vivax malaria in the United Kingdom using two markedly different modelling approaches. Malar J 9:70 123. Pritchard GC, Forbes AB, Williams DJL, SalimiBejestani MR, Daniel RG (2005) Emergence of fasciolosis in cattle in East Anglia. Vet Rec 157: 578–582 124. Dujardin JC, Campino L, Canavate C, Dedet JP, Gradoni L, Soteriadou K, Mazeris A, Ozbel Y, Boelaert M (2008) Spread of vector-borne diseases and neglect of leishmaniasis, Europe. Emerg Infect Dis 14:1013–1018 125. Gould EA, Higgs S, Buckley A, Gritsun TS (2006) Potential arbovirus emergence and implications for the United Kingdom. Emerg Infect Dis 12: 549–555 126. Shaw SE, Langton DA, Hillman TJ (2008) Canine leishmaniosis in the UK. Vet Rec 163:253–254

Climate Change Effects on Infectious Diseases 127. Depaquit J, Naucke TJ, Schmitt C, Ferté H, Léger N (2005) A molecular analysis of the subgenus Transphlebotomus Artemiev, 1984 (Phlebotomus, Diptera, Psychodidae) inferred from ND4 mtDNA with new northern records of Phlebotomus mascittii Grassi, 1908. Parasitol Res 95:113–116 128. La Ruche G, Souares Y, Armengaud A, PelouxPetiot F, Delaunay P, Despres P, Lenglet A, Jourdain F, Leparc-Goffart I, Charlet F, Ollier L, Mantey K, Mollet T, Fournier JP, Torrents R, Leitmeyer K, Hilairet P, Zeller H, Van Bortel W, Dejour-Salamanca D, Grandadam M, Gastellu-

121 Etchegorry M (2010) First two autochthonous dengue virus infections in metropolitan France, September 2010. Euro Surveill 15:2–6 129. Eitrem R, Vene S (2008) Chikungunya fever-a threat for Europeans. A review of the recent literature. Parasitol Res 103:S147–S148

Books and Reviews International Panel on Climate Change (2007) Climate change 2007: impacts, adaptation and vulnerability. Cambridge University Press, Cambridge

Malaria Vaccines Matthew B. Laurens and Christopher V. Plowe Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA

Article Outline Glossary Definition of the Subject Introduction The Malaria Life Cycle Pathogenesis and Disease Epidemiology Immunity Early Malaria Vaccines Obstacles to Malaria Vaccines Preerythrocytic Vaccines Blood Stage Vaccines Placental Malaria Vaccines Transmission-Blocking Vaccines Multistage, Multi-Antigen Vaccines Whole-Organism Vaccines Future Directions Bibliography

with malaria parasites under carefully controlled conditions. Immunogenicity The ability of a vaccine to produce specific immune responses (usually antibodies) that recognize the vaccine antigen. Preerythrocytic Stages of the malaria parasite that are injected by a mosquito and develop in the liver before emerging into the blood where they can cause symptoms. Vaccines targeting preerythrocytic stages are intended to prevent infection altogether and, if highly effective, would also prevent disease and block transmission. Sexual stage The male and female forms of malaria parasites that are responsible for transmission through mosquitoes. Vaccines directed against sexual stages are intended to prevent malaria transmission. Subunit vaccine A vaccine based on a small portion of the organism, usually a peptide or protein. Vaccine resistance The ability of malaria parasites to escape strain-specific immune responses by exploiting genetic diversity to increase the frequencies of nonvaccine-type variants in a population or to evolve new diverse forms. Whole-organism vaccine A vaccine based on an attenuated or killed whole parasite.

Glossary Adjuvant A substance added to a vaccine to stimulate a stronger or more effective immune response. Blood stage The stage of the malaria parasite life cycle responsible for clinical symptoms. Vaccines that target the blood stage are intended to prevent disease and death but that do not prevent infection and may not affect malaria transmission. Challenge trial Experimental Phase 1/2 clinical trial in which healthy volunteers receive a malaria vaccine and are exposed to the bites of malaria-infected mosquitoes or injected

Definition of the Subject Vaccines are the most powerful public health tools mankind has created, and research toward malaria vaccines began not long after the parasite responsible for this global killer was discovered and its life cycle described more than 100 years ago. But parasites are bigger, more complicated, and wilier than the viruses and bacteria that have been conquered or controlled with vaccines, and an effective malaria vaccine has remained elusive. High levels of protective efficacy were achieved in

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_536 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2022 https://doi.org/10.1007/978-1-4939-2493-6_536-3

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crude early experiments in animals and humans using weakened whole parasites, but the results of more sophisticated modern approaches using molecular techniques have ranged from modest success to abject failure. In a major milestone, a subunit recombinant protein vaccine that affords in the neighborhood of 25–50% protective efficacy against malaria has been licensed but not yet widely deployed. Incremental improvements on this vaccine are possible and worth pursuing, but the best hope for a malaria vaccine that would improve prospects for malaria eradication may lie with the use of attenuated whole parasites and powerful immune-boosting adjuvants.

Introduction The malaria parasite is thought to have killed more human beings throughout history than any other single cause [1]. Today, along with AIDS and tuberculosis, malaria remains one of the “big three” infectious diseases, every year exacting a heavy toll on human life and health in parts of Central and South America, large regions of Asia, and throughout most of sub-Saharan Africa, where over 90% of malaria cases and deaths occur [2]. In the middle of the twentieth century, the availability of the long-acting insecticide dichlorodiphenyltrichloroethane (DDT) and the safe and effective antimalarial drug chloroquine provided the basis for optimism that global eradication was possible. Although malaria was eliminated in several countries around the margins of the malaria map, factors including the emergence of DDT-resistant mosquitoes and chloroquine-resistant parasites, as well as waning economic and political support led the campaign to stall by the late 1960s, resulting in the rapid resurgence to previous disease levels in many locations [3]. For the next 30 years, the spread of drugresistant malaria and donor fatigue contributed to an overall lack of progress against the disease. But starting in the late 1990s, new tools, including long-lasting insecticide-impregnated nets and highly efficacious combination drug therapies,

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led to a wave of successes, including dramatic reductions in disease burden in some areas and complete elimination of malaria in others [4]. These successes have stimulated a renewed sense of optimism about prospects for global eradication [5]. If it is to succeed, the renewed drive toward country-by-country elimination and eventual worldwide eradication of malaria will require powerful new tools, importantly including malaria vaccines that produce protective immune responses that surpass those acquired through natural exposure to malaria [6]. Successful global or regional campaigns to eradicate smallpox, polio, measles, and rubella have all relied on vaccines. Those for yellow fever, hookworm, and yaws— and for malaria—which have relied on nonvaccine measures such as vector control or drug treatment have all failed [7]. While the odds of successful global malaria eradication are long even with an ideal malaria vaccine, they are virtually nil without one. In the meantime, even a modestly effective vaccine could substantially reduce the continuing heavy burden of malariaattributable disease (229 million annual cases) and death (409,000 annual deaths) [2]. After nearly a century of malaria vaccine research, today, one modestly effective vaccine based on a parasite surface protein was given favorable review by European regulators in 2015, but has not yet been recommended for widespread use by the World Health Organization as it continues to be evaluated in Africa. This vaccine, RTS,S, continues to undergo costbenefit evaluation in pilot implementation studies in three sub-Saharan African countries that will aim to generate evidence for a policy recommendation for its use [8]. Many other vaccine candidates have fallen short and been abandoned before reaching this stage, although several are in early stages of clinical development. The chief reasons that it has taken this long to get this far are that malaria parasites replicate and propagate through an extremely complex life cycle involving vertebrate hosts and an insect vector, and that they have evolved a repertoire of mechanisms for evading both natural and vaccine-induced immunity.

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This review focuses on key themes that have emerged over 85 years of malaria vaccine research and development and on a few key examples of malaria vaccines that have reached the stage of testing for efficacy in clinical trials in humans. Several recent review articles listed after the primary bibliography explore malaria immunology and preclinical vaccine development in more detail.

The Malaria Life Cycle Malaria is a potentially fatal parasitic disease transmitted to humans and other vertebrate animals by mosquitoes. Species of Plasmodium that cause malaria disease primarily in humans include: P. falciparum, P. vivax, P. ovale, and P. malariae. P. knowlesi infects mainly nonhuman primates but also can infect and sicken humans [9], and hundreds of other malaria species infect other mammals, reptiles, and birds. Because it is responsible for most severe malaria and deaths, P. falciparum has been the target of most vaccine development efforts and is the main focus of this review. However, in many parts of the world, P. vivax is the predominant species, and it causes more severe malaria than has sometimes been appreciated [10]. A vivax malaria vaccine would be highly beneficial, especially if it interrupted transmission. The malaria life cycle begins when the female Anopheles mosquito injects sporozoites from her salivary gland into the skin as she takes a blood meal (Fig. 1). The worm-like sporozoites—about 7 microns in length, or as long as an erythrocyte is wide—invade liver hepatocytes, each sporozoite multiplying over several days into tens of thousands of tiny (about 1 micron in diameter) merozoites packed into a single infected hepatocyte. These preerythrocytic stages cause no clinical signs or symptoms. A highly efficacious preerythrocytic vaccine would thus completely block infection, preventing parasites from reaching the blood and causing disease, and also preventing transmission. Rupturing hepatocytes release showers of merozoites into the circulation, initiating the blood

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stage of malaria infection that is responsible for disease. Merozoites quickly invade erythrocytes and undergo asexual multiplication, dividing, growing, bursting from the erythrocyte, and reinvading in a periodic pattern with each cycle lasting 2 days (3 days in the case of P. malariae), until interrupted by host immunity, drug treatment, or death. Malaria vaccines that target the blood stage are thought of as anti-disease vaccines and would be expected to prevent or reduce clinical illness but would not prevent infection. Some blood stage parasites develop into male and female gametocytes. These sexual forms are taken up by the mosquito vector during a blood meal, mate to form a brief diploid stage, and then develop through further haploid stages and migrate from the gut to the salivary glands (Fig. 1). Each mating pair of gametocytes yields up to 1000 infectious sporozoites, which are injected into the host to complete the transmission cycle. Vaccines targeting the sexual stages would prevent neither infection nor disease in the vaccinated individual and are thought of as transmission-blocking vaccines. Highly efficacious preerythrocytic or blood stage vaccines that prevent sexual reproduction would also block transmission, so the term “transmissionblocking” need not refer exclusively to vaccines against sexual or mosquito stages of the parasite. The term “vaccines that interrupt transmission” (VIMT) encompasses all vaccines that interrupt transmission, whatever stage they target [11].

Pathogenesis and Disease While semi-immune individuals who have been repeatedly exposed can be chronically infected with malaria and experience no symptoms of illness, malaria infection of a nonimmune person (e.g., young infants or travelers) usually causes an acute illness characterized by fever, chills, aches, and other flu-like symptoms. In a minority of cases, for reasons that are not well understood, more severe illness can develop. The clinical syndrome of falciparum malaria originates with changes in the infected erythrocytes. After they invade, blood stage P. falciparum parasites

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Malaria Vaccines, Fig. 1 Life cycle of malaria and stages targeted by vaccines. Source: PATH Malaria Vaccine Initiative. (1) Malaria infection begins when an infected female Anopheles mosquito bites a person, injecting Plasmodium parasites, in the form of sporozoites, into the bloodstream. (2) The sporozoites pass quickly into the human liver. (3) The sporozoites multiply asexually in the liver cells over the next 7–10 days, causing no symptoms. (4) The parasites, in the form of merozoites, burst from the liver cells. (5) In the bloodstream, the merozoites invade red blood cells (erythrocytes) and multiply again until the cells burst. Then they invade more erythrocytes. This cycle is repeated, causing fever each time parasites break free and invade blood cells. (6) Some of the infected

blood cells leave the cycle of asexual multiplication. Instead of replicating, the merozoites in these cells develop into sexual forms of the parasite, called gametocytes, that circulate in the bloodstream. (7) When a mosquito bites an infected human, it ingests the gametocytes, which develop further into mature sex cells called gametes. (8) The gametes develop into actively moving ookinetes that burrow into the mosquito’s midgut wall and form oocysts. (9) Inside the oocyst, thousands of active sporozoites develop. The oocyst eventually bursts, releasing sporozoites that travel to the mosquito salivary glands. (10) The cycle of human infection begins again when the mosquito bites another person

effectively hijack the host cell and its machinery, expressing their own proteins on the surface of the host erythrocyte. Highly variant protein receptors

called P. falciparum erythrocyte membrane proteins (PfEMP1) are encoded by a large, diverse family of up to 60 var genes in each parasite

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genome [12, 13]. PfEMP1 are expressed on the surface of infected red blood cells in clumps known as knobs, which are responsible for adherence of parasitized erythrocytes to the vascular endothelium, resulting in sequestration in tissue blood vessels [14]. The ability of infected red blood cells to adhere to endothelium and the hemozoin they release there serves to activate endothelial receptors that increase sequestration and potentially overstimulate the host immune response [15]. Indeed, children with severe malaria have been found to have increased whole blood transcriptomic signatures of inflammatory pathways [16]. The ability of falciparum malaria to sequester plays a critical role in disease severity—the other human malarias do not appear to sequester and, therefore, are not associated with most of the severe manifestations seen with falciparum malaria [17]. Infected red blood cells cytoadhere and sequester in the microcirculatory compartments of organs, most notably in the brain and placenta, leading to disease, and most abundantly in the spleen, causing splenomegaly. Localization of infected red blood cells to the brain can lead to a form of severe malaria known as cerebral malaria, which can result in death through brain swelling and brain stem dysfunction [18]. Cerebral malaria presents initially with coma, and parasite sequestration in brain tissue manifests with characteristic ocular findings including retinal hemorrhages [19]. Autopsy studies in children with cerebral malaria found ring hemorrhages associated with small vessel blood clots in areas where infected red blood cells were sequestered [20]. PfEMP1 variants capable of binding specifically to brain endothelial cells have been identified [21] and are associated with cerebral malaria [22]. In the placenta, the PfEMP1 variant VAR2CSA binds to the host receptor chondroitin sulfate A that is unique to placental tissue [23], causing a local inflammatory response that leads to maternal hypertension, stillbirth, premature birth, fetal growth retardation, and low birthweight. Sequestered, infected red blood cells not only interfere with microcirculatory blood flow but also hide outside the reach of host defense mechanisms. Infected red blood cells lose their deformability and compromise blood flow in

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small capillaries and venules [24] and through the splenic sinusoidal filtration system [25]. The presence of variant surface antigens leads to immune responses that appear both to harm the human host as well as to lead to the eventual development of protective immunity. Immunity to severe malaria develops rapidly, after only a few infections [26], possibly due to antibody responses that protect against a relatively conserved subset of PfEMP1 variants that are associated with severe malaria. In contrast, the slow acquisition of immune protection against uncomplicated malaria over years of repeated exposure to malaria is thought to represent the accumulation of protective immune responses to a repertoire of diverse antigens, probably including both PfEMP1 and the surface proteins that are the targets of most vaccine candidates [27]. For placental malaria, immunity accumulates with successive pregnancies, such that primigravidae women are most susceptible to placental malaria [28].

Epidemiology The epidemiology of malaria is determined primarily by the patterns and intensity of malaria transmission, which in turn drives the prevalence of malaria infection and the incidence of different forms of malaria disease. In low-transmission settings with unstable malaria, there is a potential for epidemics when transmission recurs or increases, either as a result of reintroduction in a population not recently exposed to malaria, or owing to changing climactic or environmental conditions that favor contact between humans and malariatransmitting Anopheles mosquitoes. Outbreaks can occur when malaria-naïve populations such as transmigrants, miners, or soldiers are exposed to malaria, causing high rates of disease [29]. Depending largely on the degree of host immunity, the manifestations of malaria infection can range from completely asymptomatic parasitemia to mild disease that can be treated on an outpatient basis with oral drugs, and to acute catastrophic life-threatening illness requiring intensive medical care. Very young infants are thought to be protected from malaria disease by maternal

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antibodies and persistent hemoglobin F [30]. While they may be infected congenitally or by mosquito bites in the first days or weeks of life, infants typically do not experience clinical disease until they are a few months old [31]. Following this brief period of relative insusceptibility in early infancy, protective immunity against malaria disease is acquired through repeated exposure and is therefore related to transmission intensity. Where malaria transmission is moderate (average of one or more infected mosquito bites per month) or high (two or more infected mosquito bites per week; up to more than one per day in some areas), the risk period for death from malaria is highest in infants and young children who are in the process of developing acquired immunity [32]. In a typical moderate- or hightransmission setting in sub-Saharan Africa, most severe malaria is experienced by children aged less than 5 years; children aged up to 10–12 years experience frequent episodes of uncomplicated malaria; and older teenagers and adults, while still often infected, rarely experience symptoms of malaria illness. Severe anemia is more frequent in the youngest infants, while cerebral malaria tends to peak in children aged 3–4 years who have experienced previous malaria episodes, suggesting that an overly exuberant immune response contributes to the pathogenesis of cerebral malaria. In contrast, in low-transmission settings, persons of all ages are susceptible to infection and uncomplicated malaria, although the risk of exposure is often higher in younger males in high-risk occupations such as miners or plantation workers [33]. Where transmission is low, as it is in much of Southeast Asia and Latin America, most who are infected become sick, and the risk for severe malaria persists throughout life. Semi-immune adults living in higher transmission areas such as much of sub-Saharan Africa, although they remain susceptible to asymptomatic parasitemia, are protected against clinical malaria disease, rarely becoming ill even when persistently infected [32]. This protective immunity is lost after a few years in the absence of exposure. Acquired immunity is also diminished in pregnancy, in those women pregnant with their first

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child are susceptible to severe P. falciparum disease from placental malaria because they lack immunity to placenta-specific cytoadherence proteins. As placental immunity develops in subsequent pregnancies, there is a reduced risk of adverse effects of malaria in pregnancy on the mother and fetus [34]. Based on these epidemiological patterns, the primary populations targeted for malaria vaccines are infants and young children in areas of moderate and high transmission who bear the greatest burden of disease and death, and women of childbearing age in these same areas. Malaria-naïve travelers and military troops would also benefit from a malaria vaccine. As more countries move toward malaria elimination and global eradication is considered [35], the general population of malaria-endemic areas may be vaccinated to drive down transmission [6, 11].

Immunity Insights into malaria immunity come not only from studies in various animal models including birds, rodents, and nonhuman primates but also from important studies in humans, including classic passive transfer experiments [36, 37] and early studies of malaria therapy for neurosyphilis [38, 39], as well as immunoepidemiological studies that aim to identify correlates of clinical protection [40, 41]. Both humoral and cellular factors contribute to acquired immune protection against malaria. Broadly speaking, cellular immune responses are thought to be more important in controlling the preerythrocytic stages of malaria infection [42], and antibodies are thought to block erythrocyte invasion to suppress blood stage infection [36, 40]. For these reasons, cellular immune responses are typically emphasized in the development of preerythrocytic vaccines and antibody responses in the development of blood stage vaccines. However, despite nearly 100 years of human and animal research, the basis of protective immunity against malaria is still poorly understood, and no specific immune response has been established as an essential correlate of

Malaria Vaccines

clinical protection, complicating malaria vaccine development. New genomic tools have the potential to improve understanding of malaria immunity and may aid in vaccine development. For example, while it has long been known that there is some degree of strain specificity to malaria immunity, the discovery of large families of genes encoding highly variable surface antigens that mediate cytoadherence and immune evasion [14] has led to models for explaining the slow acquisition of protective immunity as a process of building up a repertoire of variant-specific immune responses until protection is in place against the full range of locally prevalent variants. As next-generation sequencing technologies continue to improve their ability to generate sequence from clinical samples and to assemble genomes and map variant sequences to a reference genome [43], genomic epidemiology studies that relate parasite genotypes to clinical risk and allele-specific immune responses will permit testing of the hypotheses generated by such models [44, 45]. In another approach, high-density protein arrays permit serological profiling of large numbers of serum samples against thousands of recombinant malaria proteins [46]. When this protein array was used to identify P. falciparum proteins that were differentially recognized by the sera of children who were resistant to clinical malaria, several previously unknown antigens were identified as possibly being important in acquired immunity, providing possible new vaccine targets [47]. High-density peptide microarrays that represent diverse whole protein epitopes have further advanced our understanding of strainspecific antibody responses and immunogenic epitopes [48]. In addition to acquired immunity, several host genetic factors offer some degree of protection against malaria, generally not by preventing infection but by reducing the risk of clinical illness or severe disease. Sickle cell trait [49] and other hemoglobinopathies [50–53] are more prevalent in populations at risk of malaria because they afford protection against clinical malaria. Various other human genetic polymorphisms associated with the host immune response [54] and with

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host-parasite interactions [55, 56] have also been correlated with susceptibility to clinical malaria in genetic association studies.

Early Malaria Vaccines In 1880, Charles Louis Alphonse Laveran, a 33-year-old French Army doctor working in Algeria, discovered motile worm-like parasites, later understood to be exflagellating male gametes, in the blood of a feverish soldier [57]. Seven years later, Ronald Ross established that malaria parasites were transmitted to birds by the bites of infected mosquitoes [58]. The tremendous public health benefit that would be provided by an effective malaria vaccine was quickly appreciated, and from the 1930s to the 1970s, malaria vaccine researchers used primarily birds (including ducklings, canaries, chickens, and turkeys) and occasionally monkeys as model systems, and inactivated or killed whole parasites or parasite extracts as vaccines, often accompanied by immune-boosting adjuvant systems. This wave of vaccine development research crested in the late 1940s as World War II ended and attention shifted to the global campaign to eradicate malaria using drugs and anti-vector methods, and only resurged in the late 1960s when it became apparent that eradication was not possible with existing tools. Early work focused on attenuated wholeparasite vaccines. Working in India, Russell and Mohan protected chickens from mosquito challenge with P. gallinaceum by immunizing them with sporozoites inactivated by ultraviolet light [59]. In 1945, the Hungarian-American immunologist Jules Freund (of Freund’s complete adjuvant fame) and colleagues reported that they had successfully protected ducks against intravenous challenge with the avian malaria P. lophurae by immunizing them with formalin-inactivated malaria-infected blood cells and an adjuvant system consisting of a lanolin-like substance, paraffin oil, and killed tubercle bacilli [60]. They used a similar vaccine formulation to protect rhesus monkeys against P. knowlesi challenge [61]. These pioneering studies demonstrated two

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important principles that remain highly relevant to contemporary malaria vaccine development efforts, namely that good protective efficacy can be achieved with whole-organism vaccines and that strong immune-boosting adjuvants can achieve levels of protection that match or exceed those acquired through repeated natural exposure. In a thoughtful and prescient paper published in 1943 describing protection of ducklings when a bacterial toxin adjuvant was added to a killed blood stage vaccine, Henry Jacobs briefly cited an abstract presented by W. B. Redmond at the 1939 annual meeting of the American Society of Parasitologists, writing that “Redmond. . .noted some protection against bird malaria when he vaccinated with irradiated parasites” [62]. Redmond’s abstract described using a frozen killed vaccine but made no mention of inactivation by irradiation [63]. Unfortunately, Redmond never published this work, but one can speculate that he may have described using some form of a radiation-attenuated P. lophurae vaccine preparation in his presentation at the 1939 meeting, anticipating subsequent work using this approach, including attenuation by irradiation of both blood stages [64, 65] and later and more famously of sporozoites [66, 67]. Columbia University scientists, who were aware of “inconclusive” earlier studies reported in the German medical literature, described in 1946 their own unsuccessful attempts to protect humans against intravenous challenge with P. vivax using a vaccine consisting of formalintreated and freeze-thawed blood containing 65–150 million blood stage P. vivax parasites [68]. No adjuvant was used in these earliest human studies, which may partially explain the disappointing results. Several of these historical threads came together in a series of major breakthroughs in the late 1960s and early 1970s. First, Ruth Nussenzweig and Jerome Vanderberg at New York University reported in 1967 that intravenous immunization with irradiated P. berghei sporozoites could protect mice against subsequent intravenous challenge with viable sporozoites [66]. This advance was quickly translated into human trials of radiationattenuated P. falciparum sporozoites delivered by

Malaria Vaccines

the bites of infected, irradiated mosquitoes [69, 70]. In these and subsequent malaria challenge trials, 90% of volunteers who were immunized with radiation-attenuated sporozoites by receiving at least 1000 infected bites over several sessions were fully protected against infection [71]. All unvaccinated volunteers acquired malaria from the bites of nonirradiated mosquitoes. These pioneering studies, first done by University of Maryland investigators in prisoners [72], provided proof that humans could be protected against infection with deadly P. falciparum through immunization, and spurred the identification of specific sporozoite proteins that could serve as antigens for subunit vaccines, as described in the following sections. At around this time, two major advances ushered in the modern era of malaria vaccine development: the advent of molecular biology and the ability to clone, sequence, and express parasite genes in heterologous expression systems such as bacteria and yeast; and the development of methods for growing P. falciparum parasites in continuous in vitro culture [73, 74], providing a reliable and reproducible source of malaria parasites, proteins, and genes.

Obstacles to Malaria Vaccines Why is there still no licensed malaria vaccine after 85 years of vaccine development research and evaluation of more than 70 vaccine candidates [75] in preclinical and clinical testing? Obstacles slowing progress toward an effective malaria vaccine include the size and complexity of the parasite, its genetic diversity, the efficiency of its amplification, the incomplete and temporary nature of naturally acquired immunity, and the fact that in addition to providing protection, immune responses also contribute to pathogenesis. Furthermore, parasite material must generally be obtained from infected hosts or mosquitoes, and only one species, P. falciparum, can be grown in continuous culture. Finally, validated immune correlates of protection are lacking, so candidate vaccines can only be downselected by conducting costly efficacy trials in humans.

Malaria Vaccines

The P. falciparum genome has about 23 million bases of DNA organized into 14 chromosomes and about 5000 genes [76]. This is orders of magnitude larger than the genomes of the viruses and bacteria to which vaccines have been successfully developed. This complexity and the large number of gene products provide the means and materials needed for the highly complex life cycle stages in vertebrate hosts and mosquitoes (Fig. 1). Moreover, mutation during mitotic reproduction in the haploid liver and blood stages and genetic recombination during the diploid sexual reproductive stages in the mosquito result in extensive genetic diversity that is driven by selection pressure from the immune system, as well as by drugs and, when they are deployed, potentially by vaccines [77]. All of this complexity and diversity greatly complicates the choice of candidate antigens for vaccine development. At least 18 different forms of one leading blood stage antigen [78] and more than 200 variants of another [79] have been documented in a single African village. If vaccines targeting these antigens generate immune responses that are insufficiently cross-protective, vaccines based on just one or two genetic variants are unlikely to be broadly efficacious [77]. To date, the choices of which variants of target antigens to include in malaria vaccines have largely been made without consideration of the frequencies at which these variants are present in natural populations. Careful molecular epidemiological studies can pinpoint which of the many polymorphisms in some of these antigens are the most important determinants of strain-specific natural immunity [78, 79], and this approach may help inform the design of polyvalent or chimeric vaccines that protect against diverse parasite strains [77]. Immunization with whole parasites of a given life cycle stage or with stage-specific proteins typically protects against only that life cycle stage, hence the notion of vaccines that prevent infection, disease, or transmission by targeting the different stages (Fig. 1). While a highly efficacious preerythrocytic vaccine would prevent not only infection, but by doing so also prevent disease as well as transmission [6], even a single surviving sporozoite could theoretically result in

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a full-blown infection, severe disease, and transmission. This is because of the parasite’s ability to multiply rapidly—one sporozoite gives rise to tens of thousands of merozoites emerging from the liver about a week and a half after a mosquito bite, and each merozoite multiplies roughly tenfold during the 48-h blood cycle, quickly resulting in billions of parasites circulating in the body. In reality, the rate at which parasites amplify their numbers is determined not just by the parasite’s maximum reproductive capacity but also by host defenses and other factors. The ability of a partially efficacious preerythrocytic vaccine to reduce the risk of clinical malaria illness [80, 81] supports the idea that there is some benefit from slowing the rate of parasite reproduction short of complete prevention of blood stage infection—a so-called leaky vaccine, perhaps better thought of as an injectable bednet. Most successful vaccines emulate naturally acquired protective immunity, preventing infection or illness caused by pathogens (e.g., measles) that typically generate strong and long-lasting immune protection after a single exposure in nonvaccinated individuals. As described above, the naturally acquired protective immunity to malaria is hard won and short lived. An effective malaria vaccine would need to produce stronger immune responses more quickly than those that develop even under intense continuous natural exposure to malaria, and a vaccine intended to prevent infection will need to surpass natural immunity, which gradually protects against clinical illness but does not completely prevent infection. Because the host immune response contributes to malaria pathogenesis, a vaccine could theoretically increase the risk of harmful inflammatory responses to subsequent infection, especially for vaccines directed against the blood stages that are responsible for pathology. One blood stage malaria vaccine based on the P. falciparum merozoite surface protein 1 (MSP1) protected monkeys against lethal infection but then resulted in lifethreatening anemia following subsequent exposure to malaria [82]. No such postvaccination anemia has been observed in malaria-exposed adults [83, 84] or children [85, 86] in African trials of a different MSP1 malaria vaccine. A Phase

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2 trial of a blood stage malaria vaccine based on a different antigen, the apical membrane antigen 1 (AMA1), reported a possible increased risk of anemia in vaccinated malaria-exposed children in an unplanned post hoc analysis [87]. No increased risk of anemia has been seen in trials with a more highly immunogenic AMA1 vaccine tested in similar populations [88–90]. Vigilance for untoward inflammatory responses to malaria vaccines or to postvaccination malaria infection will continue to be an important aspect of clinical malaria vaccine development, especially for blood stage vaccines.

Preerythrocytic Vaccines The only licensed malaria vaccine, RTS,S/AS01, targets the preerythrocytic circumsporozoite protein (CSP) of P. falciparum. The gene encoding CSP was the first P. falciparum gene cloned [91], and as the major surface protein coating sporozoites, CSP was immediately of great interest as a vaccine candidate. Thought to be important in sporozoite development and motility [92], CSP contains a central repeat region that elicits antibody responses [93], flanked on each side by nonrepetitive regions containing T cell epitopes [91]. Antibodies directed against the central repeat region cause the protein coat to slough off and block invasion of hepatocytes, suggesting that vaccine-induced antibodies might prevent infection [94]. Early synthetic CSP vaccines based on the repeat region using aluminum hydroxide as an adjuvant were poorly immunogenic, although a few individuals who achieved high antibody titers were protected against experimental challenge with homologous sporozoites [95, 96]. Seroepidemiological studies failed to find an association between anti-CSP antibodies and protection against infection [97], however, and the addition of adjuvants that produced higher antibody levels did not result in improved efficacy [98], leading the developers of RTS,S to include the flanking regions containing T cell epitopes in subsequent versions of the vaccine. The final form of RTS,S is comprised of the central repeat region (R) and T cell epitopes (T) using the hepatitis

Malaria Vaccines

B surface antigen (S) as a carrier matrix, and co-expressed in Saccharomyces cerevisiae with additional S, hence “RTS,S.” The clinical development of RTS,S has included progressive improvements in adjuvant systems, resulting in improved efficacy both in experimental challenges [99–101] and in clinical trials in malariaexposed adults [102] and children [80, 103]. The licensed formulation includes the liposomalbased adjuvant system AS01, which contains the immunostimulants monophosphoryl lipid A and QS21, a saponin derivative extracted from the bark of the South American soap bark tree Quillaja saponaria. The development of RTS,S supports the notion that strong adjuvants are a necessary component for efficacious subunit protein malaria vaccines. RTS,S/AS01 (and its immediate forebears with oil-in-water versions of the AS0-adjuvant system) was the first malaria vaccine to demonstrate meaningful levels of clinical protection in field trials. Trials in children and infants who are naturally exposed to malaria demonstrated efficacy against clinical disease in the range of 30–56% and up to 66% against infection, and a good record of safety and tolerability [80, 103, 104]. A large Phase 2 trial in 1465 Mozambican children showed 26% efficacy against all malaria episodes over nearly 4 years, 32% against first or only episode of clinical malaria, and 38% in preventing severe clinical episodes [105]. The magnitude of the protective effect after nearly 4 years was thus modest, but importantly, there was no evidence of a postimmunization “rebound” effect—a theoretical concern that the vaccine might interfere with the natural acquisition of protective immunity. Based on these demonstrations of a level of efficacy that is well below levels of protection expected for vaccines against other common pathogens but rare, good news for malaria vaccines, RTS,S/AS01 advanced to Phase 3 testing in 15,259 children and infants in seven African countries. Efficacy against clinical malaria in the year after vaccination with three doses of RTS,S was 31% in infants 6–12 weeks of age and 56% in children 5–17 months of age [106]. Extended follow-up after a fourth booster dose administered

Malaria Vaccines

18 months after the first provided clinical efficacy of 26% in infants over a 38-month period and 36% in children over a 48-month period. An increased risk of febrile seizures in the week after vaccination was documented in children who received RTS,S as compared with the control group, but this effect was transient and without long-term sequelae. The cost-effectiveness of licensing and deploying a malaria vaccine with efficacy in the range of 25–50% is debated, but where the malaria burden remains high, as in much of subSaharan Africa, such a vaccine is likely to be sought and used. After their review of the Phase 3 results, the World Health Organization Strategic Advisory Group of Experts on immunization and the Malaria Policy Advisory Group recommended pilot implementation studies to assess the feasibility of administering four doses of RTS,S to children 5–17 months of age, and to assess the overall impact on child mortality. These studies are ongoing and are expected to provide guidance for implementation of routine RTS,S vaccination in endemic areas. Strategies being investigated to improve the efficacy of RTS,S/AS01 include a delayed and fractional third dose booster schedule that showed 87% efficacy in malaria-naïve adults who underwent malaria challenge [107], and a candidate called R21 that uses the same CSP protein as RTS,S but is expressed alone in yeast to form particles, increasing the ratio of CSP to hepatitis B surface antigen S [108]. Highly encouraging first results of R21 testing in children living in a malaria-endemic area showed 77% efficacy over 1 year [31], and efficacy testing will continue through a second malaria transmission season in late 2021. These early findings suggest that a next-generation RTS,S vaccine that provides a high level of protection is achievable. Several other preerythrocytic vaccine candidates have progressed through various stages of preclinical and clinical development [75], including recombinant subunit protein vaccines as well as DNA vaccines and viral vectored vaccines [106]. Even though some of these have generated seemingly good humoral and especially cellular immune responses when formulated with strong adjuvants [109], protective efficacy has not been

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achieved in clinical testing in humans. Primeboost approaches using DNA vaccines or viral vectors have also resulted in improved immunogenicity including cell-mediated responses in some participants and, in some cases, in measurable delays in time to infection in experimental sporozoite challenge trials [110]. Although primeboost vaccine strategies have failed to demonstrate meaningful protection in published clinical efficacy trials, one report is more promising, with 27% sterile protection provided by DNA prime and viral vector boost using both CSP and the blood stage antigen AMA1 [111]. Efforts to improve these approaches to get more consistent cellular immune responses and higher levels of protection may be hampered by variability in host responses to vaccination. Other novel approaches such as a virus-like particle encoding CSP using a duck hepatitis B virus as scaffold are showing promise in preclinical testing [112]. A monoclonal antibody CIS43 targeting a CSP junctional epitope was recently isolated and found to be highly effective for passive protection [113]. This epitope was combined to a second epitope derived from another efficacious monoclonal antibody L9 [114] that targets a separate CSP epitope as the basis of a candidate preerythrocytic vaccine, also showing promising results in early preclinical testing [115]. Mining of genomic and proteomic data continues to identify additional new preerythrocytic vaccine candidate proteins, some of which have shown promising results in early preclinical testing in animal models [116].

Blood Stage Vaccines Most blood stage malaria vaccine candidates are based on antigens that coat the surface of the invasive merozoites and/or that are involved with the process of erythrocyte invasion, in hopes of generating antibodies that block invasion and curtail parasite replication in the blood, reducing the risk or severity of clinical illness. The merozoite surface protein 1, or MSP1, was the first and best characterized of many proteins on the merozoite surface that are being targeted for

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vaccine development. MSP1 undergoes cleavage into four fragments that remain on the merozoite surface as a complex. Before erythrocyte invasion, the entire MSP1 complex is shed except for the C-terminal 19-kDa fragment (MSP119), which remains on the surface as the merozoite enters the erythrocyte [117]. Naturally acquired antibodies to MSP119 inhibit erythrocyte invasion and are associated with protection from clinical malaria in field studies [118, 119], supporting its potential as a vaccine candidate. Studies of recombinant MSP1 vaccines in monkeys were encouraging [120]. An MSP119-based vaccine on the same adjuvant platform as RTS,S produced antibodies in Malian adults that recognized MSP1 from diverse strains of P. falciparum [83], but had no protective efficacy against clinical malaria in Kenyan children [86]. Comparison of the degree of homology with the vaccine strain of MSP1 sequences in the infections experienced by children in the vaccine and control groups will clarify the extent to which the genetic diversity of MSP1 accounted for this lack of efficacy. The apical membrane antigen 1, or AMA1, resides in the apical complex of the merozoite [94] before being processed and moving to the surface as the merozoite is released from the infected erythrocyte [121], where it is thought to play a role in erythrocyte invasion [122]. Proteomic studies have shown that AMA1 is also expressed in the sporozoite stage [123], suggesting that it may play a similar role in hepatocyte invasion. People living in malariaendemic areas produce antibodies to AMA1 that can inhibit erythrocyte invasion in vitro [124] and that are associated with protection in field studies [125]. Studies in animal models show strain specificity in the inhibitory activity of anti-AMA1 antibodies [124], and these results have been corroborated by subsequent allelic exchange experiments [126, 127]. Sequencing of the gene encoding AMA1 in samples from a single Malian village identified more than 200 unique AMA1 variants in about 500 P. falciparum infections [79], raising the daunting prospect that a 200-valent AMA1 vaccine might be required to achieve broad protective efficacy. However, molecular epidemiological

Malaria Vaccines

analyses showed that a group of just eight polymorphic amino acids lying adjacent to the presumed erythrocyte-binding site on AMA1 were responsible for strain-specific naturally acquired immunity, suggesting that a vaccine comprised of as few as ten “serotypes” of AMA1 might be sufficient to protect against 80% of unique variants. Two AMA1 vaccines were the first to reach the stage of efficacy trials in humans. A bivalent vaccine with two different forms of AMA1 adjuvanted with aluminum hydroxide failed to provide any protection against parasitemia or clinical malaria [87], and molecular analyses of preand postimmunization infections turned up no evidence of strain-specific efficacy or selection of nonvaccine variants [128]. A monovalent AMA1 vaccine formulated with the same adjuvant system as that used with RTS,S was more highly immunogenic [89] in a similar population of African children. Although this vaccine did not prevent infection after experimental sporozoite challenge [129] and showed marginal overall efficacy against clinical malaria, it demonstrated strong strain-specific efficacy, reducing the risk of clinical malaria caused by parasites with AMA1 homologous to the vaccine strain by more than 60% [90]. This encouraging result suggests that it may be possible to develop a more broadly efficacious multivalent or chimeric nextgeneration AMA1 vaccine, and efforts are being made to do this [130–133]. Another blood stage vaccine candidate, reticulocyte-binding protein homolog 5 (RH5), is part of the parasite’s red blood cell invasion machinery. Unlike AMA1, RH5 lacks vaccine strain–limited genetic diversity as evidenced by genomic studies and immunology experiments that support cross-protection against heterologous strains [134]. Initial testing in humans demonstrated safety and confirmed its cross-protective capacity against eight different laboratory strains [135]. RH5 is one of a three-part red blood cell invasion complex whose individual components are being advanced as a three-antigen blood stage vaccine whose components may act synergistically [136]. Proteomic screening has led to discovery of another candidate blood stage antigen

Malaria Vaccines

that is required for infected red blood cells to release progeny, called P. falciparum schizont egress antigen 1 (PfSEA-1) [137]. Tanzanian infants with high anti-PfSEA-1 antibody experienced 51% fewer cases of severe malaria when compared to those with low antibody levels [138]. PfSEA-1 vaccines have not yet been tested in humans, but hold promise as either a standalone vaccine against severe malaria, or as a component of a multi-antigen vaccine. The P. falciparum proteins that are expressed on the surface of infected erythrocytes and that mediate cytoadherence and immune evasion and contribute to pathogenesis would seem to be attractive candidates for anti-disease vaccines. These PfEMP1 proteins are encoded by a large family of diverse var genes [14] with up to 60 variants in each parasite genome. Designing a vaccine that would be broadly protective against such an extraordinarily polymorphic target is likely to be very difficult. One possible approach to overcome this difficulty may be the identification of conserved epitopes that are nevertheless immunogenic [136, 139]. Because a single PfEMP1 that is somewhat less polymorphic, VAR2CSA, mediates cytoadherence in placental malaria, prospects for a PfEMP1-based pregnancy-associated malaria vaccine may be better, and research toward this goal is underway, as described in the following section. Multistage, multi-antigen vaccines that include blood stage components are discussed in a subsequent section.

Placental Malaria Vaccines Placental malaria occurs when the PfEMP1 variant VAR2CSA binds to the host receptor chondroitin sulfate that is unique to placental tissue [23]. A vaccine that targets VAR2CSA could potentially block this syndrome, as supported by field studies that demonstrate lower risk of low birth weight associated with high maternal titers of anti-VAR2CSA antibody [140]. Studies of primigravidae versus multigravida women demonstrate the ability to acquire immunity to VAR2CSA with successive pregnancies [141].

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Vaccine development efforts for placental malaria to date have been focused on VAR2CSA. Two VAR2CSA candidate vaccines containing different sequences from distinct antigenic regions have advanced to clinical trials in malaria-exposed and malaria-naïve populations and could potentially be combined in a single vaccine [142]. Results from these first-in-human studies suggest that these candidate vaccines can induce an immune response against VAR2CSA similar to the vaccine strain but have little or no activity against variant strains [143, 144]. Current efforts to improve on these groundbreaking clinical trials to improve cross-protection against heterologous strains are ongoing. Ultimately, an effective PM vaccine may require multiple antigens that target the most highly conserved regions of VAR2CSA [145].

Transmission-Blocking Vaccines Transmission-blocking vaccines are vaccines that are specifically intended to block transmission by targeting molecules that are unique to gametocytes, the male and female forms that are taken up during a blood meal and that mate in the mosquito midgut, or that target subsequent mosquito stages. Antibodies directed against such targets are capable of blocking the development of mosquito stages, thus interrupting transmission [146]. In a rare example of a vaccine designed to target multiple species, a vaccine based on mosquito-stage proteins in both P. falciparum (Pfs25) and P. vivax (Pvs25) was shown to produce dose-dependent antibodymediated transmission-blocking activity [147]. However, the vaccine, which was formulated with the powerful adjuvant Montanide ISA 51, was unacceptably reactogenic. Subsequent formulations of the P. falciparum component lacked efficacy and/or immunogenicity [148, 149]. Another conserved antigen that could serve as a vaccine to prevent transmission of both P. falciparum and P. vivax is the Anopheline alanyl aminopeptidase N (AnAPN1) [150]. Highly immunogenic epitopes of this protein have been identified and manufactured and show potent transmissionblocking activity in mice [151]. Other candidate

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transmission-blocking vaccines include Pfs48/45, a membrane-bound protein on the surface of gametocytes that requires conformation-specific folding achieved with either a Lactococcus [152] or baculovirus expression system [153]. A separate candidate is Pfs230, another gamete surface protein that recently demonstrated greater ability to block transmission greater than Pfs25 alone or in combination with Pfs25 [154]. Another candidate Pfs47 holds significant promise as it contains a domain that is both highly conserved and able to generate antibodies that block malaria transmission in mice [155]. Subsequent characterization of the Pfs47 protein revealed that it facilitates parasite survival by shielding gametocytes from the mosquito immune system [156]. With the renewed call for global malaria eradication [5, 35], transmissionblocking vaccines are being increasingly emphasized. In one novel and promising approach, vaccines that target mosquito molecules are being contemplated in hopes of avoiding selection pressure within the host that favors “vaccine-resistant” parasites [157]. Although not traditionally thought of as transmission-blocking vaccines, highly efficacious preerythrocytic vaccines that provide sterile immunity would also interrupt transmission. Because they would completely prevent infection, such vaccines would also have the important added benefit of preventing disease caused by the blood stages. In the context of malaria elimination, a highly efficacious preerythrocytic vaccine would thus be the product development target for “vaccines that interrupt transmission” [11].

Multistage, Multi-Antigen Vaccines Compared to the viral and bacterial human pathogens for which effective vaccines exist, malaria parasites are big and complex and elicit equally complex and multifaceted immune responses. In retrospect, it may not be surprising that so many candidate vaccines that target just a single variant of a single antigen have failed to demonstrate clinical efficacy, especially against heterologous natural challenge. Several attempts have been made to improve on the efficacy of single-antigen vaccines by developing multistage, multi-antigen vaccines.

Malaria Vaccines

One of the earliest was SPf66, a synthetic vaccine consisting of peptides derived from the blood stage antigen MSP1 linked by the central repeat of the preerythrocytic antigen CSP. While reports of efficacy in initial trials in South America generated great excitement [158], subsequent studies in Africa and Asia showed no significant protective efficacy [159–161]. In another approach that yielded disappointing results, vaccinia virus was used as a vector to express seven P. falciparum genes, but both immunogenicity and efficacy were limited [117, 162]. Attempts to develop DNA vaccines with from two to as many as 15 P. falciparum genes [163] were likewise unsuccessful. One candidate known as GMZ2 is a fusion of two blood stage antigens that elicited high immunogenicity when tested in malaria-exposed adults and children [164, 165], though subsequent testing in African children 1–5 years of age showed 14% efficacy [166]. A next-generation vaccine that generates higher immunity may lead to better protection. A separate multi-antigen vaccine is the multiple epitope (ME) thrombospondin-related adhesion protein (TRAP), consisting of fused B cell, CD4 and CD8 T cell epitopes of P. falciparum liver stage antigens. Although ME-TRAP did not demonstrate consistent efficacy in field trials in endemic areas [167, 168], modifications of vector delivery and vaccine regimen are being explored [169]. Based on the modest efficacy of RTS,S against clinical malaria and evidence that a blood stage vaccine using a similar adjuvant system can produce strain-specific efficacy [90], it is reasonable to believe that it may be possible to construct a multistage, multi-antigen recombinant protein that improves on the efficacy of RTS,S [170]. However, it seems not unlikely that vaccines that target 2, 5, or even 15 of the 5000 gene products will still fall short of the high levels of protection seen with radiation-attenuated whole-organism vaccines when delivered through the bites of infected mosquitoes [71].

Whole-Organism Vaccines Even though live attenuated vaccines were the earliest and remain some of the best vaccines

Malaria Vaccines

against other pathogens, and even though birds and monkeys had been protected by live attenuated malaria parasites in the earliest vaccine studies [60, 61], the protection seen with irradiated sporozoites in the early 1970s [69] was interpreted not as a direct path to a malaria vaccine but as proof that a vaccine was possible and as justification for the ensuing decades of research aimed at identifying the “right” vaccine antigen or heterologous expression system. The limited success of these Sisyphean research efforts led to reevaluation of the dogma that it would be impossible to manufacture an attenuated sporozoite vaccine in mosquitoes [71]. A radiation-attenuated, metabolically active, nonreplicating sporozoite vaccine has been manufactured in and purified from aseptically raised mosquitoes [67] and was evaluated for safety and efficacy in experimental sporozoite challenge trials in humans. The goal was to administer by needle essentially the same immunogen—albeit aseptic, purified, and cryopreserved—that had previously demonstrated 90% protective efficacy in the form of sterilizing immunity when delivered by the bites of at least 1000 irradiated infected mosquitoes. When administered by intradermal or subcutaneous routes, the sporozoite vaccine did not have significant protective efficacy [171]. Contemporaneous studies showed that the vaccine sporozoites were highly immunogenic in monkeys when administered intravenously but not subcutaneously [171]. The likeliest explanation for this result was thought to be that sporozoites delivered into the skin by needle injection in a comparatively large volume of fluid were unable to reach the liver with efficiency approaching that of sporozoites injected either by a mosquito probing for small blood vessels or intravenously. Subsequent trials found that intravenous administration of the whole-organism sporozoite vaccine in humans achieved unprecedented 100% efficacy against malaria challenge [172]. Subsequent studies in malaria-naïve individuals have documented durable heterologous protection [173–175]. A field trial in Mali demonstrated 52% efficacy in adults [176]. Increased dosing resulted not in higher but in lower efficacy in Tanzanian adults [177]. A study in Kenyan infants and children did not demonstrate vaccine efficacy [178]. Efforts to optimize dose and regimen for adults and children are ongoing.

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Another whole-organism approach pairs administration of live, nonattenuated sporozoites that can cause clinical malaria, conjointly with a curative dose of an antimalarial drug that acts exclusively on the parasite’s blood stage, typically chloroquine. As the live sporozoites advance through replication cycles in the liver, they express a multitude of antigens to generate local liver immunity. Resulting blood stage parasites are then killed by the antimalarial drug when they reach the erythrocytic stage. The approach was pioneered with mosquito bite delivery of sporozoites, resulting in both short- and longterm protection against malaria challenge [179, 180]. Later studies using cryopreserved sporozoites injected intravenously achieved similarly high levels of protection against challenge with homologous parasites [181], and modest protection against heterologous challenge [182]. Subsequent studies of this approach in Tanzania demonstrated 55% efficacy against homologous malaria challenge, compared to 27% efficacy of the intravenously administered attenuated sporozoite vaccine in the same population, raising the intriguing possibility that constituents of mosquito saliva boost the immune response to sporozoites [183]. Optimization efforts for this approach are ongoing. Genetically attenuated P. falciparum sporozoites that carry gene deletions to arrest parasite growth in hepatocytes have shown promise in preclinical testing and early clinical trials. The first human study of this approach using a vaccine delivered by mosquito bite lacked two genes and led to breakthrough clinical malaria infection [184]. A next-generation vaccine lacking three genes was then tested, demonstrating no breakthrough infections [185], and has advanced to testing with malaria challenge. A separate genetically attenuated sporozoite vaccine lacking two different genes was tested in the Netherlands, showing limited efficacy [186]. Next steps for genetically attenuated vaccines include knockout of genes expressed later in the parasite’s liver stage so that a maximum of antigens are expressed to stimulate host immunity. Table 1 summarizes the different approaches to malaria vaccine development described in this chapter.

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Malaria Vaccines

Malaria Vaccines, Table 1 Approaches to malaria vaccine development Preerythrocytic vaccines CSP based LSA1 based DNA based Viral vectored Virus-like particle Bloodstage vaccines MSP1 AMA1 RH5 PfSEA-1 PfEMP-1 Placental malaria vaccines VAR2CSA Transmissionblocking vaccines Pfs25 and Pvs25 AnAPN1 Pfs48/45 Pfs230 Pfs47 Multistage multiantigen Spf66 GMZ2 Whole-organism vaccines PfSPZ vaccine PfSPZ-CVac

GAPKO

Description

References

Based on the circumsporozoite protein that coats sporozoites; RTS,S and R21 are examples Liver stage antigen 1, a parasite protein expressed by liver stage parasites Introduces DNA encoding parasite antigen that is produced by human cells Virus encoding parasite antigen infects cells that then produce the antigen Displays multiple copies of antigen on virus particle surface

[80, 95, 96, 98– 108, 115, 187] [109] [111]

Merozoite surface protein 1, expressed on the surface of merozoites Apical membrane antigen 1 plays a role in red blood cell invasion

[83, 86, 117–120] [79, 87, 89, 90, 121–133] [134–136]

Reticulocyte-binding protein homolog 5 (RH5), part of the parasite’s red blood cell invasion machinery P. Falciparum schizont egress antigen, necessary for infected red blood cells to releasing progeny P. Falciparum erythrocyte membrane protein-1

[110, 111] [112]

[137, 138] [14, 136, 139].

PfEMP-1 variant that binds to host receptor chondroitin sulfate A that is unique to placental tissue

[142–145]

Mosquito-stage proteins Anopheline alanyl aminopeptidase N, another mosquito-stage protein Membrane-bound protein on the surface of gametocytes Gametocyte surface protein Gametocyte surface protein

[147–149] [150, 151] [152, 153] [154] [155, 156]

Synthetic peptide vaccine consisting of peptides derived from the blood stage antigen MSP1 and the preerythrocytic antigen CSP Fusion of two blood stage antigens

[158–162]

Radiation-attenuated, metabolically active, nonreplicating sporozoite vaccine Paired administration of live, nonattenuated sporozoites, conjointly with a curative antimalarial drug that acts exclusively on the parasite’s blood stage Genetically attenuated knockout P. falciparum sporozoites

Future Directions While nearly 50 of the 87 countries with malaria transmission are actively trying to eliminate malaria, it is generally agreed that global malaria eradication is not possible without either global

[164–169]

[172–178] [179–183]

[184–186]

economic development to the levels that permitted malaria elimination in the USA, Europe, and the former Soviet Union or new tools such as a highly efficacious malaria vaccine that interrupts transmission [11]. The powerfully adjuvanted preerythrocytic single-antigen recombinant protein

Malaria Vaccines

vaccine, RTS,S/AS01, significantly reduces the clinical burden of malaria in African children [106], but it does not prevent infection, and, somewhat surprisingly, its effect on transmission, if any, has not been reported. While the results of a field trial of a similarly adjuvanted blood stage vaccine [104] as well as early studies using different constructs to present the CSP antigen [108, 112] provide a rationale and potential pathways for pursuing improvements on RTS,S and similar vaccines, the dire public health need for a highly efficacious malaria vaccine calls for more than incremental improvements. Whole-parasite vaccines may be the radically different new (and yet very old) approach that is needed. Challenges remain to produce and deliver such a vaccine, but none that are insurmountable. Research from the 1940s and many more recent studies point to the importance of strong adjuvants for subunit as well as for wholeorganism vaccines, and wide access to immunogenic and safe adjuvants will be important for accelerating malaria vaccine development. Researchers are focusing now on the painfully elusive goal of achieving a high degree of efficacy, but the ideal vaccine would be not only safe and efficacious but also thermostable and protective for a long period of time after a single immunization—characteristics that will not be easy to achieve but which might be possible through pharmaceutical technologies such as controlled release formulations or new platforms including self-amplifying RNA delivery technology resembling the highly successful mRNA-based vaccines against COVID-19. A highly efficacious malaria vaccine would be a significant addition to current tools used for malaria control and eradication. These additional tools are listed in Table 2 together with selected references as a starting point for further reading. Preliminary results of the RTS,S pilot implementation studies are expected in late 2021, at which time a policy recommendation on broader use is expected from the World Health Organization. Introduction into routine childhood vaccination schedules by individual countries may follow. This will be a magnificent accomplishment that will mark not the end of the road for

139 Malaria Vaccines, Table 2 Additional tools for malaria control and eradication Tool Diagnostics

Drug development

Intermittent preventive or presumptive treatment for malaria in infants, children, and pregnant women

References 1. Mbanefo A, Kumar N (2020) Evaluation of malaria diagnostic methods as key for successful control and elimination programs. Trop Med Infect Dis 5(2):102 2. Zainabadi K (2021) Ultrasensitive diagnostics for low-density asymptomatic Plasmodium falciparum infections in low-transmission settings. J Clin Microbiol 59(4): e01508–20 1. Antonova-Koch Y, Meister S, Abraham M, Luth MR, Ottilie S, Lukens AK, Sakata-Kato T, Vanaerschot M, et al. (2018) Open-source discovery of chemical leads for nextgeneration chemoprotective antimalarials. Science 362(6419): eaat9446 2. Malaria Drug Accelerator Consortium (2021) MalDA, accelerating malaria drug discovery. Trends Parasitol S1472–4922(21) 1. Gutman J, Kovacs S, Dorsey G, Stergachis A, Ter Kuile FO (2017) Safety, tolerability, and efficacy of repeated doses of dihydroartemisinin-piperaquine for prevention and treatment of malaria: a systematic review and meta-analysis. Lancet Infect Dis 17(2):184–193 2. Desai M, Hill J, Fernandes S, Walker P, Pell C, Gutman J, Kayentao K, Gonzalez R, Webster J, Greenwood B, Cot M, Ter Kuile FO (2018) Prevention of malaria in pregnancy. Lancet Infect Dis 18(4):e119-e132 3. Saito M, Briand V, Min AM, McGready R (2020) Deleterious effects of malaria in pregnancy on the developing fetus: a review on prevention and treatment with antimalarial drugs. Lancet Child Adolesc Health 4(10):761–774 4. Ashley EA, Poespoprodjo JR (2020) Treatment and prevention of malaria in children. Lancet Child Adolesc Health 4(10): 775–789 (continued)

140

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Malaria Vaccines, Table 2 (continued) Tool Vector control

References 1. Killeen GF (2020) Control of malaria vectors and management of insecticide resistance through universal coverage with nextgeneration insecticide-treated nets. Lancet 395(10233): 1394–1400 2. Sougoufara S, Ottih EC, Tripet F (2020) The need for new vector control approaches targeting outdoor biting Anopheline malaria vector communities. Parasit Vectors 13(1):295 3. Benelli G, Beier JC (2017) Current vector control challenges in the fight against malaria. Acta Trop 174:91–96

malaria vaccine development but a critical milestone on the path leading toward the malaria vaccine that the world needs. Acknowledgments CVP’s malaria vaccine work has been supported by the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health, the US Department of Defense, the Doris Duke Charitable Foundation, and by the Howard Hughes Medical Institute. MBL’s malaria vaccine work has been supported by the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health, the U.S. Department of Defense, the Howard Hughes Medical Institute, the Maryland Industrial Partnerships Program, and the Bill and Melinda Gates Foundation. The authors acknowledge the US Agency for International Development, the Walter Reed Army Institute of Research, GlaxoSmithKline Biologicals, Sanaria Inc., the US Military Malaria Vaccine Program—Naval Medical Research Center, the Malaria Research and Training Center of the University of Bamako, Mali, and the Center National de Recherche et de Formation sur le Paludisme and the Groupe de Recherche Action en Santé, both of Ouagadougou, Burkina Faso, for collaboration on malaria vaccine trials.

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Saverino E, Fink M, Cosi G, Gondwe L, Studer F, Styers D, Seder RA, Schindler T, Billingsley PF, Daubenberger C, Sim BKL, Tanner M, Richie TL, Abdulla S, Hoffman SL (2019) Increase of dose associated with decrease in protection against controlled human malaria infection by PfSPZ vaccine in Tanzanian adults. Clin Infect Dis. https://doi.org/10. 1093/cid/ciz1152 Steinhardt LC, Richie TL, Yego R, Akach D, Hamel MJ, Gutman JR, Wiegand RE, Nzuu EL, Dungani A, Kc N, Murshedkar T, Church LWP, Sim BKL, Billingsley PF, James ER, Abebe Y, Kariuki S, Samuels AM, Otieno K, Sang T, Kachur SP, Styers D, Schlessman K, Abarbanell G, Hoffman SL, Seder RA, Oneko M (2019) Safety, tolerability, and immunogenicity of PfSPZ vaccine administered by direct venous inoculation to infants and young children: findings from an age de-escalation, dose-escalation double-blinded randomized, controlled study in western Kenya. Clin Infect Dis. https://doi.org/10. 1093/cid/ciz925 Roestenberg M, Teirlinck AC, McCall MB, Teelen K, Makamdop KN, Wiersma J, Arens T, Beckers P, van Gemert G, van de Vegte-Bolmer M, van der Ven AJ, Luty AJ, Hermsen CC, Sauerwein RW (2011) Long-term protection against malaria after experimental sporozoite inoculation: an openlabel follow-up study. Lancet 377(9779):1770–1776. https://doi.org/10.1016/S0140-6736(11)60360-7 Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJ, van Gemert GJ, van de Vegte-Bolmer M, van Schaijk B, Teelen K, Arens T, Spaarman L, de Mast Q, Roeffen W, Snounou G, Renia L, van der Ven A, Hermsen CC, Sauerwein R (2009) Protection against a malaria challenge by sporozoite inoculation. N Engl J Med 361(5):468–477. https://doi.org/ 10.1056/NEJMoa0805832 Mordmuller B, Surat G, Lagler H, Chakravarty S, Ishizuka AS, Lalremruata A, Gmeiner M, Campo JJ, Esen M, Ruben AJ, Held J, Calle CL, Mengue JB, Gebru T, Ibanez J, Sulyok M, James ER, Billingsley PF, Natasha KC, Manoj A, Murshedkar T, Gunasekera A, Eappen AG, Li T, Stafford RE, Li M, Felgner PL, Seder RA, Richie TL, Sim BK, Hoffman SL, Kremsner PG (2017) Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 542(7642):445–449. https://doi.org/ 10.1038/nature21060 Walk J, Reuling IJ, Behet MC, Meerstein-Kessel L, Graumans W, van Gemert GJ, Siebelink-Stoter R, van de Vegte-Bolmer M, Janssen T, Teelen K, de Wilt JHW, de Mast Q, van der Ven AJ, Diez Benavente E, Campino S, Clark TG, Huynen MA, Hermsen CC, Bijker EM, Scholzen A, Sauerwein RW (2017) Modest heterologous protection after plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial. BMC Med 15(1):168. https://doi.org/10.1186/ s12916-017-0923-4

Malaria Vaccines 183. Jongo SA, Urbano V, Church LWP, Olotu A, Manock SR, Schindler T, Mtoro A, Kc N, Hamad A, Nyakarungu E, Mpina M, Deal A, Bijeri JR, Ondo Mangue ME, Ntutumu Pasialo BE, Nguema GN, Owono SN, Rivas MR, Chemba M, Kassim KR, James ER, Stabler TC, Abebe Y, Saverino E, Sax J, Hosch S, Tumbo AM, Gondwe L, Segura JL, Falla CC, Phiri WP, Hergott DEB, Garcia GA, Schwabe C, Maas CD, Murshedkar T, Billingsley PF, Tanner M, Ayekaba MO, Sim BKL, Daubenberger C, Richie TL, Abdulla S, Hoffman SL (2021) Immunogenicity and protective efficacy of radiation-attenuated and chemo-attenuated PfSPZ vaccines in Equatoguinean adults. Am J Trop Med Hyg. 104(1):283–293. https://doi.org/10.4269/ajtmh.20-0435 184. Spring M, Murphy J, Nielsen R, Dowler M, Bennett JW, Zarling S, Williams J, de la Vega P, Ware L, Komisar J, Polhemus M, Richie TL, Epstein J, Tamminga C, Chuang I, Richie N, O'Neil M, Heppner DG, Healer J, O'Neill M, Smithers H, Finney OC, Mikolajczak SA, Wang R, Cowman A, Ockenhouse C, Krzych U, Kappe SH (2013) First-inhuman evaluation of genetically attenuated plasmodium falciparum sporozoites administered by bite of anopheles mosquitoes to adult volunteers. Vaccine 31(43):4975–4983. https://doi.org/10.1016/j.vaccine.2013.08.007 185. Kublin JG, Mikolajczak SA, Sack BK, Fishbaugher ME, Seilie A, Shelton L, VonGoedert T, Firat M, Magee S, Fritzen E, Betz W, Kain HS, Dankwa DA, Steel RW, Vaughan AM, Noah Sather D, Murphy SC, Kappe SH (2017) Complete attenuation of genetically engineered plasmodium falciparum sporozoites in human subjects. Sci Transl Med 9(371). https://doi.org/10.1126/scitranslmed. aad9099 186. Roestenberg M, Walk J, van der Boor SC, Langenberg MCC, Hoogerwerf MA, Janse JJ, Manurung M, Yap XZ, Garcia AF, Koopman JPR, Meij P, Wessels E, Teelen K, van Waardenburg YM, van de Vegte-Bolmer M, van Gemert GJ, Visser LG, van der Ven A, de Mast Q, Natasha KC, Abebe Y, Murshedkar T, Billingsley PF, Richie TL, Sim BKL, Janse CJ, Hoffman SL, Khan SM, Sauerwein RW (2020) A double-blind, placebo-controlled phase 1/2a trial of the genetically attenuated malaria vaccine PfSPZ-GA1. Sci Transl Med 12(544). https:// doi.org/10.1126/scitranslmed.aaz5629 187. Datoo MSMNHS A, Traoré O, Rouamba T, Bellamy D, Yameogo P, Valia D, Tegneri M, Ouedraogo F, Soma R, Sawadogo S, Sorgho F, Derra K, Rouamba E, Orindi B, Ramos-Lopez F, Flaxman A, Cappuccini F, Kailath R, Elias S, Mukhopadhyay E, Noe A, Cairns M, Lawrie A, Roberts R, Valéa I, Sorgho H, Williams N, Glenn G, Fries L, Reimer J, Ewer KJ, Shaligram U, Hill AVS, Tinto H (2021) High efficacy of a low dose candidate Malaria vaccine, R21 in 1 adjuvant matrixM™, with seasonal administration to children in

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Books and Reviews Beeson JG, Kurtovic L, Dobaño C, Opi DH, Chan JA, Feng G, Good MF, Reiling L, Boyle MJ (2019) Challenges and strategies for developing efficacious and long-lasting malaria vaccines. Sci Transl Med 11(474):eaau1458 Birkett AJ, Moorthy VS, Loucq C, Chitnis CE, Kaslow DC (2013) Malaria vaccine R&D in the decade of vaccines: breakthroughs, challenges and opportunities. Vaccine 18(31 Suppl 2):B233–B243 Bruder JT, Angov E, Limbach KJ, Richie TL (2010) Molecular vaccines for malaria. Hum Vaccin 6:54–77 Desowitz RS (1991) The malaria capers: more tales of parasites and people, research and reality. Norton, New York Dinglasan RR, Jacobs-Lorena M (2008) Flipping the paradigm on malaria transmission-blocking vaccines. Trends Parasitol 24:364–370 Draper SJ, Angov E, Horii T, Miller LH, Srinivasan P, Theisen M, Biswas S (2015) Recent advances in recombinant protein-based malaria vaccines. Vaccine 33(52):7433–7443 Duffy PE, Sahu T, Akue A, Milman N, Anderson C (2012) Pre-erythrocytic malaria vaccines: identifying the targets. Expert Rev Vaccines 11(10):1261–1280 Duffy PE, Gorres JP (2020) Malaria vaccines since 2000: progress, priorities, products. NPJ Vaccines 5(1):48 Heppner DG (2013) The malaria vaccine–status quo 2013. Travel Med Infect Dis 11(1):2–7 Good MF, Stanisic DI (2020) Whole parasite vaccines for the asexual blood stages of plasmodium. Immunol Rev 293(1):270–282 Hill AV, Reyes-Sandoval A, O'Hara G, Ewer K, Lawrie A, Goodman A, Nicosia A, Folgori A, Colloca S, Cortese R, Gilbert SC, Draper SJ (2010) Prime-boost vectored malaria vaccines: progress and prospects. Hum Vaccin 6:78–83 Hoffman SL, Vekemans J, Richie TL, Duffy PE (2015) The march toward malaria vaccines. Vaccine 33(Suppl 4): D13–D23 Hviid L (2010) The role of plasmodium falciparum variant surface antigens in protective immunity and vaccine development. Hum Vaccin 6:84–89 Itsara LS, Zhou Y, Do J, Grieser AM, Vaughan AM, Ghosh AK (2018) The development of whole Sporozoite vaccines for plasmodium falciparum Malaria. Front Immunol 9:2748 Langhorne J, Ndungu FM, Sponaas AM, Marsh K (2008) Immunity to malaria: more questions than answers. Nat Immunol 9:725–732 Miura K (2016) Progress and prospects for blood-stage malaria vaccines. Expert Rev Vaccines 15(6):765–781 Moorthy VS, Kieny MP (2010) Reducing empiricism in malaria vaccine design. Lancet Infect Dis 10:204–211

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Sustainable Radical Cure of the Latent Malarias J. Kevin Baird Eijkman-Oxford Clinical Research Unit, Eijkman Institute of Molecular Biology, Jakarta, Indonesia Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK

Article Outline Glossary Definition of the Subject Introduction Malaria Disease Treatment The 8-Aminoquinolines G6PD Deficiency as a Chemotherapeutics Problem Military Medicine Repurposed Tafenoquine Beneficiaries Technological Imperatives Perceptions of the Malaria Problem Future Directions Conclusions Bibliography

Glossary Patent malaria infection by active blood stages of the plasmodia responsible for acute attacks of a febrile illness detectable by microscopic or immunochromatographic testing. Sub-patent malaria infection by active blood stages of the plasmodia but in numbers too low to allow detection and, most often, without acute illness. Latent malaria infection by dormant stages of the plasmodia responsible for human malaria

that awaken to provoke renewed attacks of malaria and onward transmission. Chronic malaria patent, sub-patent, or latent malaria sustained for prolonged periods with or without repeated attacks at short intervals causing additional health problems like anemia or stunting. Primary infection inoculation of a human by infectious sporozoites via an anopheline mosquito bite. Relapse patent malaria originating from a latent infection by the plasmodia. 8-aminoquinolines the only class compounds having activity against latent hepatic stages of the plasmodia represented by plasmochin, primaquine, and tafenoquine as hypnozoitocidal therapies, the latter two in clinical use. Glucose-6-phosphate dehydrogenase (G6PD) deficiency the most common inherited human enzymopathy resulting in hemolytic sensitivity to 8-aminoquinoline therapies. Radical cure the administration of therapies the terminate both acute malaria (blood schizontocides) and latent malaria (hypnozoitocides).

Definition of the Subject Protozoan parasites in the genus Plasmodium include at least five species that naturally infect humans and provoke an acute febrile illness called malaria. Three of those species also exhibit latent phases of infection where hosts experience no illness whatsoever over periods ranging from several weeks to years or, in one of those species, sometimes lasting decades. Dormant liver stages called hypnozoites occur in the two species of lesser periods of latency and often involve multiple attacks. The treatment of those latent hepatic infections, called radical cure, imposes significant technical and clinical challenges involving therapies that are dangerously toxic in some patients having a common inherited enzyme deficiency. In the settings where almost all of these malarias

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_896 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2021 https://doi.org/10.1007/978-1-4939-2493-6_896-3

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occur – in the impoverished rural tropics – these challenges have proven practically insurmountable, resulting in poor access to radical cure. This chapter examines the technical character of those obstacles and the ways and means by which sustainable approaches to overcoming them have evaded us. Doing so may inform technical strategies for attacking latent reservoirs of infection that may otherwise frustrate aspiration for the elimination or eradication of the malarias as a significantly harmful human illness.

Introduction Technology applies scientific knowledge to practical purposes. In medicine, the cardinal purpose of technology is the prevention or reversal of physical harm to people. The revolution in biological technology that occurred through the twentieth century has profoundly improved the practice of medicine and the state of human health. Great complexity characterizes that progress, with it often occurring opportunistically and unevenly across multitudes of niches and endeavors according to abilities and needs. Societies and the technologists they nurture naturally assign priorities in managing the focus of resources and human energy across that front of progress. How a particular medical problem is perceived and managed thus defines both its urgency and the character of its technological

Sustainable Radical Cure of the Latent Malarias

solution. That character, in turn, defines practice and equity of access to its benefits. The human condition is thus unevenly elevated by technological progress. A solution to a medical problem may bypass many by inaccessibility or impracticality, and is thus less relevant to those beyond its reach. Organ transplantation, as an illustrative example, is a sophisticated technology that is not accessible by most of humanity. Its tremendous benefits are enjoyed by the relative few having access. A more equitable distribution of technological benefit defines the ideal of sustainable technologies in health. By that ideal, we strive to develop technological advances that become relevant to most of the people who may benefit from them. In the context of serious problems of tropical health, we may think of North and South disparities where access to the benefits of technological progress favors the North but need weighs more heavily on the South. This chapter explores a specific example of the difficulty of realizing the ideal of sustainable tropical health – radical cure of the latent malarias. Chemotherapeutics that solve that problem – the 8-aminoquinoline antimalarials – were discovered nearly a century ago, but even today the vast majority of people who would benefit from those treatments do not have safe access to them. Examining how that occurred across the long and eventful technological history of the 8-aminoquinolines (Fig. 1) informs not only addressing that specific

Sustainable Radical Cure of the Latent Malarias, Fig. 1 Key events in the technological history of the 8-aminoquinoline antimalarial drugs

Sustainable Radical Cure of the Latent Malarias

Sustainable Radical Cure of the Latent Malarias, Fig. 2 Global distribution of the incidence of P. vivax malaria in 2017 indicating the geographical distribution

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of need of sustainable radical cure of malaria. Taken from Battle et al. [xx] with permission of The Lancet

Sustainable Radical Cure of the Latent Malarias, Fig. 3 Life cycle of the plasmodia capable of hepatic latency and antimalarial classes. Taken from Baird [3] with permission of Clinical Microbiology Reviews

problem in a technological sense but also offers insight to the blind creation of consequential disparities in access to the benefits of technology.

Malaria Malaria infects hundreds of millions of people each year, and several hundred thousand do not survive [1]. What makes these numbers tragically extraordinary – and explicit regarding inequities in access to the benefits of technologies – is the fact that malaria is wholly preventable and

treatable with available understanding and tools. The burden of acute malaria weighs almost exclusively on residents of the impoverished rural tropics [2] (Fig. 2). We know the mechanics of this disease and its transmission in excruciatingly detailed granularity, and we possess insecticides and chemotherapies that kill the agents of malaria transmission and illness (Fig. 3). How and why this knowledge fails us is a conspicuously important question rooted in the proposition of sustainable technologies. Exploring this question and problem requires appreciation of the biological complexity of malaria.

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The plasmodia that infect humans as intermediate hosts do so through their definitive hosts, mosquito species within the genus Anopheles. Historically, transmission of malaria has occurred anywhere between the polar circles where anophelines may appear, even if only during relatively very brief periods of warmth [4]. Today we tend to think of malaria as an exclusively tropical disease, and indeed it occurs across almost the entirety of those perpetually warm latitudes. Nonetheless, endemic transmission still occurs at temperate latitudes like the Korean Peninsula, and outbreaks may occur at sites beyond the tropics where it has been absent for many decades [5, 6]. Whether in the tropics or cooler latitudes, some plasmodia are biologically equipped for variable periods of dormancy in having both proliferative disease-causing states (called patency) and clinically silent quiescent states (called latency) of infection. All of the plasmodia follow a highly complex life cycle involving multiple discreet forms in both the human and mosquito hosts (Fig. 3). If an infection-receptive species of anopheline mosquito ingests a male and a female gametocyte from human blood, sexual union and meiotic recombination of those forms occurs in the mosquito gut. The product of that union, an ookinete, penetrates the gut wall and attaches to its coelomfacing side as an oocyst. Through a process called sporogony, the oocyst generates several thousand forms infectious to humans called sporozoites which migrate to the salivary glands of the mosquito a week or two following the sexual union. These pass to another human host at blood meal, when they infect hepatocytes within minutes of inoculation. After a week or two of development called hepatic schizogony, each sporozoite yields thousands of forms called merozoites that are equipped to invade red blood cells. Each merozoite then multiplies asexually to produce one-half to two dozen merozoites (blood schizogony) through 24, 48 or 72 hr cycles associated with acute febrile illness. A minority of merozoites commit to developing into terminal (nondividing) sexual gametocytes, forms which are clinically silent and virtually dormant until ingested by a suitable mosquito.

Sustainable Radical Cure of the Latent Malarias

Hepatic development is not limited to that immediate schizogony. In some species, there are two forms of invasive sporozoites: tachysporozoites and bradysporozoites [7]. The latter develop as described, whereas the former become dormant parasites called hypnozoites (Fig. 3). Two species of plasmodia that naturally infect humans are known to form hypnozoites: Plasmodium vivax and Plasmodium ovale. Another species also forms hypnozoites in the Asian macaques it naturally infects; Plasmodium cynomolgi is an important experimental animal model for hepatic latency in human malaria. These species thus have a latent phase of infection in the liver that persists for weeks or several years before activating and provoking renewed and often multiple attacks of acute malaria and onward transmission [8, 9]. Another species exhibiting latency, Plasmodium malariae, does so by wholly unknown means and may become patent many decades after the primary infection [10]. Latency in malaria is an important reservoir of infection in humans. In areas of relatively high transmission, the majority of acute attacks of vivax malaria originate not from recent mosquito bites but from awakened hypnozoites (approx. 80%) [11, 12]. That rate is lower in areas of very low transmission [13]. The prevalence of the latent malarias cannot be known because no technology can confirm the presence of hypnozoites in the livers of outwardly healthy people. Nonetheless, those attack rate estimates accord with both a prevalence of latency in substantial excess of that observable as patent infections and the known biology of the infection (Fig. 4). For instance, although the ratios of tachysporozoites to bradysporozoites in any given inoculation of about a dozen sporozoites varies by strain-specific geographies [14, 15], each mosquito-borne primary attack is likely to seed the liver with three or more hypnozoites. In other words, a single infectious bite is likely to result in repeated attacks of acute malaria over the weeks, months, and several years that follow without further involvement of a mosquito. Models of P. vivax transmission suggest that eliminating the latent reservoir of infection would rapidly lead to its collapse [16, 17].

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Sustainable Radical Cure of the Latent Malarias, Fig. 4 The biology of latency and patency in P. vivax malaria. Taken from Baird [3] with permission of Clinical Microbiology Reviews

Disease The most common form of any of the malarias in humans is sub-patent and largely asymptomatic, as it typically occurs in areas of endemic transmission [18, 19]. Naturally acquired immunity through repeated infection dampens parasite biomass and the illness it provokes [20], although more subtle effects on health with chronic infection occur [21, 22]. Severe anemia, for example, is a dominant cause of mortality attributable to malaria in highly endemic zones [23, 24]. Repeated attacks of P. vivax malaria in an area of eastern Indonesia came with higher rates of all-cause mortality than among patients who had experienced acute P. falciparum malaria [25]. The chronically infected and asymptomatic do not seek treatment and they figure prominently in the sustained transmission of malaria in their communities [26, 27]. People in those communities who are vulnerable to acute malaria are thus exposed and become ill, sometimes seriously or fatally so. Acute malaria is a nonspecific febrile illness typically punctuated by daily paroxysms of spiking fever with drenching sweats followed by bouts of shaking chills [28]. Headache, achiness, malaise, and nausea and vomiting typically also occur. Uncomplicated acute malaria typically proves completely debilitating. Depending on the species involved and comorbidities, progression to varied states of

severe and complicated malaria may occur within hours, days, or weeks of onset of illness. Those states include neurological (seizures, coma), respiratory (acute respiratory distress), renal or hepatic (injury and dysfunction), hematological (severe anemia or thrombocytopenia), and circulatory (shock) [29]. Survival in complicated malaria may be improbable without intensive care at hospital.

Treatment Plasmodia in humans are represented by distinct forms of variable susceptibility to varied classes of antimalarial drugs. Figure 3 illustrates these classes in the context of those forms. A diagnosis of uncomplicated, acute malaria demands prompt administration of therapies aimed at arresting blood schizogony and progression to severe and complicated malaria. The blood schizontocides dominate the antimalarial armamentarium in terms of available options, most of those being the artemisinins combined with therapies like piperaquine, pyronaridine, or mefloquine (ACTs), along with therapies like atovaquone-proguanil, chloroquine, and now rarely used quinine combined with some antibiotics [30]. Prompt administration of these drugs arrests the progression of illness and recovery occurs within a few days. Intravenous artesunate

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is applied for severe and complicated malaria in order to arrest progression of the infection, but recovery often hinges on clinical management of the varied and serious complications having already arisen from it [31]. Chemotherapeutic clearing of the sexual and latent forms of malaria requires the additional administration of gametocytocidal and hypnozoitocidal classes of drugs. In both instances, only a single chemical class of compounds is available for that clinical application, the 8-aminoquinolines.

The 8-Aminoquinolines Plasmochin The 8-aminoquinolines occupy an important space in the history of medicine, representing the first rationally synthesized antimicrobial agents. The discovery of them at the Bayer laboratory at Elberfeld, Germany, in the 1920s employed methylene blue as a lead compound screened for blood schizontocidal activity against Plasmodium relictum in finches on industrial scale [30]. The 8-aminoquinoline called plasmochin proved 60-times more active than quinine in that model and came to see clinical use by 1926 for therapy of acute vivax malaria (it had poor activity against falciparum malaria). Its gametocytocidal properties were almost immediately recognized, as was its apparent activity against what was called “delayed attacks” of vivax malaria (latent stages were suspected but not confirmed until decades later). Avian malarias like P. relictum lack hypnozoites, and activity against latent forms was not the intent of discovery at Elberfeld. That activity occurring in plasmochin was wholly serendipitous. Plasmochin also quickly became known as a dangerous drug causing a threatening acute hemolytic anemia in about 10% of non-Caucasian patients in what seemed an idiosyncratic manner. Seeking to mitigate this problem, investigators augmented lower doses of plasmochin with quinine. Sinton and colleagues [32, 33] working in India optimized this approach. In so doing, they stumbled upon what is today called the radical cure of vivax malaria, i.e., the administration of

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a blood schizontocide (quinine) together with a hypnozoitocide (plasmochin) in order to clear patent and latent stages of the infection. The hemolytic toxicity problem was not solved, however, and plasmochin ultimately never saw wide use in endemic areas or significant impacts. It was appropriately viewed as too dangerous to use outside of the hospital setting [34]. Primaquine Soon after the outbreak of a broader war in the Pacific during late 1941, the Americans quickly came to understand malaria in that region as a very significant threat to their military operations [35]. The loss of the island of Java and its cinchona plantations in the East Indies (modern Indonesia) in early 1942 cut off the Allies from that supply of quinine (amounting to more than 95% of the global supply). Having developed no synthetic alternatives, the Americans appropriated German synthetic antimalarials and intellectual property, manufacturing and deploying plasmochin and atabrine (another Bayer product from the Elberfeld laboratory), for the prevention and cure of malaria in their troops [36]. Within months, however, atabrine was discovered to exacerbate the already problematic toxicity of plasmochin and in 1943, the US Surgeon General recommended against its use for radical cure [37]. As a key piece of its broader work, the wartime Board for Coordination of Malarial Studies seated at the National Science Foundation in Washington, DC, undertook a discovery effort focused on 8-aminoquinolines and radical cure [38]. Among the hundreds of 8-aminoquinolines screened for toxicity in rodents and macaques, several dozen were evaluated for safety and efficacy in clinical trials at two American prisons through the 1940s [39]. Primaquine (first synthesized in 1945 and put into humans in 1948) became registered for clinical use in radical cure in 1952. It was combined with chloroquine, yet another compound discovered at Elberfeld in the 1930s. Primaquine also came with hemolytic toxicity in some patients. As the developers of plasmochin had done, those of primaquine adopted relatively small daily doses as means of

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mitigating that toxicity problem, i.e., adult doses of 15 mg or 30 mg daily for 14 days [40]. Tafenoquine Satisfaction with the primaquine experience against P. vivax on the Korean Peninsula at war in the early 1950s gave way to dissatisfaction by the American military health authorities with their long experience in Vietnam through the 1960s. Primaquine performed poorly in preventing delayed attacks of P. vivax and its toxicity limited usefulness [41]. Another military discovery effort was undertaken to improve on primaquine and came to be dominated by 8-aminoquinolines. Toxicity screens in rats, dogs, and macaques were accompanied by screening for hepatic schizontocidal activities (not to be confused with hypnozoitocidal activity, Fig. 2) against Plasmodium berghei or Plasmodium yoelii in rodents (which lack hypnozoites). In general, hypnozoitocides also exhibit hepatic schizontocidal activity, whereas most hepatic schizontocides do not exhibit hypnozoitocidal activity. The most promising compounds advanced to hypnozoitocidal efficacy screening employing P. cynomolgi in rhesus macaques. That model, which was not available to the US Army during their 1940s screening efforts until very late [42], would allow the later discovery program the confidence to bring only a single candidate compound to clinical development in human trials. In 1992, the US Army registered the 8-aminoquinoline called WR238605 for experimental use in humans with the US Food and Drug Administration (FDA) as an investigational new drug (IND). It would later be called tafenoquine and in 2018 earn registration for clinical use in radical cure of vivax malaria and, in a separate application by another sponsor, for the chemoprevention of all malarias [43, 44]. The remarkable 26 years of continuous clinical development of tafenoquine speaks to the extraordinary complexity and difficulty of that pathway, and it merits consideration here as a technological problem. The 8-aminoquinoline screening program discovered tafenoquine in 1978, and in addition to having threefold greater therapeutic activity

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against relapse in the P. cynomogli model, tafenoquine had two extraordinary properties for an 8-aminoquinoline: (1) it was very slowly eliminated, with a plasma half-life counted in weeks (as opposed to the hours of plasmochin or primaquine elimination) and (2) it exhibited good activity against asexual forms of P. falciparum at therapeutic levels (whereas plasmochin or primaquine have almost no such activity) [45, 46]. These properties drove the pivoting of the US Army 8-aminoquinoline discovery program from the original goal of improving on primaquine in radical cure of P. vivax to one of chemoprevention of P. falciparum. The US Army progress report on tafenoquine development expressed, “P. falciparum malaria is considered the most important malaria parasite because it causes high morbidity and mortality. . . There is currently no effective weekly or monthly malaria prophylactic drug available to U.S. or coalition forces. . .” [47]. The notion of what the military developers colloquially called “fire and forget” chemoprevention (long-lasting protection from a single dose) of that malaria dominated strategic thinking as they approached clinical development during late 1980s. In reviewing the available technical reports of 8-aminoquinoline screening for efficacy against P. cynomolgi relapse in that era [45, 46], the presence of one compound, WR242511, stands out in two key respects: (1) its efficacy was more than three times greater than tafenoquine (ten-fold greater than primaquine) [45, 48] and (2) it was the sole 5-alkyloxy (hexyl) derivative of primaquine among the series of 5-phenoxy derivatives composing the late preclinical finalist compounds (Fig. 5). The 5-phenoxy substitution delivered the slow elimination character of these compounds [49]. WR242511, in contrast, had an elimination half-life (in macaques) of only about 24 hours [50]. Nodiff et al. [45] at the US Army development laboratory in 1991 expressed of WR242511, “. . .an application is being prepared for IND approval of (145).” It is not clear why that IND and trials in humans did not materialize. The strategic weighing that must have occurred reverberates today.

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Sustainable Radical Cure of the Latent Malarias, Fig. 5 The 8-aminoquinoline preclinical finalists identified by the US Army during the 1980s. Taken from Baird [3] with permission of Clinical Microbiology Reviews

After a decade of clinical development aimed at chemoprevention, the program stalled with the finding of ophthalmic vortex keratopathy among most subjects (deployed Australian soldiers) taking weekly tafenoquine for 6 months [51]. Years would pass before that effect would be affirmed as transient and inconsequential [52]. The US Army and their commercial development partner, GlaxoSmithKline (GSK, UK) also faced ethical challenges in conducting the required phase 3 trials in subjects living in endemic areas [53]. Faced with these formidable obstacles of toxicity and ethics in chemoprevention (later overcome), the developers turned to the less challenging indication for radical cure, and in 2008 entered into partnership with the Medicines for Malaria Venture (MMV, Switzerland) to pursue it. The slow elimination character of tafenoquine that had driven its selection for chemoprevention evolved into the advantage of single dose radical cure (versus the 14 daily dosing regimens of plasmochin or primaquine). In 2018, 40 years after the discovery of tafenoquine, GSK successfully applied to the US FDA to register that indication [43].

G6PD Deficiency as a Chemotherapeutics Problem Today we understand glucose-6-phosphate dehydrogenase (G6PD) deficiency to be the most diverse and common inherited enzymopathy of humans [54]. Many dozens of distinct single nucleotide polymorphisms occur all along this gene located on the X-chromosome, endowing it with the great complexities of both intrinsic diversity and sexlinked inheritance. It affects over 400 million people and is most common in malaria endemic nations where it occurs at an average prevalence of 8% [55, 56] (Fig. 6). G6PD catalyzes the ratelimiting reaction of the hexose monophosphate shunt, which in the red blood cell is the sole means of generating reducing equivalents that neutralize oxidative stresses like those created by introduced compounds [57] (Fig. 7). People with the most common variants of G6PD deficiency live wholly normal and healthy lives unless challenged with specific drugs, foods, or infections that provoke sometimes serious acute hemolytic anemia [58].

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Sustainable Radical Cure of the Latent Malarias, Fig. 6 Global prevalence of G6PD deficiency in malaria-endemic countries indicating the geographic

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distribution of risk of hemolytic toxicity with 8-aminoquinoline therapies against latent malaria. Published by Howes et al. [52] under Creative Commons

Sustainable Radical Cure of the Latent Malarias, Fig. 7 G6PD catalysis linked to the generation of reducing equivalents that maintain a healthy reducing cytosol in red blood cells (RBC) via glutathione sulfydryl (GS) redox equilibrium. Impaired G6PD activity is the core metabolic lesion in acute hemolytic anemia induced by 8-aminoquinoline antimalarials

Plasmochin and primaquine at therapeutic doses provoked life-threatening acute hemolytic anemia in a minority of patients [59, 60]. It was the work of the developers of primaquine in American prisons that revealed an inherited deficiency in the enzymatic activity of glucose-6phosphate dehydrogenase (G6PD) in red blood cells as the basis of this toxicity problem [61]. That discovery came in 1956, 4 years after the registration of primaquine. The technological understanding needed to inform rational selection of less hemolytically toxic compounds was absent during the development of primaquine. Those developers were nonetheless aware of the problem and knew that primaquine exhibited

hemolytic toxicity in prisoner volunteers who were also sensitive to plasmochin [62]. They also understood the 8-aminoquinolines as a class offered only relatively thin margins with respect to therapeutic indices, i.e., ratios of therapeutic to toxic dose exposures [38]. The compound offering the greatest therapeutic activity, they reasoned, would probably impose the least risk of hemolytic toxicity by virtue of lower dosing. They further understood the prolonged dosing strategy employed with plasmochin, i.e., relatively small daily doses as a means of delivering an effective total dose, would mitigate the hemolytic toxicity of the rapidly eliminated 8-aminoquinolines [63]. In the clinic, the same strategy would allow

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cessation of dosing with onset of acute hemolytic anemia as a means of limiting the harm done. The prisons where the developers of primaquine worked during the 1940s and 1950s involved the use of about 2000 inmates as volunteers in their clinical trials. Most of them were Caucasian and only 18 (14 of those were African-Americans) [64] were known to be sensitive to 8-aminoquinolines. The discovery work on G6PD deficiency and hemolytic sensitivity was thus limited to those very few subjects. However, the military developers satisfied themselves with the safety of their new drug with shipboard studies of American soldiers being repatriated from the Korean Peninsula at war during the early 1950s [65, 66]. Several hundred thousand were given compulsory presumptive treatment with primaquine at 0.25 mg/kg/day (15 mg) for 14 days. Approximately 10% of those troops were African Americans, so one may surmise that at least several thousand G6PD-deficient troops were indeed exposed to therapeutic doses of primaquine. The investigators reported no serious reactions to the treatment, only mild and transient signs of hemotoxicity in a small minority. The US Army, and soon thereafter the World Health Organization (WHO), thus earnestly believed primaquine (at that anti-relapse dose) did not pose a serious threat to patients of unknown G6PD status. Decades would pass before this view would be acknowledged as overly optimistic and sometimes dangerously untrue. As early as the 1960s, evidence of clinically important diversity in G6PD deficiency and sensitivity to primaquine emerged. Work done by Italian hematologists in that era with Caucasian patients having the Mediterranean variant of G6PD deficiency showed extreme sensitivity to primaquine relative to that in African Americans [67, 68]. The exposure to primaquine was considered potentially threatening to life, because unlike African Americans having nearly normal G6PD activity in younger red blood cells, those Caucasians had almost no detectable G6PD activity in red blood cells of any age [69]. Whereas African Americans could be tolerized to primaquine with complete hematologic recovery during sustained daily dosing

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[70], the Caucasians could not. Later work demonstrated the dominance of Mediterranean or Mediterranean-like variants among people outside of Africa, especially in South and East Asia where P. vivax occurs in greatest numbers [56]. Only recently has WHO guidance on primaquine therapy explicitly warned against therapy in patients of unknown G6PD status [71]. The erroneous perspective of relatively harmless primaquine persisted through the latter half of the twentieth century, the period through which the early development of tafenoquine occurred. Scientists at the US Army’s Walter Reed Army Institute of Research led the 8-aminoquinoline preclinical discovery program within the Division of Experimental Therapeutics (WRAIR-ET) through the 1970s and 1980s [72]. Though armed with understanding of G6PD deficiency and the hemolytic toxicity of 8-aminoquinolines, those developers did not have the means of selecting compounds of lesser hemolytic toxicity. During the period 1981–1984, the author labored at WRAIR-ET as a technician devoted to the task of developing that instrument [73–76], working effectively alone and garnering the lasting impression of having been the sole minor resource committed to that objective. The hemolytic toxicity of the candidate 8-aminoquinolines did not register as a priority, and preclinical and clinical development would proceed without any understanding of the hemolytic toxicity of the candidate 8-aminoquinolines relative to primaquine. Only in 2017 did investigators in Thailand at last develop evidence indicating that therapeutic dosing with tafenoquine was approximately as hemolytic as that of primaquine (300 mg single dose vs. 210 mg total dose over 14 days of daily dosing, respectively) in healthy heterozygous female volunteers having the Mahidol variant of G6PD deficiency [77]. After nearly a century of repeated cycles of discovery and development – and of seeing the 8-aminoquinolines having minimal impacts on malaria as a problem of public health – the hemolytic toxicity of this class remains effectively unexplored by the technologists managing those efforts. No survey or understanding of

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8-aminoquinoline structures sheds any light on variable hemolytic toxicity among them. Only a lone single-subject trial in the 1950s showed a mere shift in the placement of a methyl group on the alkyl side chain of primaquine (to create a Russian compound called quinocide) substantially increased hemolytic toxicity [70]. In striving to understanding what appears to be conspicuous neglect of a serious problem, we must examine 8-aminoquinoline development during the past 75 years as an enterprise of military medicine.

Military Medicine The development of tafenoquine is extraordinary in several important respects with regard to G6PD deficiency as a safety problem. Whereas the developers of primaquine for radical cure employed a strategy of toxicity mitigation rooted in greatest therapeutic index and prolonged dosing, the developers of tafenoquine ultimately followed neither approach. Instead, they selected a compound that was not the most active, and they adopted a single dose as a decisive advantage. WR242511 was more than three times as active as tafenoquine against latent malaria [45, 48], and it was also sevenfold more active against blood stages of P. falciparum [46]. Understanding the abandonment of WR242511 in favor of tafenoquine requires examining the play of military imperative. The US Army enthusiasm for an agent of chemoprevention of malaria offering prolonged protection with relatively infrequent dosing found expression as early as 1948 [78]. As tafenoquine and other 8-aminoquinolines discovered during the 1970s progressed through preclinical experimentation, the good activity of tafenoquine against blood stages of P. falciparum came as a surprise for a drug of this class [45, 46]. In this era, this species was viewed as the lone lifethreatening malaria faced by American soldiers operating in endemic zones [79]. While the original intent of the 8-aminoquinoline discovery program of the 1970s – improving on primaquine for radical cure [41, 45] of what was then viewed as relatively benign P. vivax – was never wholly

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abandoned, it became secondary to chemoprevention as an objective [79–83]. Strategic steering towards the military priority of chemoprevention made the selection of tafenoquine sensible and inevitable. Its slow elimination and activity against P. falciparum in the blood (and liver) superbly met the requirements of that envisioned purpose. The disadvantages those features imposed on the drug as an agent of radical cure – lower therapeutic index and prolonged toxic exposures in G6PD-deficient patients – logically mattered less to the military technologists and strategists managing this program. The G6PD liability of otherwise useful drugs was a manageable problem for the military by virtue of their intrinsic close medical oversight – all soldiers and sailors, wherever deployed for duty, are never far from medical aid. As those developers expressed of the G6PD liability of tafenoquine for chemoprevention, “All U.S. service members are currently screened for G6PD deficiency upon entry into service.” [83]. The developers of primaquine likewise expressed of their efforts, “The primary purpose of the investigations has been, at all times, to satisfy the specific needs of the armed services.” [38]. Be it G6PD screening or access to transfusion with hemolytic crisis, American military men and women are protected from the serious harm 8-aminoquinolines may cause. The military medical people would view hemolytic toxicity as a problem, but not one that should dominate or delay development progress because it would be unlikely to harm the intended beneficiaries of their endeavors.

Repurposed Tafenoquine As has been explained, the US Army and their GSK partner encountered formidable obstacles in registering tafenoquine for chemoprevention, i.e., an unexpected ophthalmic toxicity issue and a revision to the Helsinki Declaration that made phase 3 trials of malaria chemoprevention almost impossible to conduct [52, 53]. They paused, reassessed, and pivoted back to an indication for radical cure of P. vivax, engaging MMV as a partner in 2008 (the US Army later resumed

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development for chemoprevention with another commercial partner). They pursued registration of tafenoquine for single-dose radical cure as a distinct advantage over the 2 weeks of daily dosing with primaquine. Adherence to full primaquine dosing is indeed a serious problem that greatly impacts its effectiveness in practice in military and civilian populations [84]. A single dose radical cure, it was reasoned, would effectively solve that problem. As advantageous as single dose therapy may be in practice, it also imposed a deeper problem with respect to what was in 2008 a supposition of a G6PD liability with tafenoquine – the drug had not yet been assessed in such patients. Unlike primaquine, there can be no clinical retreat with dosing cessation with adoption of a single dose strategy. If G6PD-deficient patients are unwittingly dosed with tafenoquine, they would likely experience full and prolonged hemolytic reactions (of as yet unknown severity). This explains the strict requirement to screen patients for G6PD deficiency (quantitatively) prior to administering tafenoquine [85]. This may not challenge the capacities of military care givers, but it does those in the rural endemic tropics.

Beneficiaries Setting aside the urban transmission of malaria in South Asian cities enabled by the adaptation of Anopheles stephensi to those environments [86], malaria is an overwhelmingly rural health problem. Access to professionally staffed clinics across most of the rural tropics is limited by often difficult distances [87]. In the specific instance of access to malaria diagnosis and treatment, national malaria control programs (NMCPs) often engage lay residents of remote villages as malaria workers providing those specialized services supported by materials, guidance, and training from NMCP professionals [88]. Those materials consist of easy-to-use immunochromatographic rapid diagnostic tests (RDTs) for malaria and frontline blood schizontocidal therapies (typically ACTs) to arrest progression of the acute attack. That kit has not

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included the capacity for G6PD screening or 8-aminoquinoline therapies against latent malaria. Patients diagnosed with P. vivax are typically referred to clinic for management of anti-relapse therapy. How many patients actually travel to the clinic for that care is not known, and likely hinges on the distances involved. Further, most of those clinics do not offer G6PD screening [89]. If primaquine is offered without such screening, as many NMCPs still recommend [90], the patient would require at least several days of clinical monitoring in order to ensure safety. In brief, management of the risk of hemolytic toxicity with primaquine exposure keeps the treatment out of the hands of village malaria workers, and it requires the patient to report to clinic for G6PD screening or several days of supervised care. The advent of tafenoquine for radical cure raises this bar of safety considerably higher. No patient can safely receive the treatment without first ascertaining G6PD activity status by quantitative testing (>70% of normal G6PD activity) [43, 44, 85]. The standard means of this test, a spectrophotometric biochemical assay in a properly outfitted laboratory, requires sophisticated equipment, expensive and labile reagents, along with a relatively high level of laboratory skill. In most developing malaria-endemic nations, such testing is only available at top-tier, large referral hospitals or is not available at all [89]. This limitation largely explains the limited impacts of primaquine on P. vivax. The notoriously poor adherence to primaquine is not driven solely by its duration of 14 days, but also by its reputation as a dangerous drug [91, 92]. NMCPs may be reasonably reluctant to undertake vigorous advocacy for improved adherence due to the concern of harming G6PD-unknown patients in doing so. The hemolytic toxicity of primaquine drove the prolonged dosing strategy and inhibits expression of the importance of consuming all 14 daily doses. Tafenoquine achieving impacts will require solving this problem of access and advocacy with far more accessible point-of-care quantitative G6PD testing technologies. Those are indeed becoming available [93, 94]. It may be unlikely to see these kits used by village malaria workers,

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but at least relatively nearby clinics of referral for P. vivax could be thus equipped for safe singledose tafenoquine radical cure. This is a reasoned strategy and hope for greatly improving sustainable access to safe and effective radical cure. The repurposing of tafenoquine from military to civilian users imposed the necessity of success in equity of access to G6PD screening among those beneficiaries. Given the technical and practical difficulty of safe practice in applying tafenoquine, some may presume that the hemolytic toxicity problem was addressed and maximally mitigated during its development. However, the strategic decision-making and actions taken by the US Army 40 years ago suggests otherwise.

Technological Imperatives Applying radical cure as the dominant strategic imperative would have demanded choosing the compound of greatest therapeutic activity as an important means of mitigating toxicity. At that stage, this would have been the only means of doing so. That compound would have been WR242511 having a minimal effective dose less than 0.10 mg/kg (per day for 7 days in the P. cynomolgi model) compared to tafenoquine at 0.315 mg/kg [45, 48]. We cannot today know if that selection would have resulted in a significantly improved hemolytic safety profile for radical cure, but lower dosing would have made it more probable. That exploration was trumped by the appeal of tafenoquine to developers focused on an imperative of chemoprevention with infrequent dosing. We cannot fault the US Army developers in that era for following their own military imperatives and rationale, but we can and should examine how those translated to and impacted the distinct imperatives operating in malaria-endemic communities. The US Army and its commercial partner adapted to events during the early 2000s [52, 53] that ultimately pivoted their development pathway priority from chemoprevention and back to radical cure. A development reset involving a return to preclinical candidates perhaps better

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suited to radical cure, if considered, does not appear to have been done. The intent of obtaining an IND for WR242511 for clinical development, expressed in 1991 [45], never materialized. The commitment to clinical development of tafenoquine alone had been irreversibly made and that compound was later pragmatically adapted to an indication for radical cure. The developers aligned the slow elimination character of tafenoquine, absent in WR242511, to an ideal of single dose radical cure. The relative hemolytic toxicity of the 8-aminoquinolines in preclinical development at WRAIR-ET was not known during the period of preclinical development. That of tafenoquine would not be known until the 39th year of its 40-year development pathway [77]. The safe management of hemolytic primaquine within the framework of military medicine largely explains the conspicuously low priority assigned to mitigating the problem. Hemolytic tafenoquine as chemoprevention or radical cure is suited to military personnel and other travelers enjoying routine access to the superb medical care that protects them from that harm. As for people living in the impoverished rural tropics, in contrast, the hemolytic toxicity problem looms far larger as a problem of safe access to the potentially very substantial clinical and public health dividends of radical cure. Achieving these impacts for tafenoquine now hinges on accessibility and practicality of new technologies for the assessment of G6PD status at point-of-care [95].

Perceptions of the Malaria Problem The perception of malaria as a threatening infection weighs on the calculus of Northern technology and Southern need discussed through this chapter. This is especially true of P. vivax malaria. Through the latter half of the twentieth century, contemporary malariology accepted the view of that malaria as relatively mild and only rarely fatal, whereas P. falciparum stood alone as a lifethreatening infection and as an overwhelmingly dominating imperative in research [96]. The classical and definitive text of malariology edited by

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Mark Boyd and published in 1949 gave those perceptions of these malarias authoritative weight [97]. Chapters in that book authored by S.F. Kitchen plainly expressed P. vivax as intrinsically benign, with only modest levels and progress of parasitemia relative to P. falciparum. Kitchen pointed to the self-limiting character of P. vivax by its obligate preference for reticulocytes for invasion and blood schizogony. Kitchen nonetheless detailed P. vivax infections (most of them in patients undergoing malaria therapy for neurosyphilis) resulting in severe anemia, respiratory distress, hyperpyrexia, seizures, intractable vomiting, and astasis. These conditions were apparently manageable in Kitchen’s clinic and, as such, seen by him as not particularly threatening. Kitchen did not mention any fatalities in his patients with induced P. vivax infections, but elsewhere mortality due to acute vivax malaria occurred in 5–15% of these patients [96]. In a hindsight that includes understanding of profound tropisms of P. vivax for extravascular spaces of marrow and spleen [98–100], along with reports of severe and complicated vivax malaria in patients hospitalized in endemic zones [101– 104], we may begin to understand Kitchen’s misperception of the consequences of this infection. P. vivax is certainly not intrinsically benign and, without prompt rescue therapy, very often progresses to those threatening conditions. In clinics of superb quality managing acute illness in previously malaria-naïve patients, P. falciparum is a more rapidly progressive illness that more often proves fatal than with P. vivax [105]. In the endemic rural tropics, on the other hand, inadequately treated P. vivax (as in Kitchen’s neurosyphilis patients) also progresses to equally threatening conditions. By Northern perspectives shaped by access to good care, the dichotomy of benign versus malignant malarias stands, but the Southern experience with the same infections is very different – any malaria may progress to threatening states where limited access to good care is the rule. The management of hemolytic toxicity in 8-aminoquinolines echoes as similarly misperceived with respect to broader contexts inclusive of the rural impoverished tropics. G6PD

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screening and access to emergency transfusion define the risk and consequences of that problem. These distinctions of context and perception define the hard work of now accommodating tafenoquine as radical cure to Southern reality. The hemolytic toxicity problem of that drug must be managed by implementing what amounts to Northern capabilities for doing so, i.e., access to G6PD screening in the first instance, and sufficient access to clinical rescue when hemolytic crisis inevitably occurs by accidental dosing. This is the immediate priority for sustainable radical cure, but it is not the only avenue of possible progress.

Future Directions The most remarkable technological aspect of this nearly century-long history is the almost complete ignorance of relationships between 8-aminoquinoline structures and relative hemolytic toxicities. We do not know if radical cure without G6PD liability in 8-aminoquinolines is a possibility because it has never been systematically explored as a development priority. The explanation for how this particular niche of scientific endeavor escaped attention and progress lies in what was an unacknowledged gap between Northern perspective and Southern need in the US Army priorities at play in their preclinical development of tafenoquine. Consequently, providers of clinical care and public health services in malarious developing nations face the challenge of broadening access to a drug capable of delivering very substantial benefits while also managing risk of significant harm with that access. This is precisely the challenge described of plasmochin nearly 90 years ago [34]. Today the many people guiding the applications of technology aimed at easing the burden of malaria globally look to greatly improving access to G6PD screening technologies as the means of overcoming the problem of hemolytic tafenoquine. Success in this approach offers immediate health dividends and may ease the difficulty of realizing the aspired elimination of malaria transmission in many zones by the end of

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this decade. We have a single dose radical cure in hand, and maximizing its utility by universal safe access must be pursued to the best of our abilities. On the other hand, a presumption of success in that endeavor should be set aside in favor of a strategic plan for coping with a latent malaria problem persisting well into the remaining decades of this century. In an objective historical and technological sense, we may rationally view the possibility of nonhemolytic 8-aminoquinonline radical cure as unexplored scientific ground of unknown promise. That exploration may begin with initiating the undone task of elucidating the molecular mechanics of 8-aminoquinoline hemolytic toxicity in G6PD-deficient patients. That understanding, in turn, may empower the rational ranking and selection of 8-aminoquinolines of minimal hemolytic toxicity and maximal activity. This exploration need not start de novo – we have dozens of 8-aminoquinolines tested in humans, and many dozens more tested in macaques over the past 75 years. Some of these compounds may be considered very promising in terms of safety (hemolytic toxicity aside) and good efficacy against latent malaria. The very substantial evidence needed to obtain an IND for human trials of experimental therapeutics may be already nearly complete for many 8-aminoquinolines. The compound known as WR242511, the extraordinarily active compound left behind by the US Army in favor of tafenoquine, is one example. Although that compound proved eightfold more active than primaquine in generating methemoglobinemia in beagles, when administered at even sublethal and lethal doses in macaques it generated almost no methemoglobinemia [106]. An excess of methemoglobin occurs in red blood cells under oxidative stress and may correlate with redox conditions that lead to druginduced acute hemolytic anemia. The apparent absence of methemoglobin in macaques (an animal that does show methemoglobinemia with other 8-aminoquinolines) exposed to WR242511 suggests the possibility of therapeutic activity without oxidative stress on red blood cells. That experiment [106] was reported in 2008, nearly two decades after the identification

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of WR242511 as a candidate for IND and trials in humans [45]. The sublethal and lethal toxicities (renal and hepatic) observed in those macaques seems belated for a compound that progressed so far in preclinical development. A reexamination of 8-aminoquinolines optimized for radical cure, and in light of understanding structure-hemolytic toxicity relationships among them, may be a way forward to antirelapse therapies without a G6PD liability. Acknowledging the hemolytic toxicity of 8-aminoquinolines as a serious Southern problem neglected by Northern technologists may be the essential first step in that direction.

Conclusions The technological march of progress in 8-aminoquinoline therapies over the past century was dominated by imperatives defined by the specific contexts of military medicine. Those imperatives set aside the hemolytic toxicity of these compounds as an acknowledged but unimportant problem. We inherited chemotherapeutics for latent malaria that are potentially both very powerful and dangerous implements in combatting that serious challenge to tropical health. Today, we may further develop sustainable technologies for G6PD screening that mitigate the danger of available therapeutics. In addition, we may finally undertake a scientific exploration aimed at neutralizing that problem by creating and using therapeutic indices of hemolytic toxicity in further discovery efforts with the 8-aminoquinolines. Either course, and the lasting necessity of each after a century of development, illustrate the challenges in achieving sustainable technology for tropical health.

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Further Reading Beadle C, Hoffman SL (1993) History of malaria in the United States naval forces at war: world war I throught the Vietnam conflict. Clin Infect Dis 16:320–329 Coatney GR (1963) Pitfalls in a discovery: the chronicle of chloroquine. Am J Trop Med Hyg 12:121–128 Condon-Rall ME (1995) The role of the US Army in the fight against malaria, 1940–1944. War Soc 13:91–111 Greenwood D (1995) Conflicts of interest: the genesis of synthetic antimalarial agents in peace and war. J Antimicrob Chemother 36:857–872 Joy RJT (1999) Malaria in American troops in the south and Southwest Pacific in World War II. Med Hist 43: 192–207 Masterson KM (2014) The malaria project: the US Government’s Secret Mission to find a miracle cure. Penguin, New American Library

Waterborne Plant Viruses of Importance in Agriculture Walter Q. Betancourt Water and Energy Sustainable Technology Center, University of Arizona, Tucson, AZ, USA

Article Outline Glossary Definition of the Subject Introduction Virus Groups Transmitted Through Terrestrial and Aquatic Environments Future Directions Bibliography

Glossary Aphids Insects of the order Hemiptera Contraseason Opposite to the normal season trend Cucurbits General definition for members of the family Cucurbitaceae that includes economically important species including cucumber (Cucumis sativus) and melon (Cucumis melo L) Debris The remains of natural material Host-switching The evolutionary change of the host-specificity of a pathogen Metagenomics The study of a collection of genetic material from a mixed community of organisms Phytoviruses General definition for plant viruses Resistant-breaking virus Viruses capable of infecting new hosts as a result host-range expansion from mutations Virome The assemblage of viruses that is investigated and described by metagenomics sequencing Virulent Term used to describe the degree of pathogenicity caused by a virus

Viruliferous Virus-carrying, especially insects that infest edible crops

of

Definition of the Subject Water-mediated transmission of plant viruses represents a major concern for sustainable food and agricultural systems. Numerous plant pathogenic viruses have been recovered from environmental waters with species members of the family Tombusviridae and Virgaviridae dominating the list of waterborne viruses that can infect plants. However, there are additional members of virus families including Alphaflexiviridae, Bromoviridae, Secoviridae, Potyviridae, Tymoviridae, and Solemoviridae that are transmitted mechanically and have been frequently detected in surfaces waters for irrigation as well as in untreated and treated wastewater metagenomic libraries. The potential for environmental-mediated transmission of these viruses largely depends on their persistence within terrestrial and aquatic environments. Several epidemics generated by novel viruses that spilled over from reservoir species or new variants of classic viruses that show new pathogenic and epidemiological capabilities have harmed global agriculture production in recent decades. This chapter provides an overview of key aspects related to water-mediated transmission of plant viruses of importance in agriculture.

Introduction Numerous plant viruses (i.e., phytoviruses) capable of infecting different cell types from a wide range of plant species have been characterized. Cucumber mosaic virus (Genus: Cucumovirus of the Bromoviridae family), for instance, has an extremely broad host range, infecting plants in 85 families and more than 1000 species [1]. Plant viruses are responsible for considerable

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_1096 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2022 https://doi.org/10.1007/978-1-4939-2493-6_1096-1

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economic losses which represents a threat for sustainable agriculture [2]. Estimates of several billion dollars economic losses due to plant viral diseases have been reported [3]. During the last decade, Plant Virologists compiled a “Top 10” list of the most scientifically and economically significant plant viruses [4] (Table 1). Honorable mentions for viruses just missing the Top 10 included Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus, and Tomato bushy stunt virus. Notwithstanding, emerging diseases with rapid increases in incidence, geographical scope, and/or pathogenicity tend to have a greater impact [2]. Several epidemics generated by novel viruses that spilled over from reservoir species or new variants of classic viruses that show new pathogenic and epidemiological capabilities have harmed global agriculture production in recent decades [5]. Since plant viruses need to overcome the physical barrier of a cell wall, they enter their host cells either via mechanical inoculation or the infection is mediated by vectors like insects, nematodes, or even fungi [6]. Despite the fact that plant viruses can be transmitted through a variety of ways, aphids are the most common and essential vectors of many plant viruses [7–11]. In addition, a Waterborne Plant Viruses of Importance in Agriculture, Table 1 Top 10 plant viruses. The table represents the ranked list of plant viruses voted for by plant virologists associated with Molecular Plant Pathology Rank 1 2 3 4 5 6 7 8 9 10

Virus Tobacco mosaic virus (TMV) Tomato spotted wilt virus (TSWV) Tomato yellow leaf curl virus (TYLCV) Cucumber mosaic virus Potato virus Y (PVY) Cauliflower mosaic virus (CaMV) African cassava mosaic virus (ACMV) Plum pox virus (PPV) Brome mosaic virus (BMV) Potato virus X (PVX)

Author of virus description Karen-Beth G. Scholthof Scott Adkins Henryk Czosnek Peter Palukaitis Emmanuel Jacquot Thomas Hohn and Barbara Hohn Keith Sanders Thierry Candresse Paul Ahlquist Cynthia Hemenway

variety of plant viruses from various genera are harbored in and transmitted through the soil. In this case, viruliferous resting spores of zoosporic vectors (e.g., plasmidiophorids, fungi, and nematodes) play an important role in the transmission of soil-borne viral diseases of plants [12, 13]. The distribution of these viruses is often locally limited, since they cannot rely on their vectors to transport them over long distances, however, once established at a site they may be very persistent [13]. The soil itself becomes a reservoir of infection between crops. The control of soil-borne viral diseases with conventional chemical or agronomic methods is challenging because virulent vectors may be widespread underground. Hence, disease control measures are mainly dependent on natural plant resistance resources. Meanwhile, the spread of resistant-breaking viruses is also a serious threat to crop production in agricultural systems [6, 12]. There is also evidence that plant viruses may have potential for host-switching to human or higher animals via recombination with vertebrate-infecting viruses [14, 15]. This chapter focuses largely on environmentalmediated transmission of plant viruses of importance in agriculture; those plant viruses that are not vector-transmitted, including members of the Tombusviridae and Virgaviridae families as well as members of the Alphaflexiviridae and Bromoviridae families. The potential for environmental-mediated transmission of these depends largely on their persistence within terrestrial and aqueous environments [16]. Viruses that affect plants have an extremely high stability. Tomato bushy stunt virus (TBSV; family Tombusviridae; genus Tombusvirus) and Pepper mild mottle virus (PMMoV; family Virgaviridae; genus: Tobamovirus) retain their infectivity after ingestion and subsequent exposure at 37  C to the enzymes of the human alimentary tract, the bile salts, and the wide fluctuations in pH (from 3.0 to 8.8) [17, 18]. PMMoV has been shown to survive standard food processing, such as in the manufacture of hot chili sauce and powdered chili. In addition, Pepino mosaic virus (PepMV; family Flexiviridae; genus: Potexvirus) can survive in plant debris and on contaminated surfaces, where it can remain infectious for several weeks

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Virus Groups Transmitted Through Terrestrial and Aquatic Environments

are related by the presence of a conserved RNAdependent RNA polymerase [21, 34]. The virions from the genera Tombusvirus have a rounded outline, a granular surface and a diameter of about 32–35 nm. Individual virus species have a relatively limited natural host range. Members can infect either monocotyledonous or dicotyledonous plants, but no species can infect both at the same time. The geographical distribution of viral species can range from extensive to limited, and it is strongly tied to the presence of the specific host [21, 34]. Infection is initiated via mechanical inoculation (piercing of leaf or root tissues with viruscontaminated material) or through soil-borne vectors. Infection is frequently limited to the root system, but when hosts are infected systemically, viruses spread throughout the plant tissues. Viruses are frequently found in natural habitats, such as surface waters and soils. As soil-borne viruses, both tombusviruses and aureus viruses, accumulate to high levels in infected plants, which are easily transmitted to healthy plants grown in virion-containing soils. The geographic spread of a species can range from extensive to limited. The majority of species are found in temperate climates (https://talk.ictvonline.org/), however Tombusviruses have been found in a wide range of locales, including North and South America, various regions in Europe, Algeria, and the Mediterranean [34].

Family Tombusviridae Members of the Tombusviridae family reside within the realm Riboviria which includes three subfamilies (Procedovirinae, Regressovirinae, and Calvusvirinae) and 16 genera (Alphacarmovirus, Alphanecrovirus, Aureusvirus, Avenavirus, Betacarmovirus, Betanecrovirus, Dianthovirus, Gallantivirus, Gammacarmovirus, Macanavirus, Machlomovirus, Panicovirus, Pelarspovirus, Tombusvirus, Umbravirus, and Zeavirus), each of which has a member species count ranging from 1 to 17. Tombusvirus is the type genus of the family Tombusviridae and is closely related to two other genera in this family, Aureusvirus and Zeavirus (Table 3). All Tombusviruses have uncapped positive sense, single-stranded RNA [(+)ssRNA] genomes encapsulated in icosahedral virions that

Family Virgaviridae There are 100 accepted members of the Virgaviridae family, which are classified into seven genera (Furovirus, Goravirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus, and Tobravirus) that show distinct host ranges, genome organization and modes of transmission [35, 36]. The family Virgaviridae consists of rodshaped, positive sense, single-stranded [(+) ssRNA] viruses infecting mostly plants [15]. Virions are non-enveloped and rod-shaped with a diameter of about 20 nm and predominant lengths that depend upon the genus [35]. Biologically, the viruses have a wide range of characteristics. Virgaviruses are transmitted in nature through pollen and seed as well as by soil-borne vectors such as plasmodiophorid and nematode

[19]. Melon necrotic spot virus (MNSV; family Tombusviridae; genus: Carmovirus) can attach to the outer covering of the aquatic zoospores of the chytrid fungus Olpidium bornovanus and can remain viable in the soil for several years [20]. Moreover, the virions of Tobamoviruses, Tombusviruses, and Tymoviruses are also resistant to elevated temperatures (thermal inactivation occurs above 80  C), organic solvents, and nonionic detergents [21]. Members of the Bromoviridae family are also resistant to elevated temperatures, however to a lesser extent (60  C) than the Tombusviruses and Tomaboviruses. These plant viruses along with members of the Alphaflexiviridae, Bromoviridae, Secoviridae, Potyviridae, Tymoviridae, and Solemoviridae families that are transmitted mechanically have been frequently detected in surfaces waters for irrigation by electron microscopy as well as in untreated and treated wastewater metagenomic libraries (Table 2), indicating their environmental persistence and resilience to wastewater treatment processes [26–31]. The latter has important implications in water reuse applications for agricultural irrigation since recycled water may still harbor infectious plant viruses [25, 31–33].

178

Waterborne Plant Viruses of Importance in Agriculture

Waterborne Plant Viruses of Importance in Agriculture, Table 2 Studies demonstrating the occurrence of waterborne plant viruses Target viruses Infectious tobamoviruses

Infectious TomaboPotex and Tombusvirus groups

Infectious plant viruses

Plant virus diversity in wastewater

Infective cereal viruses

Method Centrifugation (1 L: irrigation ditches and drainage canals) and ultracentrifugation for virus concentration. Infectivity assay: sets of three to seven plants of tobacco (Nicotiana ta-bacum cv. Xanthi), pigweed (Chenopodium quinoa), thorn apple (Datura stramonium), wheat (Triticum aestivum cv. Muszelka) and barley (Hordeum vulgare cv. Conchita) were mechanically inoculated with water samples. Identification performed by transmission electron microscopy Ultrafiltration (50 L: forest water) (Amicon-DiafloHollow-Fiber-DC 10 LA) followed by secondary concentration by Amicon-H 10 P 100-20 cartridge. Infectivity assay: inoculation of water concentrate to Chenopodium quinoa. Identification by transmission electron microscopy plus serological and agar gel double diffusion test for identification Centrifugation (5000 g) (1 L: river water) followed by resuspension of sediments in phosphate buffer for concentration. Infectivity assay: inoculation of water concentrates to Chenopodium quinoa followed by immunosorbent electron microscopy tests for identification Prefiltration (0.8 mm) untreated and treated wastewater followed by filtration by convective interaction media (CIM) quaternary amine monolithic columns. Detection performed by RT-qPCR and metagenomics analyses

0.5 L water from irrigation ditches and draining canals surrounding cereal fields subjected to lowspeed centrifugation (10,000 rpm, 10 min). Supernatant subjected to ultracentrifugation (30,000 rpm, 2 h) followed by inoculation of both supernatant and sediment into barley (Hordeum vulgare) and wheat (Triticum aestivum) as well as in pigweed (Chenopodium quinoa) plants. Virus identification performed by transmission electron microscopy, DAS-ELISA, and multiplex reverse transcription – polymerase chain reaction (RT-PCR)

vectors [36]. Members of the Virgaviridae family have been found in a wide variety of herbaceous, monocotyledonous, and dicotyledonous plant species (Table 3), although their host range is

Viruses identified Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV)

References [22]

Potexviruses, Tobamoviruses, Tombusviruses

[23]

Tobacco Mosaic virus and Cucumber mosaic virus

[24]

Pepper mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Cucumber green mild mottle virus, Tomato brown rugose fruit virus, Pepino mosaic virus, Melon necrotic spot virus, White clover mosaic virus, Southern bean mosaic virus, Sitke waterborne virus, Carnation Italian ringspot virus, Watermelon mosaic virus Brome mosaic virus (BMV)

[25]

[26]

usually limited. Viruses such as BSMV, PCV, PMTV, TMV, ToMV, TRV, and others in the Virgaviridae family are responsible for large quantitative and qualitative yield losses in a

Waterborne Plant Viruses of Importance in Agriculture

179

Waterborne Plant Viruses of Importance in Agriculture, Table 3 List of species within the family Tombusviridae (positive sense RNA viruses) grouped by genera (type species of each genus is indicated in bold) [21]

Virus

Subfamily/Genus

Length (nt)

Exemplar RefSeq Accession Number

Exemplar GenBank Accession Number

Exemplar Isolate

Artichoke mottled crinkle virus

Tombusvirus

4789

NC_001339

X62493

(AMCVBari)

Carnation Italian ringspot virus Cucumber Bulgarian latent virus Cucumber necrosis virus Cymbidium ringspot virus Eggplant mottled crinkle virus Grapevine Algerian latent virus Havel river virus Lato river virus Limonium flower distortion virus Moroccan pepper virus

4763

NC_003500

X85215

(CIRV-IT)

4576

NC_004725

AY163842

(CBLVBulg)

4701

NC_001469

M25270

4733

NC_003532

X15511

4767

NC_023339

FJ977166*

(CNVCan) (CymRSVUK) (EMCV-Is)

4731

NC_011535

AY830918

(GALV-JA)

2088a

AY370535*

1231a

NC_038690 ND NC_038691

4772

NC_020073

AF540886*

(HRV-S) (LRV-IT) (LFDVLim2) (MPV-IT)

Neckar River virus

1305a 4789

NC_038927 NC_030452

AY500887* AF290026*

4744

NC_005285

AJ607402

1238a

NC_038692

AY500881*

1194a

NC_038693

AY500886*

4776

NC_001554

M21958

4742 4094

LC_373507 NC_007729

4431

NC007816

CLSV

4421

NC005287

JCSMV

4293

NC009533

MWLMV

4415

NC000939

PoLV

4464

NC022895

YSV

Pelargonium leaf curl virus Pelargonium necrotic spot virus Petunia asteroid mosaic virus Sikte waterborne virus Tomato bushy stunt virus Gertian virus A

Maize necroc Zeavirus streak virus Cucumber leaf Aureusvirus spot virus Johnsongrass chloroc stripe mosaic virus Maize white line mosaic virus Pothos latent virus Yam spherical virus Elderberry aureusvirus 1

Unassigned aureusvirus

1134a

AY500882*

(NRV-GR) (PLCVKoenig) (PNSVUPEV) (PAMVLim1) (SWBVLim6) (TBSVcherry) GVA

MNeSV

ElAV1

(continued)

180

Waterborne Plant Viruses of Importance in Agriculture

Waterborne Plant Viruses of Importance in Agriculture, Table 3 (continued)

*Sequences do not comprise the complete genome. aNo. sgRNAs; genus demarcation criteria uses number of protein-coding sgRNAs only; protein-coding sgRNAs only for genus demarcation. Satellite RNAs associated with viruses in the family Tombusviridae Artichoke mottled crinkle virus satellite RNA Black beet scorch virus satellite RNA

[AY394497]

Carnation Italian ringspot virus satellite RNA Cymbidium ringspot virus satellite RNA

[D00720]

Panicum mosaic virus satellite RNA

[M17182]

Pelargonium leaf curl virus satellite RNA Petunia asteroid mosaic virus satellite RNA Tobacco necrosis virus small satellite RNA

[E03054]

Tomato bushy stunt virus satellite RNA (several types)

[AF022787-8][FJ666076]

Satellite RNAs associated with viruses in the family Bromoviridae Cucumber mosaic virus satellite RNA (several types)

[X69136]

Peanut stunt virus satellite RNA

[Z98197]

Satellite RNAs associated with viruses in the genus Umbravirus† Carrot mottle mimic virus satellite RNA

[EU914919]

Groundnut rosette virus satellite RNA*

[Z29702]

Pea enation mosaic virus satellite RNA

[U03564]

Tobacco bushy top virus satellite RNA*

[AF510392]

* These may be regarded as a satellite-like RNAs as they appear to be essential components of a disease complex. † These in turn depend upon viruses in the family Luteoviridae for their encapsidation and transmission.

variety of crops, although the economic relevance of other family members is restricted, poorly understood, or unimportant [36]. Mechanical inoculation is the only known method of transmission for those viruses within the genus Tobamovirus. There are no known invertebrate vectors for Tobamoviruses. Tobamovirus virions are non-enveloped, rod-shaped particles of approximately 18  300 nm with a central hollow core 4 nm in diameter. The genomes of tobamoviruses consist of one single-stranded positive-sense 6.3–6.8 kb

RNA. Within the genus, there are now 38 species recognized (Table 4). Several viruses that have recently been sequenced are likely members of novel species in the genus [37]. Tobamoviruses infect a wide spectrum of plants. Overall, the 38 species recognized and the three tentative members infect both dicotyledonous and monocotyledonous plants. TMV and ToMV are considered serious diseases for vegetable production over the world and cause significant losses in many solanaceous crops, particularly in greenhouses where employees move around. Plants

Waterborne Plant Viruses of Importance in Agriculture

181

Waterborne Plant Viruses of Importance in Agriculture, Table 4 Definitive and tentative species members of the genus Tobamovirus (positive sense RNA viruses), family Virgaviridae [37] Species name Bell pepper mottle virus Brugmansia mild mottle virus Cactus mild mottle virus Clitoria yellow mottle virus Cucumber fruit mottle mosaic virus Cucumber green mottle mosaic virus Cucumber mottle virus Frangipani mosaic virus Hibiscus latent Fort Pierce virus Hibiscus latent Singapore virus Kyuri green mottle mosaic virus Maracuja mosaic virus Obuda pepper virus Odontoglossum ringspot virus Opuntia virus 2 Paprika mild mottle virus Passion fruit mosaic virus Pepper mild mottle virus Plumeria mosaic virus Rattail cactus necrosis-associated virus Rehmannia mosaic virus Ribgrass mosaic virus Streptocarpus flower break virus Sunn-hemp mosaic virus Tobacco latent virus Tobacco mild green mosaic virus Tobacco mosaic virus Tomato brown rugose fruit virus Tomato mosaic virus Tomato mottle mosaic virus Tropical soda apple mosaic virus Turnip vein-clearing virus Ullucus mild mottle virus Wasabi mottle virus Yellow tailflower mild mottle virus Youcai mosaic virus Zucchini green mottle mosaic virus Tentative species Chara corallina Canada virus Chara corallina Australia virus Hoya chlorotic spot virus

Acronym BPMoV BrMMoV CMMoV ClYMoV CGMoMV CFMoMV CuMoV FrMV HLFPV HLSGV KGMoMV MaMV ObPV ORSV OV2 PaMMoV PfMV PeMMoV PlMV RCNaV

Accession # NC009642 NC010944 NC011803 NC016519 NC002633 NC001801 NC008614 NC014546 NC025381 NC008310 NC003610 NC008716 NC003852 NC001728 NC040685 NC004106 NC015552 NC003630 NC026816 NC016442

Length (nt) 6375 6381 6449 6514 6562 6424 6485 6643 6431 6485 6514 6794 6507 6618 6453 6524 6791 6357 6688 6506

Status Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete

ReMV RMV SFBV SHMV TLV TMGMV TMV ToBRFV ToMV ToMoMV TSAMV TuVCV UMMoV WMoV YTMMoV YMoV ZGMoV

NC009041 NC002792 NC008365 NC043383 NC038703 NC001556 NC001367 NC028478 NC002692 NC022230 NC030229 NC001873 ND NC003355 NC022801 NC004422 NC003878

6395 6311 6279 4683 1415 6355 6395 6393 6383 6398 6350 6311

Complete Complete Complete Incomplete Incomplete Complete Complete Complete Complete Complete Complete Complete

6298 6379 6303 6513

Complete Complete Complete Complete

Orobanchacee Plantaginaceae Gesneriaceae Leguminosae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Brassicaceae Brassicaceae Brassicaceae Taxaceae Brassicaceae Cucurbitaceae

CCCAV CCAUV HCSV

MK521928 AEJ33768 NC034509

9593 9065 6386

Complete Complete Complete

Characeae Characeae Apocynaceae

may become infected by rub inoculation of virions containing substances like Insect, animals, and humans visiting the fields [37].

Natural host family Solanaceae Solanaceae Cactaceae Fabaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Apocynaceae Malvaceae Malvaceae Cucurbitaceae Passifloraceae Solanaceae Orchidaceae Solanaceae Solanaceae Passifloraceae Solanaceae Apocynaceae Cactaceae

Tobamoviruses comprise one of the more intensively studied plant virus genera [37]. Members of this genus including PMMoV and

182

Waterborne Plant Viruses of Importance in Agriculture

CGMoMV have been frequently found in highly treated effluents [38–41] and recharged groundwater for reuse applications [42]. The presence of PMMoV and CGMoMV in surface waters has been proposed as a water quality monitoring tool to determine human fecal contamination in surface waters [31, 43, 44] irrigation waters [45] virus transport in managed aquifer recharged systems for potable reuse applications [42, 46]. Family Bromoviridae Plant viruses in the Bromoviridae family are trisegmented, single-stranded, positive-sense RNA viruses with a genomic size of roughly 8 kb. Virions have a variety of morphologies (spherical or bacilliform) with icosahedral symmetry and a diameter of 26–35 nm (genera Anulavirus, Bromovirus, Cucumovirus, and Ilarvirus), or bacilliform (genera Alfamovirus, Ilarvirus, and Oleavirus) with dimensions of 18–26 nm by 30–85 nm. Virions are spread mechanically, in/on pollen, and by insect vectors on

a non-persistent basis. Members of this family (Table 5) cause disease epidemic in fruit, vegetable, and fodder crops such as tomato, cucurbits, bananas, and alfalfa [48]. Brome mosaic virus (BMV) is the type member of the genus Bromovirus in the family Bromoviridae. Virions can be transmitted in the field by human activities involved in agricultural production, such as machinery trampling [49]. BMV infects monocotyledonous cereal crops like barley, maize, rice, wheat, and sorghum, as well as model plants like Brachypodium distachyon and Setaria viridis. Previous studies demonstrated Brome mosaic virus (BMV) infections in symptomatic plants inoculated with concentrated water samples from irrigation ditches and drainage canals surrounding fields in Southern Greater Poland. Following virus concentration via centrifugation and ultracentrifugation of water samples, virus identification was performed by biological, electron microscopic, serological, and molecular methods [26].

Waterborne Plant Viruses of Importance in Agriculture, Table 5 List of genera and species within the family Bromoviridae (positive sense RNA viruses) [47] GenBank accession no. Genus Alfamovirus Anulavirus Tentative species Bromovirus

Cucumovirus

Ilarvirus

Species Alfalfa mosaic virus Amazon lily mild mottle virus Pelargonium zonate spot virus Cassava Ivorian bacilliform virus

Acronym (AMV) (ALMMoV) (PZSV) (CsIBV)

RNA 1 (P1) NC_001495 NC_018402 NC_003649 NC_025482

RNA 2 (P2) NC_002024 NC_018403 NC_003650 NC_025483

RNA 3 (MP and CP) NC_002024 NC_018404 NC_003651 NC_025484

Broad bean mottle virus Brome mosaic virus Cassia yellow blotch virus Cowpea chlorotic mottle virus Melandrium yellow fleck virus Spring beauty latent virus Cucumber mosaic virus Gayfeather mild mottle virus Peanut stunt virus Tomato aspermy virus Subgroup 1 Ageratum latent virus Blackberry chlorotic ringspot virus Parietaria mottle virus Privet ringspot virus Strawberry necrotic shock virus

(BBMV) (BMV) (CsYBV) (CCMV) (MeYFV) (SBLV) (CMV) (GMMoV) (PSV) (TAV)

NC_004006 NC_002026 NC_006999 NC_003543 NC_013266 NC_004120 NC_002034 NC_012134 NC_002038 NC_003837

NC_004007 NC_002027 NC_007000 NC_003541 NC_013267 NC_004121 NC_002035 NC_012135 NC_002039 NC_003838

NC_004008 NC_002028 NC_007001 NC_003542 NC_013268 NC_004122 NC_001440 NC_012136 NC_002040 NC_003836

(ALV) (BChRSV)

NC_022127 NC_011553

NC_022128 NC_011554

NC_022129 NC_011555

(PaMoV) (PrRSV) (StNSV)

NC_005848 NC_027928 NC_008706

NC_005849 NC_027929 NC_008707

NC_005854 NC_027930 NC_008708 (continued)

Waterborne Plant Viruses of Importance in Agriculture

183

Waterborne Plant Viruses of Importance in Agriculture, Table 5 (continued) GenBank accession no. Genus

Oleavirus

Species Tobacco streak virus Subgroup 2 Asparagus virus 2 Citrus leaf rugose virus Citrus variegation virus Elm mottle virus Lilac ring mottle virus Spinach latent virus Tomato necrotic streak virus Tulare apple mosaic virus Subgroup 3 Apple mosaic virus Blueberry shock virus Lilac leaf chlorosis virus Prunus necrotic ringspot virus Subgroup 4 Fragaria chiloensis latent virus Prune dwarf virus Unassigned species American plum line pattern virus Apple necrotic mosaic virus Cape gooseberry virus 1 Humulus japonicus latent virus Tea plant line pattern virus Olive latent virus 2

Acronym (TSV)

RNA 1 (P1) NC_003844

RNA 2 (P2) NC_003842

RNA 3 (MP and CP) NC_003845

(AsV2) (CiLRV) (CVV) (EMoV) (LRMoV) (SpLV) (ToNSV) (TAMV)

NC_011808 NC_003548 NC_009537 NC_003569 EU919668a NC_003808 NC_039075 NC_003833

NC_011809 NC_003547 NC_009538 NC_003568 NC_038777 NC_003809 NC_039074 NC_003834

NC_011807 NC_003546 NC_009536 NC_003570 NC_038776 NC_003810 NC_039076 NC_003835

(ApMV) (BlSV) (LiLChV) (PNRSV)

NC_003464 NC_022250 NC_025477 NC_004362

NC_003465 NC_022251 NC_025478 NC_004363

NC_003480 NC_022252 NC_025481 NC_004364

(FCLV) (PDV)

NC_006566 NC_008039

NC_006567 NC_008037

NC_006568 NC_008038

(APLPV) (ANMV) (CGV1) (HuJLV) (TPLPV) (OLV)

NC_003451 NC_040469 NC_040393 NC_006064 NC_040435 NC_003673

NC_003452 NC_040471 NC_040392 NC_006065 NC_040436 NC_003674

NC_003453 NC_040470 NC_040394 NC_006066 NC_040437 NC_003671

Other species within the Bromoviridae family that are easily mechanically transmissible and therefore with potential for waterborne transmission include Alfalfa mosaic virus, which has an extensive host range and is one of the most significant viruses in the cultivation of pasture legumes [50]. Family Alphaflexiviridae Members of the Alphaflexiviridae family of plant viruses (Table 6) are transmitted mechanically and several species within the genus Potexvirus (Potato virus X) have been isolated from rivers and ponds [23]. Plant viruses of the Alphaflexiviridae family have flexuous filamentous virions that are 470–800 nm long and 12–13 nm wide. Alphaflexiviruses contain a 5.4–9 kb positivesense RNA genome that is single-stranded. They infect plants and fungi that infect plants [51, 52].

Family Solemoviridae This is a recently recognized family which consists of the previously unassigned genera Sobemovirus and Polemovirus [53]. Members of the Solemoviridae (Table 7) are very stable icosahedral virions of 25–34 nm in diameter with single-stranded genomic and subgenomic (sgRNA) RNA. Virus stability depends greatly on pH and the availability of divalent cations, Ca2+ and Mg2+ as well as by protein-protein interactions between subunits and by viral coat protein (CP)-RNA interactions. Virions may be spread mechanically, however many of them can also be spread by beetles (Sobemovirus – Positive Sense RNAViruses – Positive Sense RNAViruses (2011) – ICTV (ictvonline.org). These viruses have a high thermal inactivation point near 80–90  C [54]. Rice yellow mottle virus

184

Waterborne Plant Viruses of Importance in Agriculture

Waterborne Plant Viruses of Importance in Agriculture, Table 6 List of genera, virus species, acronyms, NCBI accession # and Genome length (nt) in the family Alphaflexiviridae (positive sense RNA viruses) [51] Genus/Species name Potexvirus Actinidia virus X Allium virus X Alstroemeria virus X Alternanthera mosaic virus Asparagus virus 3 Babaco mosaic virus Bamboo mosaic virus Cactus virus X Cassava common mosaic virus Cassava virus X Clover yellow mosaic virus Cymbidium mosaic virus Foxtail mosaic virus Hosta virus X Hydrangea ringspot virus Lettuce virus X Lily virus X Malva mosaic virus Mint virus X Narcissus mosaic virus Nerine virus X Opuntia virus X Papaya mosaic virus Pepino mosaic virus Phaius virus X Pitaya virus X Plantago asiatica mosaic virus Potato aucuba mosaic virus Potato virus X Schlumbergera virus X Senna mosaic virus Strawberry mild yellow edge virus Tamus red mosaic virus Tulip virus X Turtle grass virus X Vanilla virus X White clover mosaic virus Yam virus X Zygocactus virus X Allexivirus Alfalfa virus S Arachis pintoi virus Blackberry virus E Garlic mite-borne filamentous virus Garlic virus A Garlic virus B

Acronym

Accession no.

Length (nt)

AVX AlVX AlsVX AltMV AV3 BabMV BaMV CVX CsCMV CsVX CYMV CymMV FoMV HVX HdRSV LeVX LVX MaMV MVX NMV NVX OpVX PapMV PepMV PhVX PiVX PlaMV PAMV PVX SchVX SenMV SMYEV TRMV TVX TGVX VVX WClMV YVX ZyVX

NC028649 NC012211 NC007408 NC007731 NC003400 NC036587 NC001642 NC002815 NC001658 NC034375 NC001753 NC001812 NC001483 NC011544 NC006943 NC010832 NC007192 NC008251 NC006948 NC001441 NC007679 NC006060 NC001748 NC004067 NC010295 NC024458 NC003849 NC003632 NC011620 NC011659 NC030746 NC003794 NC016003 NC004322 NC040644 NC035205 NC003820 NC025252 NC006059

6888 7118 7009 6607 6985 6692 6366 6614 6376 5879 7015 6227 6151 6528 6185 7212 5823 6858 5914 6955 6582 6653 6656 6450 5816 6677 6128 7059 6435 6633 6775 5966 6495 6056 6278 6295 5845 6158 6624

AVS ApV BVE GarMbFV GarV-A GarV-B

NC034622 NC032104 NC015706 NC038864 NC003375 NC025789

8349 7599 7718 762a 8660 8336 (continued)

Waterborne Plant Viruses of Importance in Agriculture

185

Waterborne Plant Viruses of Importance in Agriculture, Table 6 (continued) Genus/Species name Garlic virus C Garlic virus D Garlic virus E Garlic virus X Shallot virus X Vanilla latent virus Lolavirus Lolium latent virus Mandarivirus Citrus yellow vein clearing virus Indian citrus ringspot virus Platypuvirus Donkey orchid symptomless virus Botrexvir Botrytis virus X Sclerodarnavirus Sclerotinia sclerotiorum debilitation-associated RNA virus Unassigned species Insect-associated alphaflexivirus 3 Euonymus yellow vein virus Potexvirus sp. Insect-associated alphaflexivirus 1 Insect-associated alphaflexivirus 2 a

Acronym GarV-C GarV-D GarV-E GarV-X ShVX VLV

Accession no. NC003376 NC022961 NC004012 NC001800 NC003795 NC035204

Length (nt) 8405 8424 8451 8106 8832 7462

LoLV

NC010434

7674

CYVCV ICRSV

NC026592 NC003093

7531 7560

DOSV

NC022894

7838

BVX

NC005132

6966

SSDaRV

NC007415

5470

IaAlV-3 EuYVV – IaAlV-1 IaAlV-2

MN203145 NC035190 NC040842 MN203143 MN203144

796a 7279 5839 4165 868a

partial sequence. Type species in bold

(RYMV) and Papaya lethal yellowing virus (PLYV) are two species of this family that cause serious crop diseases. Members of the Sobemovirus genus are often found in surface waters and soils where they can infect plants without the assistance of vectors [54]. RYMV can be transmitted by cows, donkeys, and grass rats in irrigated rice crops. Studies have shown that cattle-mediated spread enhances the size of the virus load in the contraseason and the infection potential to infect the next crop [55]. Windmediated and soil-mediated transmission of RYMV, PLYV, and Southern bean mosaic virus (SBMV) have also been documented [56, 57].

Future Directions The increased demand for alternative sources of water supply due to decreased water supplies in the world, contamination of surface and ground water, uneven distribution and frequent droughts

caused by extreme global weather pattern changes represent a serious threat to the future of food and agriculture. Recycled water for crop irrigation is considered a vital resource for enhancement of agricultural productivity and for irrigation of residential and public areas. However, the quality of recycled water used in non-potable agricultural and non-agricultural applications must be safe for plants and soils, food safety, and public health. The frequent emergence of new viral diseases associated with international trade, climate change, and the rapid evolution of viruses requires disease management strategies that strongly depend on fast and accurate identification of the causal agent [2]. The application of nextgeneration sequencing for multiplex detection, quantification, and the discovery of novel viruses is undoubtedly an excellent tool for monitoring the emergence and re-emergence of plant viral diseases that can have devastating economic, social, and/or ecological impacts, especially in developing countries where food insecurity is an

186

Waterborne Plant Viruses of Importance in Agriculture

Waterborne Plant Viruses of Importance in Agriculture, Table 7 Members of the family Solemoviridae (positive sense RNA viruses) [53] Genus/species Sobemovirus Artemisia virus A Blueberry shoestring virus Cocksfoot mottle virus Cymbidium chlorotic mosaic virus Imperata yellow mottle virus Lucerne transient streak virus Papaya lethal yellowing virus Rice yellow mottle virus Rottboellia yellow mottle virus Ryegrass mottle virus Sesbania mosaic virus Solanum nodiflorum mottle virus Southern bean mosaic virus Southern cowpea mosaic virus Sowbane mosaic virus Soybean yellow common mosaic virus Subterranean clover mottle virus Turnip rosette virus Velvet tobacco mottle virus Polemovirus Poinsettia latent virus

Acronym

Accession no.

ArtVA BSSV CfMV CyCMV IYMV LTSV PLYV RYMV RoMoV RGMoV SeMV SNMoV SBMV SCPMV SoMV SYCMV SCMoV TRoV VTMoV

NC_017914.1 NC_029578.1 NC_002618.2 NC_027123.1 NC_011536.1 NC_001696.2 NC_018449.1 NC_001575.2 NC_027198.1 NC_003747.2 NC_002568.2 NC_033706.1 NC_004060.2 NC_001625.2 NC_011187.1 NC_016033.1 NC_004346.1 NC_004553.3 NC_014509.2

PnLV

NC_011543.1

ongoing issue [2, 58–61]. Novel landscape-scale geometagenomics approaches have been recently applied to examine relationships between agricultural land use and distributions of plant-associated viruses. These studies have revealed not only numerous unknown virus species from uncultivated plants but also how agriculture substantially influences plant virus distributions viromes across broad agro-ecological interfaces [62]. Innovative approaches for fast and accurate identification of viral plant pathogens along with innovative virus disease management strategies are indispensable to ensure sustainable food and agriculture systems.

3.

4.

5.

6.

7. 8.

Bibliography 1. Hirsch J, Moury B (2021) Cucumber mosaic virus (Bromoviridae). In: Bamford DH, Zuckerman M (eds) Encyclopedia of virology, 4th edn. Academic Press, Oxford, UK, pp 371–382 2. Rubio L, Galipienso L, Ferriol I (2020) Detection of plant viruses and disease management: relevance of

9.

10.

Vector

Aphid Beetle

Beetle

Beetle, mirid Beetle Beetle Aphid, leafminer, leafhopper, mirid Beetle Beetle, mirid Beetle, mirid

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Water-, Sanitation-, and Hygiene-Related Diseases Y. Velleman1, L. Blair2, F. Fleming3 and A. Fenwick4 1 Department for Policy and Communications, SCI Foundation, London, UK 2 Department for Programmes, SCI Foundation, London, UK 3 Department for Monitoring, Evaluation and Research, SCI Foundation, London, UK 4 Faculty of Medicine, School of Public Health, Imperial College London, London, UK

Article Outline Glossary and Acronyms Definition of the Subject Introduction Bacteria: Diarrheal Diseases – Cholera and Typhoid Bacteria: Trachoma Helminth: Guinea Worm (Dracunculiasis) Helminth: Lymphatic Filariasis Helminth: Onchocerciasis (River Blindness) Helminth: Schistosomiasis Helminth: Soil Transmitted Helminths (Ascaris, Trichuris, Hookworm, and Strongyloides Infections) Protozoa: Malaria Virus: Dengue Virus: West Nile Fever Virus: Yellow Fever Future Directions Bibliography

Waterborne disease was authored in the 1st edition by A. Fenwick, A.F. Gabrielli, M. French, and L. Savioli. We acknowledge the contribution of S. Boisson (Department for Environment, Climate and Health, World Health Organization, Geneva, Switzerland) in this edition.

Glossary and Acronyms APOC African Program for Onchocerciasis Control DALY Disability-adjusted life year – The sum of years of potential life lost due to premature mortality and the years of productive life lost due to disability. MDA Mass drug administration NTDs: Neglected tropical diseases A medically diverse set of diseases and disease groups that disproportionately affect populations living in poverty, predominantly in tropical and subtropical areas. They are termed neglected because they lack the visibility, research support, and funding of other, more high profile, infections, and health conditions. While some diseases can result in severe disease and death, others can lead to chronic suffering as well as disability and can lead to stigmatization and social exclusion of affected individuals. They impose a significant human, social, and economic burden. WHO has listed 20 diseases and disease groups as NTDs. OCP Onchocerciasis Control Program STH Soil transmitted helminths: Intestinal worms infecting humans that are transmitted through contaminated soil. WASH Water, sanitation, and hygiene Water-, sanitation-, and hygiene-related diseases Those diseases that rely on, or are heavily associated with, water for at least one stage of their life cycle or transmission, or affect populations closely related to areas of water, or are transmitted through lack of separation between humans and excreta, or lack of personal and environmental hygiene.

Definition of the Subject Diseases related to water, sanitation, and hygiene (WASH) services and practices represent a

© Springer Science+Business Media, LLC, part of Springer Nature 2023 L. M. Shulman (ed.), Infectious Diseases, https://doi.org/10.1007/978-1-0716-2463-0_547 Originally published in R. A. Meyers (ed.), Encyclopedia of Sustainability Science and Technology, © Springer Science+Business Media, LLC, part of Springer Nature 2021 https://doi.org/10.1007/978-1-4939-2493-6_547-3

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significant and substantial burden on human health that disproportionately affects those living in low-income settings. Human populations have historically been established near surface water bodies, due to their crucial importance for survival, as well as productive and recreational activities. The development of water resources to exploit their potential has undoubtedly been of great benefit to all human populations via, for example, an increase in the amount of land suitable for agriculture provided by irrigation schemes, or the provision of hydroelectric power following the construction of dams and reservoirs. However, as an unintentional consequence, such development may have unwittingly increased the extent of human waterborne infections by increasing the areas of suitable habitat for disease vectors and/or intermediate hosts, as well as attracting human populations to congregate, thereby aiding the life cycles of many infectious organisms [1–3]. Despite significant progress globally, many individuals and communities continue to rely, even if partially, on surface water for their daily needs; many more come into contact with

water from contaminated sources, exposing them to harmful pathogens, chemicals, and minerals. Irregular and poor-quality supplies, even if delivered through piped services, cause continued reliance on surface water in many parts of the world. The importance of water for human health cannot be divorced from the issue of sanitation. Approximately a third of the global population continues to lack even basic sanitation services, with a significant proportion practicing open defecation and urination at least partially, even in areas in which sanitation coverage has increased. Inadequate sanitation represents a significant risk to human health globally through multiple pathways, including through transmission of pathogens through the fecal-oral route, vector breeding in and around human excreta, sludge and black water, and through interaction with animals and related zoonoses (see Fig. 1) [4]. Hygiene is intimately linked with human health. Health-promoting practices such as handwashing with soap, food hygiene, regular bathing, laundry and household, and community cleanliness are strongly associated with ample

Water-, Sanitation-, and Hygiene-Related Diseases, Fig. 1 The health impact of unsafe sanitation. (Source: WHO (2018) [4]. The diagram highlights the role of safe sanitation systems as a primary barrier to transmission of excreta-related pathogens by showing the way in which unsafe management at each step of the sanitation chain

spreads excreta in the environment. The diagram also captures transmission routes that are not fecal-oral and shows the complex ways in which different hazards and hazardous events interrelate. It forms a conceptual basis for risk assessment and management for sanitation systems)

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supplies of safe water in or in close proximity to the home [5]. In extra-household settings such as healthcare facilities and food establishment, the availability of water at the point of use is fundamental for preventing the spread of infections.

Introduction Various water-, sanitation-, and hygiene-related diseases are listed down in Table 1. These diseases represent a diverse taxonomic group of infections stretching from viruses to multicellular macroparasitic helminth infections. A brief description of the main infectious organisms is presented in Table 2. Global Guidance for the Control and Elimination of WASH-Related Diseases Neglected Tropical Diseases

The control of neglected tropical diseases has been guided by the World Health Organization, through the establishment of the Department for the Control of Neglected Tropical Diseases. In 2012, WHO released its first NTD Roadmap 2012–2020 [21], setting control targets for six diseases, elimination targets for nine diseases, and eradication targets for two diseases. The roadmap listed five key strategies for tackling NTDs: 1. Preventive chemotherapy 2. Intensified case-detection and case management 3. Vector and intermediate host control 4. Veterinary public health at the human–animal interface 5. Provision of safe water, sanitation, and hygiene It has also been recognized that where overlaps in control approaches exist between infections, not least with regard to the targeted populations, and the frequency and mode of treatment, then an integrated approach to control can be employed which should be more cost-effective than the separate, individual, vertical control programs which have previously been employed. Indeed, there is a growing momentum behind such an approach [22–28].

191 Water-, Sanitation-, and Hygiene-Related Diseases, Table 1 Water-, sanitation-, and hygiene-related diseases Sorted alphabetically by family of infectious agent 1a Bacteria Diarrheal disease – Cholera 1c Bacteria Diarrheal disease – Typhoid and paratyphoid enteric fevers 1b Bacteria Trachoma 2a Helminth Guinea worm disease (Dracunculiasis) 2b Helminth Lymphatic filariasis (Elephantiasis) 2c Helminth Onchocerciasis (River blindness) 2d Helminth Schistosomiasis (Bilharzia) 2e Helminth Soil-transmitted helminthiasis (STH) infections – ascariasis, trichuriasis, and hookworm 3a Protozoa Malaria 4a Virus Chikungunya 4b Virus Dengue 4c Virus West Nile Fever 4d Virus Yellow Fever Sorted alphabetically by disease 4a Virus Chikungunya 4b Virus Dengue 1a Bacteria Diarrheal disease – Cholera 1c Bacteria Diarrheal disease – Typhoid and paratyphoid enteric fevers 2a Helminth Guinea worm disease (Dracunculiasis) 2b Helminth Lymphatic filariasis (lephantiasis) 3a Protozoa Malaria 2c Helminth Onchocerciasis (River blindness) 2d Helminth Schistosomiasis (Bilharzia) 2e Helminth Soil-transmitted helminthiasis (STH) infections – ascariasis, trichuriasis, and hookworm 1b Bacteria Trachoma 4c Virus West Nile Fever 4d Virus Yellow Fever

In 2015, a Global Strategy on WASH and NTDs [29] was issued by WHO to accompany the NTD roadmap, acknowledging the significant potential co-benefits of a cross sectoral approach to NTDs, and the central role played by WASH in the prevention, treatment, and care of NTDs. The strategy was complemented in 2016 by the BEST framework, developed by the NTDs NGO Network, which sets out a comprehensive approach to NTDs incorporating the aspects of behavior, environment, social inclusion, and treatment and care.

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Water-, Sanitation-, and Hygiene-Related Diseases, Table 2 Classification and burden of major WASH-related diseases affecting human health. M million, K thousand DALYs disability adjusted life years Family Bacteria

Helminth

Disease Diarrheal disease – Cholera Diarrheal disease – Typhoid and paratyphoid enteric fevers Trachoma Guinea worm disease (Dracunculiasis) Lymphatic filariasis (Elephantiasis) Onchocerciasis (River blindness) Schistosomiasis (Bilharzia)

Soil-transmitted helminthiasis (STH) infections – ascariasis, trichuriasis, and hookworm

Type and species Vibrio cholera Salmonella enterica

9.69 M [7]

Chlamydia trachomatis Dracunculus medinensis

181 K [8] 813 [9]

Cases globally 1.3–4 M cases annually [6] Prevalence: 327 K Incidence: 13 M [7] Prevalence: 1.89 M [8] 54 [10]

Wuchereria bancrofti; Brugia malayi Onchocerca volvulus

1.63 M [11]

Prevalence: 71.9 M [11]

1.2 M [12]

Prevalence: 19.1 M [12]

1.64 M [13]

Prevalence: 140 M [13]

754 K [14]

Prevalence: 446 M [14]

236 K [15]

360 M [15]

984 K [16]

Prevalence: 173 M [16]

None available 46.4 M [18]

Prevalence: 613.9 M [17]

Intestinal (Schistosoma mansoni, S. japonicum, S mekongi, S intercalatum) Urogenital (S. haematobium) Roundworm (Ascaris lumbricoides) Whipworm (Trichuris trichiura) Hookworm (Necator americanus; Ancylostoma duodenale) Strongyloides stercoralis

Protozoa

Malaria

Virus

Dengue

Cerebral (Plasmodium falciparum) Others (P. vivax; P. ovale; P. malariae; P. knowlesi) Flavivirus

West Nile Fever

Flavivirus

Yellow Fever

Arbovirus

In January 2021, a new ten-year road map [30] was issued by WHO, which included significant shifts in the global approach to NTDs: • Accountability for impact: from historical orientation towards process, with success measured based on actions taken to impact orientation measuring public health impact of NTD interventions • Programmatic approaches: from siloed [vertical] disease-specific programs that have

DALYs

2.38 M [19]

290 K [20]

Prevalence: 181 M Incidence: 231 M [18]

Prevalence: 3.39 M Incidence: 56.9 M [19] No global estimates available Prevalence: 3,050 Incidence: 111 K [20]

limited interfaces with national health care systems and adjacent sectors to Holistic, crosscutting approaches including integration across NTDs, mainstreaming in national health systems, coordinating with adjacent sectors, and strengthening country capacity and global support • Program ownership: from externally driven agenda reliant on partner support and donor funding to Country ownership and financing with NTDs integrated in national health plans and budgets and supported

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At the heart of the road map is a recognition of the fundamental need for endemic countries to develop and implement context-specific approaches to disease control, which takes into consideration the role of multiple sectors in prevention and care activities. Although several NTDs continue to benefit from cost-effective mass drug administration using donated drugs for the treatment of entire at-risk populations, there has been a move towards a more holistic approach that seeks to enhance primary prevention through environmental and behavioral control and seeks to prevent reinfection and sustain the impact of MDAs. Mosquito-Borne Diseases

Global action to address mosquito-borne diseases is guided by the WHO Global Vector Control Response 2017–2030 [31], a strategy aiming to reduce the burden and threat of vector-borne diseases through effective locally adapted sustainable vector control. The strategy is comprised of four pillars of action: (1) strengthen inter- and intra-sectoral action and collaboration; (2) engage and mobilize communities; (3) enhance vector surveillance and monitoring and evaluation of interventions; and (4) scale up and integrate tools and approaches. At the foundation of these pillars are measures to enhance vector control capacity and capability and to increase basic and applied research, and innovation. Action on malaria towards is coordinated by the WHO’s Global Malaria Programme and guided by the WHO Global technical strategy for malaria 2016–2030 [32] and the Action and Investment to defeat Malaria 2016–2030 [33] – the second Global Malaria Action Map. Progress Against Global Disease Control Targets The progress made in the control of each of these diseases to date is outlined in Table 3, alongside the aim of control and the control strategies employed. Most of the diseases below are covered by target 3.3 of Sustainable Development Goal 3 [34] to ensure healthy lives and promote wellbeing for all at all ages: “By 2030, end the epidemics of AIDS, tuberculosis, malaria and

193

neglected tropical diseases and combat hepatitis, water-borne diseases, and other communicable diseases.”

Bacteria: Diarrheal Diseases – Cholera and Typhoid Diarrheal disease is the second leading cause of death among children under five and is responsible for an estimated 525,000 deaths every year in this age group [51]. In 2019 alone, over 1.5 million deaths in all age groups were estimated to be caused by diarrheal diseases [52]. Diarrheal disease is caused by a range of pathogens including viruses, bacteria, and protozoa, most often due to ingestion through the fecal-oral route [53]. The most common etiological agents associated with moderate to severe diarrhea in low income settings include Rotavirus, Escherichia coli, cryptosporidium, shigella, although this may vary by geographical location [51]. According to recent estimates, around 60% of deaths due to diarrheal diseases in low- and middle-income countries can be attributable to inadequate WASH [54]. Diarrheal diseases include cholera (caused by Vibio cholerae) and typhoid (caused by Salmonella typhi), which are endemic in many low- and middle-income countries where poor water and sanitation conditions prevail. Both diseases can cause outbreaks and epidemic, particularly during times of crisis such as after natural disasters like floods, earthquakes, and tsunamis, or conflict. Outbreaks are more likely in crowded, temporary settings with inadequate housing and sanitation such as refugee camps and when breakdown of sanitation and water systems leads to water contamination [55]. Cholera bacteria are most often associated with waterborne transmission, but can also be transmitted by contaminated food [55]. The classic symptoms of cholera are profuse, watery diarrhea, and vomiting, which can rapidly lead to severe dehydration and death in hours if left untreated. In addition to these severe sequelae of infection, there will likely be many, unreported cases that are asymptomatic or result in nonspecific mild symptoms [56].

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Water-, Sanitation-, and Hygiene-Related Diseases, Table 3 A summary of the current status of the major water-, sanitation-, and hygiene-related infectious diseases, aims, and approaches to control. ACT artemisinin Control or elimination aim Bacteria – Diarrheal disease – Cholera 2017 target: Remove cholera as an active threat to public health by reducing the mortality resulting from cholera by 90% [35].

combination therapy, DEC diethylcarbamazine, APOC African Program for Onchocerciasis Control, MDA mass drug administration

Intervention approach

Current status of progress (2020)

Early detection and quick response to contain outbreaks at an early stage; a multisectoral approach to prevent cholera in hotspots in endemic countries; and an effective mechanism of coordination for technical support, resource mobilization, and partnership at the local and global level [35].

Cholera continues to affect at least 47 countries globally, resulting in an estimated 2.9 million cases and 95,000 deaths per year worldwide [6]. Despite its status as a notifiable disease, cholera is consistently under-reported due to concerns about trade and travel restrictions. The continued burden of cholera represents a failure in both disease control in endemic areas and outbreak prevention. A global roadmap on cholera was issued by WHO in 2017 [35].

Bacteria – Diarrheal disease – Typhoid and paratyphoid enteric fevers Minimization and control of Improving sanitation, identification outbreaks [36] and treatment of carriers, and use of typhoid vaccines to reduce susceptibility to infection [36] Bacteria – Trachoma Implementation of the WHO2012 target: global elimination by recommended SAFE Strategy 2020 [21] (Surgery; Antibiotics (azithromycin); 2021 target: elimination as a public Facial cleanliness; Environmental health problem in 64 countries improvement) [37] (100%) by 2030. (defined as (i) a prevalence of trachomatous trichiasis “unknown to the health system” of 50 years.

Polio and Its Epidemiology, Fig. 1 Timeline of milestones toward achieving polio eradication

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Polio and Its Epidemiology

Polio and Its Epidemiology, Fig. 2 Living after paralytic poliomyelitis: then (1500 BC) and now (2007). Paralytic poliomyelitis occurs after a biphasic infection where viremia in a small number of systemic infections is followed by infection of the CNS. Paralysis is a direct result of destructive replication of poliovirus in motor neurons followed by atrophy of de-enervated muscles. Both pictures represent men whose skeletal muscles have been affected by infections of nerves in the anterior horn of their spinal cord. The picture on the left (a) depicts the

earliest record of poliomyelitis in a man and comes from a stele from ancient Egypt created around 1500 BCE and is strikingly similar to the image of the man in the photograph on the right (b) who has atrophy of the right foot and leg due to polio that was taken in the Far East in 2007 ((a) Egyptian Stele at the Ny Carlsberg Glyptotek Museum, Copenhagen, Denmark (GNU free documentation License). (b) Photograph #134 Centers for Disease Control and Prevention Public Image Library [CDC/NIP/Barbara Rice])

new concepts were the establishment of a national center for treatment of poliomyelitis victims at Warm Springs, Georgia, and the use of professional fundraisers by President Roosevelt supported by others in the late 1920s. The nonpartisan National Foundation for Infantile Paralysis and the March of Dimes established in 1937 institutionalized this fundraising effort. The iron lung, developed by Drinker in 1929, and the concept of supportive rehabilitation involving the use of hot moist packs to relieve muscle spasm and physiotherapy to maintain strength of unaffected muscle fibers promoted by Kenny in the 1940s were important advances for treatment of poliomyelitis.

The study of the pathological organism that caused poliomyelitis was enabled by the discovery of a bacteria-free “filterable” etiological agent, the poliovirus, which could pass disease from one primate to another by Landsteiner and Popper in 1909. Burnet and MacNamara realized in 1931 that there was more than one type of poliovirus since exposure to some isolates did not protect against exposure to others. By 1951, the National Foundation for Infantile Paralysis concluded that there were only three serotypes of poliovirus. The study of poliovirus was aided by the following: (a) The first passages of poliovirus in a nonprimate rodent system by Armstrong in 1939

Polio and Its Epidemiology

(b) Passage in tissue cultures by Enders, Weller, and Robbins in 1949 [8] (c) Development of plaque assays for quantification of polio by Dulbecco and Vogt in 1954 [9] (d) The use of microcarrier cell systems for vaccine production by van Wezel in 1967 [10] (e) Development of monkey neurovirulence tests in 1979 [11] (f) The use of pathogen-free diploid MRC5 cells (human fetal cells derived from normal lung tissue) and permanent cell lines like Vero (a cell line prepared from the kidney of a normal adult African green monkey) for vaccine production in the early 1990s (g) Identification and cloning of the poliovirus receptor CD155 [12] (h) Development of the transgenic PVR mouse model which expresses the human poliovirus receptor as an alternative to monkeys for neurovirulence testing [13] (i) Preparation of a murine cell line, L20B, expressing the human poliovirus receptor for selective growth of poliovirus [14] (j) The development of the immunological and molecular tools (discussed in detail throughout the sections below) that provide the identity of the serotype of the isolate, distinguish whether its origin was from a vaccine or wild strain, and provide phylogenetic information on the evolutionary relationship to other isolates Advances in culturing polioviruses outlined in the previous paragraph laid the foundation for developing the vaccines that have turned polio into a vaccine-preventable disease and a candidate for eradication. Early experiments and clinical trials such as those in 1935–1936 with inactivated poliovirus by Brodie and Park [15] and attenuated live vaccine by Kolmer [16] were hampered by lack of awareness until 1951 that there were three serotypes. Afterward, effective inactivated vaccine was developed and tested by Salk and coworkers starting in 1953–1954 [17, 18], while Koprowski, Sabin, and Cox developed and tested attenuated oral vaccines in 1950 [19, 20], 1956–1957 [21], and 1958 [22], respectively. Between 1951 and 1962, 12.9 million children

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were vaccinated with Koprowski strains and 11 million with Sabin strains [20]. A number of important epidemiological observations were made during that time that continue to guide current vaccination strategies. For attenuated oral vaccines, these included (a) the first demonstration by Koprowski of interference between poliovirus serotypes during coinfection [20], (b) a demonstration that maternal antibodies did not prevent an immune response in vaccinees under 6 months of age [20], (c) the observation by Koprowski and especially Bottiger that live vaccine spread to contacts [20], (d) a demonstration of persistence of antibodies at the same levels in vaccinated children for at least 3 years [20], (e) proof of concept by Koprowski that live polio vaccine could be effective in containing large outbreaks [20], and (f) documentation of high vaccine safety with both the Koprowski and Sabin OPV strains [5, 20, 23]. Safety issues relating to both the live and inactivated viral strains will be mentioned in discussions below. After extensive evaluation in hundreds of monkeys at Baylor College of Medicine, and the Division of Biological Standards at the NIH, the Sabin strains were chosen for licensure primarily on the basis of lower neurotropism, but also based on genetic stability on passage in humans and a lower ability to spread to contacts (reviewed in Sutter et al. [5] and Furesz [23]) (also see the discussion on development of new, safer oral polio vaccine strains). Efforts to eradicate polio and to prevent reemergence are presented in detail in the following section. Initial paradigms attributed to the different properties of the individual vaccines have not always held true in all circumstances [24].

The Epidemiology of Polio Epidemiological studies to discover the means of preventing a disease usually begin with the recognition of a new pattern of similar symptoms among those affected and the establishment of a case definition. Discovering means for preventing the disease that may start before the disease agent is discovered and characterized but is certainly accelerated once this characterization becomes

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available together with the means of quantifying intervention strategies. It is much less common to start with an agent and then search for a disease as in the case of human anelloviruses [25, 26]. Human anelloviruses are small circular DNA viruses considered to be orphan viruses. They were initially discovered in a patient with hepatitis, but subsequent research indicated no causal link to hepatitis, and it has been very difficult to associate them with any other specific disease. However, this section of the review will start with a description of those physical aspects of poliovirus that have the most impact on epidemiology of the disease. This is because there is already a clear case definition for polio and poliomyelitis, polioviruses have been recognized as the causative agents of these diseases, numerous methods for characterizing poliovirus and preventing poliovirus infections have been developed and tested, and the disease is approaching elimination or eradication. Structural and Functional Organization of the Poliovirus Genome Polioviruses belong to the Picornaviridae virus family. The Picornaviridae genome consists of a single strand of positive-sense RNA approximately 7,500 nucleotides located within a protein capsid made up of 60 capsomeres that forms a virion 27–30 nm in diameter. The genome is organized from its 50 end to its 30 end into a number of functional regions (Fig. 3) that include a 50 untranslated region (50 UTR) that regulate translation and replication [27], a long open reading frame (ORF) that encodes a single large polypeptide that is cleaved after translation into four structural capsid proteins and a number of nonstructural proteins including an RNA polymerase, and a short 30 untranslated region that is attached to a poly-A tail in both viral mRNA and genomic RNA in the virion [27] (see reviews by Wimmer et al. [28], Racaniello [27] and Sutter et al. [5]). The positive-sense single strand of genomic RNA in the virion serves directly as an mRNA template for translation to viral proteins once the virion penetrates its host cell membrane. Later, it serves as a template for synthesis of a complimentary negative-sense strand. The current

Polio and Its Epidemiology

understanding of the physical and genetic aspects of polio was greatly facilitated by the development of and the current commercial availability of methods for easily extracting viral nucleic acids from poliovirus and poliovirus-infected cells and analyzing and manipulating these sequences. Some of these studies led to the unanticipated conclusion that poliovirus capsid proteins and the sequences that encode them define polioviruses, whereas all other elements in the poliovirus genome may be substituted by genomic recombination with equivalent sequences from closely related isolates of enterovirus species C in vivo and even more distantly related rhinoviruses in the laboratory as long as functionality is maintained (reviewed by Kew et al. [4]). Finally, advances in molecular biology also enabled poliovirus to be the first virus to be synthesized from nucleotides in a test tube [29, 30]. The 50 UTR was first subdivided into a highly conserved region (nucleotides 1–650) and a hypervariable region (nucleotides 651–750) based on an analysis of 33 wild-type 3 polioviruses [31]. A series of stem-loop structures with a high degree of secondary structure were proposed to be present within the conserved region by Pilipenko et al. [32] and Skinner et al. [33]. Secondary structures (stems and loops) within major domain V of the 50 UTR that are relevant for the discussion on development and clinical testing of new, safer oral polio vaccine [34] are shown in Fig. 3d. A single nucleotide substitution in one of the loop structures in stem-loop V of the 50 UTR significantly influenced the neurovirulence of poliovirus isolates from all three serotypes and affected the maximum temperature at which viral isolates replicate efficiently (see [4, 5] and discussions on poliovirus evolution). The hypervariable region appears to be much less structured, reflecting the high degree of variation and U nucleotide richness [35]. An internal ribosome entry site, IRES [36, 37], enables uncapped RNA from Picornaviridae to be translated in eukaryotic cells by host ribosomes [27]. One of the first steps in initiation of viral translation is the binding [38] of cellular RNA binding proteins PCB1 and PCB2 to stem-loop IV of the IRES. This enables the 40S ribosomal

Polio and Its Epidemiology Open reading Frame for Polyprotein

73 49

33 8 38 4 41 31 22

0

74 95 8 4

A

3'UTR

51 0 54 9 3653 70 59 85

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17 67 24 81

VPg

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AAAA (n) Capsid P1

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non-Structural P2 P3

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2BC

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3CD 3D

3A VPg 3C or 3C'

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Sabin 1

A480G G935U U2438A G2791A C2879U U6203C

5'UTR VP4 VP3 VP1 VP1 3D

Non-coding ala 65 ser lys 225 met ala 106 thr leu 134 phe tyr 73 his

Yes Yes

Sabin 2

G481A C2909U

5'UTR VP1

Non-coding thr 143 iso

Yes -

Sabin 3

C472U C2034U U2493C

5'UTR VP3 VP1

Non-coding ser 91 phe iso 6 thre

Yes Yes

D

Polio and Its Epidemiology, Fig. 3 Organization of the polio viral genome, posttranslational processing of the nascent poliovirus polyprotein, the nucleotide substitutions that differentiate attenuated oral polio vaccine strains from their neurovirulent progenitors, and genetic modifications to produce new, highly stable, hyperattenuated new strains of OPV. The RNA positive-sense strand genome of Sabin 2 based on GenBank/EMBL/DDBJ entry AY184220 (a) is covalently linked to the viral encoded protein VPg. There is a single open reading frame flanked by a 50 and a 30 untranslated sequence (UTR). An internal ribosomal entry site (IRES) in the 50 UTR allows the uncapped polio genomic RNA to

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C C 500 A G A C C G AG U C C C G 490 A A C G 510 G C G C A U 480 C G U G A C U A A UA UUAA G C U UAUAAUU 470 U G C A G A AU A A C 520 530 A A G C GC A C A

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S18

472

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C C 500 A G A C C G AG U C C C G 490 A A C G 510 G C G C A U 480 C G U G A C U A C UA UUAA G C U GA UA A UU 470 U G C A G A AU A A C 520 530 A A G C GC A C A

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C C 500 A G A C C G AG U C C C G 490 A A C G 510 G C G C A U 480 C G U G A C U A C C A U U GA G C U GGUA A C U 470 U G C A G A AU A A C 520 530 A A G C GC A C A

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Sabin 3

472

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C C 500 A G A C C G AG U C C C G 490 A A C G 510 G C G C A U 480 C G U G A C U A C C A U G GA G C U GGUGC C U 470 U G C A G A AU G A C 520 530 A A G C GC A C A

serve as mRNA for translation on host cell ribosomes. The open reading frame is translated into a single poliovirus polyprotein that undergoes a series of posttranslational proteolytic cleavages (b) while it is still being translated. Some of the intermediate products have enzymatic and/or structural functions that differ from those of the final cleavage products. Poliovirus genomic and mRNA terminates in a poly-A tail. The attenuation of neurovirulence in Sabin 2 and the other 2 serotypes, Sabin 1 (GenBank/EMBL/ DDBJ entry V01150) and Sabin 3 (GenBank/EMBL/ DDBJ entry X00925), of poliovirus strains used for the live polio vaccine results from the nucleotide and amino acid substitutions shown in (c). Reversion of these

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unit to bind to the IRES and continue the process of translation as if the RNA was a capped eukaryotic mRNA. Functional IRES elements can be interchanged among Picornaviridae [4]. Nucleotide differences in the conserved 50 UTR among different isolates were unevenly distributed [31] with changes tending to conserve the stem structures. Knowlson et al. [34] introduced nucleotide substitutions in domain V that significantly increased genetically stability and attenuation of neurovirulence. In contrast, the hypervariable region did not seem to have a highly conserved secondary structure, and nucleotide differences appeared to be more or less evenly spread throughout [31]. While the length of the hypervariable region was generally conserved suggesting an unknown function [[31], small deletions were tolerated [35]. Picornaviridae have a genome of approximately 7,200–7,400 nt with a single open reading frame (ORF). While this ORF encodes four capsid proteins and at least seven viral proteins (Fig. 3a, b), these proteins are only produced after the initial translation product, a single polypeptide, is enzymatically cleaved into smaller and smaller polyproteins during and after translation (posttranslational processing). The polypeptide is cleaved in an ordered series of steps (Fig. 3b), by viral encoded protease activity within the nascent polypeptide (self-cleavage) and in trans from viral proteases released after cleavage. Interestingly, some of the intermediate cleavage products have unique activities by themselves that contribute to the replication cycle of the virus, but which differ from those of the final cleavage products (reviewed by Racaniello [39] and Krausslich et al. [40]). Properties of the polioviral capsid proteins define the

Polio and Its Epidemiology

epidemiology of polioviruses. The most important aspects of the structure of the four capsid proteins, their assembly into capsomeres and organization within adjacent capsomeres that relate to the epidemiology of polio, will be discussed below. The 900–906 nucleotide sequence of the VP1 of polioviruses has become the minimum standard for determining the evolutionary relationship among polioviruses and the rate at which they evolve [5, 41, 42]. Many of the nonstructural proteins and intermediate cleavage products (Fig. 3b) are multifunctional and act at a number of steps in RNA synthesis (reviewed in [5, 27]). Most of the nonstructural proteins will only be mentioned in passing since equivalent nonstructural proteins from other related picornaviruses may replace all of the nonstructural viral proteins as long as functional sites including cleavage recognition sites are maintained (reviewed in [4]). The resultant chimeric recombinants behave as polioviruses. One nonstructural protein, the RNA polymerase, will be discussed in some detail because of its profound effect on polio epidemiology regardless of its source. A cis-acting replication element (CRE) located in the poly-protein encoding region of the genome (nucleotides 4447–4499 in the 2C gene of Sabin 2) is important for uridylylation of the VPg protein that is covalently linked to the poliovirus genome and is required for [43]. This unpaired terminal loop CRE element retains its function when translocated elsewhere in the genome [43, 44]. The native CRE element was mutated without changing the amino acid sequence encoded in the region, and a new CRE was introduced into the 50 UTR of candidate for some candidates for new oral poliovirus vaccines undergoing clinical trials [45, 46]. This increases genetic stability of the

ä Polio and Its Epidemiology, Fig. 3 (continued) substitutions may restore a neurovirulent phenotype for the progeny of these vaccine strains. Nucleotide substitutions are indicated by the original nucleotide of the parental strain, the nucleotide position, and the substituted nucleotide in the vaccine strain (adenine uracil, guanine, or cytosine). Amino acid substitutions are indicated by the parental amino acid, the position of the amino acid in the

final cleavage product, and the amino acid in the vaccine strain (alanine, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, and tyrosine). ((b) Based on Krausslich et al. [40] and Kitamura et al. [409]. (c) Modified from Kew et al. [4]). (d) Adapted from Fig. 1 in Knowlson et al. [34] and reproduced under the Creative Commons Attribution License

Polio and Its Epidemiology

candidates by reducing the risk of a single recombination event replacing the modifications of domain V of the 50 UTR. The secondary structure in the 30 UTR that may play a role in translation and replication of picornaviral genomic RNA has been reviewed [27]. The temperature at which serotype 1 can replicate is influenced by a single nucleotide difference between Sabin serotype 1 and its wild parent [47]. A poly-A tail is present on both genomic and mRNA that stimulates the capindependent, internal ribosome entry site (IRES)-driven translation of poliovirus RNA in a mammalian cell-free system by tenfold [48]. Both genomic and minus strand RNA are linked to the small viral encoded protein, VPg, (Fig. 3a, b) through pUpU bound to tyrosine, the third amino acid from NH terminal end of VPg, by a phosphodiester bond [49]. VPg is also present in infected cells in an unmodified form and bound to pUpU through the same 04-phosphotyrosine bond found in the covalently linked forms [49, 50]. The uridylylation of VPg takes place on the opposite side of the polymerase that binds RNA. A host encoded unlinking enzyme that cleaves the 04phosphotyrosine bond between VPg and RNA has been described [51] although its role in replication has not been established. The poliovirus encoded VPg can be replaced by VPg from echoviruses [50]. A mature infectious poliovirus consists of a single sense strand of polyadenylated RNA covalently linked to a viral encoded protein, VPg, surrounded by an icosahedral protein coat, the capsid, made up of 60 capsomeres that each contains a single copy of each of the four viral capsid proteins. Adjacent capsomeres are organized around both fivefold and threefold axes of symmetry, and the surface around these axes is organized into a series of regular protrusions and depressions (Fig. 4). The capsid structure is metastable [52] rather than rigid, and internal parts of capsid may even be transiently expressed on the surface [53] (reviewed in [27])) exposing additional epitopes such as PALTAVE in VP1 [54]. Capsid proteins are the first viral encoded proteins to appear on the nascent poliovirus polyprotein and are cleaved from the nascent polyprotein into an intermediate polyprotein, P1,

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by 2Apro, while the full-length polyprotein is still being synthesized. P1 encodes the four viral capsid proteins. P1 is processed into final cleavage products VP1 and VP3 and an intermediate cleavage product VP0. VP0 is only cleaved into VP2 and VP4 during the final stages of maturation of the virion. The protein chains of VP1, VP2, and VP3 are arranged in wedge-like structures consisting of eight anti-parallel beta-strands connected by extruding loops that interact to form the major (NAgIa, NAgIIa, and NAgIIIa) and minor (NAgIb, NAgIIb, and NAgIIIb) neutralizing antigenic epitopes [55, 56]. Capsomeres consist of a single copy of the four capsid proteins, VP1, VP2, VP3, and VP4. In the virion, a three-dimensional view of the structure of the capsomeres at the fivefold axis of symmetry reveals an elevated central plateau with a hole in the middle surrounded by a depression called the canyon [57, 58]. The fivefold axis of symmetry for type 2 poliovirus is shown in Fig. 4. The amino acids of the neutralizing antigenic sites have been mapped onto the three-dimensional structures of the viral capsid of type 2 poliovirus as colored spheres (Fig. 4). Those that are unique to the neutralizing antigenic epitopes are colored yellow. Amino acid residues in or adjacent to neutralizing epitope sites that share functionality with poliovirus receptorbinding sites have been colored red. Amino acid differences within neutralizing antigenic sites divide polioviruses into three serotypes with limited cross-reactivity [59]. Mayer et al. [60] subjected ultrafiltrationpurified polioviruses from infected money kidney cells to sucrose density ultracentrifugation. The resulting sucrose gradient was divided into four fractions that were labeled A, B, C, and D. Fraction C contained 130S noninfectious poliovirus particles, and fraction D contained 150S infectious poliovirus particles. The conformation of the epitopes and the repertoire of antibodies that they interact with depend on the stage in the life cycle of the poliovirus. Antibodies that interact with “D-antigens” on native 150S virions are different from those that bind to “C-antigens” on virus particles that transition into 135S particles when virions bind to the human poliovirus

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Polio and Its Epidemiology, Fig. 4 Type 2 poliovirus capsid organization around the fivefold axis of symmetry: the hydrophobic pocket and amino acid residues in receptor-binding and neutralizing antigenic epitopes. The hydrophobic pocket and amino acid residues in the neutralizing antigenic epitopes and receptor-binding sites of the Sabin 2 polio vaccine strain. The three-dimensional structures represent capsomeres 1–5 from human serotype 2 poliovirus, Genbank/EMBL/DDBJ entry 1eah. The backbones of the amino acid chains of the capsid proteins are represented by light blue, pale green, light orange, and magenta colored ribbons for VP1, VP2, VP3, and VP4, respectively. Amino acid residues at the surface of the hydrophobic pocket are represented by blue spheres. Amino acid residues within the epitopes recognized by neutralizing antibodies are represented by yellow spheres, those involved in receptor recognition and binding are represented by magenta spheres, and amino acid residues shared by both antigenic sites and receptor-binding sites are represented by red spheres. The figure was prepared using the MacPyMOL program (DeLano

Polio and Its Epidemiology

Scientific LLC, www.pymol.org). Figure (a) is a representation of the entire capsid of poliovirus showing the positions of the threefold (in red) and fivefold (in blue) symmetrical organization of the capsomeres. Each poliovirus capsomere (b) contains a single copy of each of the four viral capsid proteins. Five capsomeres are assembled around a fivefold axis of symmetry shown in (c) and by blue in (a). They also assemble around a threefold axis of symmetry shown in red in (a). Figure (c) represents an external view of the five capsomeres at the fivefold axis of symmetry. Figure (d) is the side view of the same five capsomeres formed by rotating the figure in (c) in the direction of the circular arrow, so that the lower structures in (c) are nearest the viewer and the internal surfaces of the capsid proteins are facing downward. Figure (e) is a transverse section of the figure in (d) at the position of the straight arrow in (c) to more clearly illustrate the topography of the surface of the virion. An animated “Interactive 3D Complement” (I3DC) for the structures in this figure appears in Proteopedia at http://proteopedia.org/w/ Polio_Epidemiology

Polio and Its Epidemiology

receptor, CD155, or after virions are treated with UV or heat (60  C for 20 min) [61–63]. This terminology has stuck, and the effective content of each serotype in OPV and IPV is expressed in D-antigen units. One result of the CD155mediated transformation but not heat-mediated transformation is externalization of the first 31 amino acids of VP1 that allows binding to liposomes [64]. Antibodies raised against “Yantigens” on untreated poliovirus produced at Yale University by Black and Melnick [65] and “H-antigens” on heat-treated poliovirus (60  C for 20 min) produced at Harvard University in 1953 by Svedmyr et al. [66] were equivalent to D- and C-antigens, respectively. Some individuals developed antibodies to D-Ag/Y-Ag of native 150S infectious poliovirus, while others also developed antibodies to the C-Ag/H-Ag of the 130S form and additional degradation products including the 80S empty capsids and denatured viral proteins ([60] and references therein). To further complicate the situation, monoclonal antibodies have been developed that only recognize D-Ag/Y-Ag, that only recognize C-Ag/H-Ag, or that recognize both D-Ag/Y-Ag and C-Ag/H-Ag ([67] and references therein). Convalescent sera react mainly with D-antigen, whereas serum from the acute phase mostly reacts with the C-antigen [60]. Antibodies that recognize C-antigens and D-antigens were purified and used to characterize different virus preparations. Viable viral progeny from tissue cultures reacted with D-antigen antibodies [68]. If IPV is accidentally frozen, it may loose significant D-antigen content, but this may not affect immunogenicity at least in a rat model [69]. D-antigen results depend on the antibodies used, e.g., monkey-, chick-, guinea pig-, mice-, or rat-produced in the ELISA test [70–76]. The rat model is the generally accepted model. The picture is complicated further by the observation that some monoclonal antibodies (MoAbs) for PV1-D-antigen ELISA react differently for different strains of PV1 [77]. Thus, D-Ag ELISA can be used to determine titers in diff preps from the same strain but probably should not be used to compare amounts of different strains. From the point of view of vaccine production, tissue culture conditions of Vero cells in bioreactors affects the yield of D-antigen-specific poliovirus [78] (from

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abstract), and similar or higher yields for sIPV strains could be obtained on the PER.C6 ( ®) cell line [79]. “Strictly spoken, the D-antigen unit represents two entities (antigenic content and immunogenic content) which are equivalent for WT-IPV but are different for Sabin-IPV” in [71]. Differences between results for D-antigen content for IPV strains and for OPV strains used in preparing IPV (sIPV) required that a new international standard be formulated [71, 80–82]. It is good to keep in mind the observation by Salk et al. [83] that “the essential factor in the preparation of such a [polio] vaccine is the inclusion of a sufficient mass of the immunizing antigen, for each of the three antigenic types of poliovirus, to induce the formation of humoral antibody and/or immunologic memory after the first dose.” Monoclonal antibodies are an alternative for characterizing polioviruses. According to the observations of Singer et al. [84], “The monoclonal antibodies developed, which bound D antigen but not C antigen, were neutralizing unless relatively weakly reactive. Those that bound C antigen only were non-neutralizing. Those that bound both C and D antigens were sometimes neutralizing. D-specific and D/C-specific neutralizing monoclonal antibodies against type-2 poliovirus protected mice on passive immunization against paralytic disease and death from the MEF strain virus. Potency measurements by ELISA using either D-specific neutralizing monoclonal antibodies or type-specific goat sera for antigen detection were sensitive and precise. Tests using C-specific monoclonal antibodies for antigen detection indicated that increased C antigen content may result in falsely elevated reactivity of animal sera with some vaccines.” Rombaut et al. [85] demonstrated that three out of the four antigenic sites were already present in 14S subunits (pentamers, assemblies of five structural capsomere units containing a single copy of viral capsid proteins 1, 2, 3, and 4) and that the 3B epitope only formed when the 14S subunits assembled into capsids. 135S particles that elute from the cell or were internalized were indistinguishable. The next step is formation of an 80S empty particle after release of VP4 and RNA is ejected or enters the host cell [63]. This transition process can be mimicked in vitro without

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interaction with CD155 by treatment at above physiological temperatures (50  C for 3 min or 60  C for 20 min) [86]. The three-dimensional view of the structure of the capsomeres at the fivefold axis of symmetry reveals an elevated central plateau with a hole in the middle surrounded by a depression called the canyon [57, 58]. The same fivefold axis of symmetry for type 2 poliovirus that illustrated the position of amino acid residues within antigenic epitopes will be used to indicate the position of amino acids that have been shown to be involved in binding the CD155 host cell poliovirus receptor. These conserved residues are located at positions on the surface of the canyon walls and include residues within and adjacent to the serotype-specific neutralizing epitopes [28, 87]. Receptor-binding residues that are within or adjacent to antigenic epitopes are colored in red, while those uniquely associated with receptor binding are colored in blue in Fig. 4. This combination of functions may have restricted the number of serotypes [87] and influence evolution in neutralizing epitopes in the absence of immune selection in individuals with primary immune deficiencies as a result of receptor-based selection for persistence of vaccine-derived polioviruses (iVDPVs). A hydrophobic pocket (Fig. 4, blue spheres) located below the canyon floor is normally occupied by pocket factors such as sphingosine-like molecules including palmitic and myristic acids and hydrophobic compounds that stabilize the capsid and enable receptor docking and whose removal is a necessary prerequisite for uncoating [27, 52, 88, 89]. Small molecules such as pleconaril, pocapavir, and isoflavines can bind in this hydrophobic pocket and exert antiviral effects by affecting the binding of the receptor or enhancing the stability of the virion and preventing uncoating [27, 90]. Because of the metastable nature of the capsid, mutations distant from the receptor and drug binding sites can compensate mutations in the respective binding sites [52, 91]. The human encoded, poliovirus receptor, CD155, belongs to the immunoglobulin supergene family and has one variable and two constant

Polio and Its Epidemiology

immunoglobulin-like domains (residues 28–337) [12].) This human encoded gene has alternative splice sites that result in two membrane-bound and two secreted isoforms [92]. The variable domain 1 penetrates the canyon and binds to amino acid residues from all three external capsid proteins, and the principal binding sites are at the bottom of the canyon above the hydrophobic pocket (Fig. 4; blue spheres) and on the outer side of the canyon rim [87, 89]. The residues of type 1 poliovirus involving receptor virion binding include residues 102–108, 166–169, 213–214, 222–236, 293–297, and 301–302 in VP1; residues 140–144 and 170–172 in VP2; and residues 58–62, 93, and 182–186 in VP3. The equivalent residues for serotype 2 poliovirus have been mapped onto the three-dimensional capsid structure as red (shared with neutralizing antigenic epitopes) and magenta spheres for those associated only with the receptor-binding sites (Fig. 4). Cryo-electron microscope studies have shown the binding of the poliovirus to the virion to be a twostep process [89]. The initial binding of the receptor to amino acid residues along the canyon wall results in little or no change in virion structure. However, this binding rapidly sets into motion conformational changes leading to the 135S or A particle state that initiates uncoating and the start of the infection cycle [52, 89] and alters viral conformational epitopes. The human poliovirus receptor has been cloned and used to establish a murine cell line, L20B, where expression of the poliovirus receptor allows infection and growth of polio from clinical and other samples but not most other human nonpolio enteroviruses [14, 93, 94]. Transgenic mice, PVR Tg-21 mice that express the human poliovirus receptor, not only support poliovirus infection and present with neurological symptoms but also allow determination of the relative neurovirulence of the isolates [13, 95–97]. Molecular analysis studies of isolates shed during persistent infections of immunodeficient patients [98, 99] and from phylogenetically related aVDPVs from environmental samples help pinpoint amino acid substitutions in capsid proteins that determine antigenicity, receptor recognition, attenuation of neurovirulence, and

Polio and Its Epidemiology

properties of the hydrophobic pocket. Other changes, some of which are at interfaces between the threefold or fivefold interfaces of capsomeres, may also affect these properties indirectly. In order for single-stranded, positive-sense genomic RNA to be incorporated into progeny of the infecting virus, a complimentary negative RNA strand must first be synthesized using the original single-stranded positive-sense RNA genome as template, and this complimentary negative strand must then be used as template for synthesis of new positive-strand RNAs. While some positive-sense copies are incorporated into progeny virions as genomic RNA, other newly synthesized positive-strand RNAs serve as templates to repeat and amplify RNA replication and/or for translation to produce more viral proteins. Eukaryotic cells that serve as the host for poliovirus replication lack a polymerase that can synthesize complimentary RNA from an RNA template. Therefore, the virus must encode its own polymerase. Since the single-stranded positive-sense RNA genome of the infecting virion is also an mRNA that is immediately translated, the virion does not have to incorporate the polymerase into the virion itself to start replication. The translation product of the 3Dpol gene (Fig. 3b) is the required RNA-primed RNA polymerase. Both the intermediate cleavage products that contain the 3Dpro and the final cleavage product are multifunctional, and the crude replication complex also contains other viral proteins and protein cleavage intermediates such as 2 BC, 2C, and 3AB as well as host proteins (reviewed in [5, 27]). The binding site for RNA template and primer is on one face of 3Dpol, and a binding site for the uridylylation of VPg, a prerequisite for covalent linking of VPg to viral RNA, is on the opposite face [27]. One important contrast between genomic DNA replication in eukaryotic host cells and genomic RNA replication in Picornaviridae relates to the fidelity of replication. Specifically, there is an elaborate proofreading mechanism combined with pathways for correcting misincorporation during replication of eukaryotic DNA that is lacking in the RNA-primed RNA polymerase complex for viral replication [100,

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101]. This leads to such a high evolutionary rate for polioviruses that it borders on error catastrophe [102, 103]. Poliovirus Infections in Cells, Individuals, and Populations This section will deal with the epidemiological aspects of poliovirus infections at three levels, infections in single cells, infections in a single individual, and infections in populations of individuals. The normal infectious cycle of a poliovirus starts with recognition and attachment to poliovirus-specific cell receptors on susceptible cells of human or closely related primate origin. It continues with penetration and uncoating, translation of viral RNA, posttranslational processing of viral polyproteins, replication of viral genomes, and assembly and maturation of progeny viruses culminating in the release of infectious polioviruses. During this process, the virus employs and modifies host cell functions to optimize viral yield. The observation that viral RNA and cDNA is infectious when transfected into permissive host cells has allowed recovery of virus from extracted genomic RNA, cloned cDNA or RNA translated from cloned DNA [104–106], genomic RNA immobilized on FTA paper (WHO 16th Informal Consultation of the Global Polio Laboratory Network, September 2010, Geneva, Switzerland), and polioviral RNA synthesized in a test tube from individual nucleotides [29, 107]. The infectious cycle has been reviewed extensively. The reader is referred to the following reviews for further reading [5, 27, 108]. Poliovirus Infections at the Level of the Host Cell All polioviruses recognize a single host cell receptor [12, 87], CD155, also known as the poliovirus receptor (PVR). Identification and cloning of the poliovirus receptor CD155 [12, 87] allowed the creation of cell lines and animal models [14, 96, 97] for the study of polioviral infections in nonprimate hosts. The interaction of virion and receptor is complex [27]. The capsid structure is dynamic allowing the transient presence of internal portions and epitopes of capsid proteins on the outer surface of the virion [27, 53] including the

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N-terminus of VP1 even before uncoating. The shape of the receptor and its position relative to the host cell membrane and the canyon on the virion into which it fits bring the fivefold axis of capsomeres in close proximity to the cell membrane [89]. The presence and maintenance of separate poliovirus lineages coinfecting immunodeficient hosts [109–111] may imply that additional receptors may be involved in poliovirus binding. Moreover, it is tempting to speculate that nucleotide substitutions that accumulate during establishment and maintenance of persistence are related to specific combinations and densities of these receptors. Conformational changes, induced shortly after the virion-receptor interaction, are required to initiate the uncoating process (reviewed in [27]). The capsid begins to disassociate during a transition to the A particle. The A particle contains the viral RNA but has lost its VP4 capsid proteins. The N-terminal of the VP1 externalizes and may insert into the plasma membrane. Viral RNA is believed to enter the cell at or near the fivefold axis through a continuous channel formed in part by VP1 that continues through the cell membrane [89]. VP4 plays a part in formation of the pore. The pore for poliovirus entry is probably not formed within endosomes [27]. Small molecules that sit in the hydrophobic pocket may influence these conformational changes without affecting receptor binding [91, 112–114]. The only viral proteins in the virion are the four capsid proteins and the VPg covalently linked to the genomic RNA. The internal ribosomal entry site (IRES) on uncoated polioviral RNA enables translation of the viral polyprotein on host cell ribosomes (reviewed in [4, 27]). VPg appears to be cleaved from this RNA and subsequently synthesized viral RNA that will be used as mRNA [51]. Nuclear trafficking of cellular proteins is downregulated shortly after infection resulting in accumulation of host nuclear proteins in the cytoplasm that could function alone or in combination with viral encoded proteins in viral RNA translation, synthesis, and packaging [112]. Downregulation may be due in part to specific degradation of two host transporters, Nup 153 and p62. A full-length polyprotein is not

Polio and Its Epidemiology

observed in spite of being encoded by the single long open reading frame since posttranslational cleavage of the polyprotein is initiated as soon as the portion encoding the 2Apro has been translated. Many of the nonstructural proteins and intermediate cleavage products are multifunctional and act directly or indirectly at a number of steps in the RNA synthesis pathway (reviewed in [5, 27]). One example is the aforementioned viral encoded protease, 2Apro, that also shuts off host protein synthesis by cleaving eIF4G. eIF4G is required for translation of capped eukaryotic mRNAs, while the C-terminal of the cleavage product enhances IRES activity [27]. 2Apro is also important for negative-strand but not positive-strand RNA synthesis [115]. Another example is the intermediate cleavage product, 3CDpro, that also participates in the posttranslational processing of the polyprotein. The last protein of the polyprotein to be translated is the 3D polymerase, 3Dpol. RNA-primed RNA synthesis is initiated once the 3D has been released from the polyprotein reviewed in [27]. VPg-pUpU or VPg itself could act as a precursor for RNA synthesis by hybridizing to template RNA [51, 116]. The binding site for template and primer is on one side and that for VPg is on the other side. A replicate intermediate is formed and consists of a positive-sense RNA with 6–8 nascent negative-strand RNAs. The negative-sense strand serves in turn as template for synthesis of a 30-fold excess of new sense strand RNAs. Full-length dsRNAs can be isolated from infected cells. Altogether, the genomic RNA is amplified up to 50,000-fold. VPg is bound to both genomic RNA and negative-sense RNA. Poliovirus and other picornaviruses employ a quasispecies reproductive strategy [100, 117] where the lack of proofreading rapidly results in a mixture of progeny with modified genomes containing randomly positioned single nucleotide substitutions. Genomic recombination is a second method of evolution where a single event results in substitutions of many nucleotides from a different poliovirus or closely related non-polio enterovirus for the equivalent sequence in the original poliovirus. The majority of single nucleotide substitutions are deleterious or neutral;

Polio and Its Epidemiology

however, some may confer a reproductive advantage for progeny for growth in the current or future host and/or for host-to-host transmission. Evolutionary changes become “fixed” by selective outgrowth of individual members of the quasispecies that pass through bottlenecks within and between hosts [118]. Two evolutionary pathways, the very high number of progeny (>10,000 per infected cell) and outgrowth by chance selection and/or a selective advantage, result in one of the highest observed rates of molecular evolution [42]. RNA is synthesized from four nucleotides, two pyrimidines (uracil and cytosine) and two purines (adenine and guanine). The most common route for polioviral evolution is by nucleotide misincorporation (single nucleotide substitution) in the absence of both proofreading and postincorporation excision-repair pathways. The nucleotide position that is substituted is probably random but may be influenced to some extent by secondary structure and the adjacent nucleotides. Quasispeciation arises from the fact that the remaining progeny retains the original nucleotide at the position of each unique substitution in an individual progeny virus, while within the cloud of progeny, each isolate may have a unique substitution at a different position in the genome. Among the isolates that make up the quasispecies, substitutions should be found at each position in the genome at an equal frequency, at least in theory. However, substitutions are much more frequently observed in some positions than in others. Two related factors contribute significantly to the nonuniform distribution of observed substitutions along the genome. The first is that almost all observations have been made with RNA extracted from the quasispecies that arose during replication of a viable virus directly in the primary host or after amplification of one or more isolates from the quasispecies in vivo in a second host or ex vivo in tissue culture. The second is that substitutions in some positions produce nonviable or less fit offspring that are eliminated during this amplification process. Sequence-specific variability, based on the individual nucleotide base and its nearest neighbors [103], and inherent characteristics of the polymerase are other factors that contribute to

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the nonuniform distribution. If misincorporations were unbiased, transversions (the substitution of a pyrimidine by a purine or vice versa) would be expected to occur at twice the rate of transition (the substitution of a purine with a purine or a pyrimidine with a pyrimidine). However, empiric observations have revealed a polymerase-based bias of approximately ten to one in favor of transitions [42]. To currently include sequence data from the genomes of nonviable progeny requires either amplification of individual genomes by a process that does not require an active poliovirus infection but that includes high fidelity with proofreading and excision-repair (reverse transcribing the genomic RNA and cloning the cDNA of all viable and nonviable poliovirus progeny into plasmids that can be amplified in bacterial strains with high fidelity, proofreading, and error correction) or by direct sequencing of individual gnomes without amplification (chip/array sequencing technology) [102, 119]. Neither approach is currently very easy to apply since both would require individually processing large numbers of genomes equivalents, although Crotty et al. were able to calculate a rate of 2.1  102 substitutions per site by direct measurement of mutations in the VP1 of 55 cloned genomes after a single cycle of in vitro virus growth. Massive parallel next-generation sequencing may offer the best approach for analyzing viable and nonviable members of a quasispecies and for sequencing minor variant in homotypic viral mixtures [120– 122]. Wild poliovirus genomes frequently recombine with polioviruses and closely related nonpolio enterovirus genomes [123, 124]. This recombination can only occur during concurrent infection of a single cell by both parental isolates. Rarely, intratypic recombination may occur even within capsid proteins [125–127]. The noncapsid regions of polioviruses are most similar in sequence to other members of the enterovirus C genotype that includes Coxsackie A virus (CAV) serotypes 1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24, and these sequences are readily shuffled among polioviruses and the other members of this group [128, 129]. In fact, polioviruses show evidence of having evolved from

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C-cluster Coxsackie A viruses and may reemerge from them after eradication [129]. Interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of circulating vaccine-derived polioviruses, cVDPVs [130]. Recombination is not site specific, does not require extensive homology between genomes at the crossover site, and most likely occurs by an exchange of templates by the synthesis of complimentary RNA by the RNA-primed RNA polymerase rather than by breakage rejoining [131]. Intratypic (same serotype) and intertypic (different serotype) recombination in in vitro occurred at 1.3  103 and 7.6  106, respectively [131], while recombinations between polio and non-polio enteroviruses (NPEVs) occurred at a frequency of 106 [129]. Administration of trivalent oral vaccine anywhere and in areas where wild polioviruses and genotype C viruses co-circulate provides the conditions for concurrent infections and polio vaccine-polio vaccine, polio vaccine-wild polio, and polio vaccineNPEV, as well as endemic wild polio-NPEV recombinations. For examples of such recombinations, see molecular analyses of isolates from the cVDPVoutbreaks in Haiti and Dominican Republic [132] and Indonesia [133] and in individual cases [134]. Molecular epidemiology is the study of disease and factors controlling the presence or absence of a disease or pathogen using molecular data (DNA, RNA, or protein sequences). The next portion of this section will concentrate on those aspects of molecular epidemiology that impact on the epidemiology of polio. Polioviruses and other enteroviruses are among the organisms with the highest rate of misincorporation ([103, 117] and reviewed in [42]). Misincorporation comes at a high cost, namely, only approximately 10% of the >10,000 progeny from a single infected cell are viable [103]. This high frequency of misincorporation helps to explain the high ratio of physical to infectious particles [135]. Studies with ribavirin [102, 136], an antiviral drug acting as a nucleoside analog, have shown that the misincorporation rate of polioviruses is close to the catastrophe

Polio and Its Epidemiology

error rate, that is, the transition point where a modest increase in misincorporation results in a drastic decrease in viability. In these experiments, a 9.7-fold increase in mutagenesis resulted in a 99.3% loss in viral genome infectivity after a single round of replication, while a less than twofold increase in the natural mutational frequency resulted in a 50% loss of viability. Fitness is based on the overall performance during viral replication [103], a complex process, involving recognition of and binding to the host cell, uncoating/entry, initiation of protein synthesis before RNA replication takes place, regulation of replication and translation once RNA replication is initiated, culminating with assembly, maturation and externalization of mature virus, and survivability until subsequent infection of the next host cell or organism. Changes can affect more than one of these processes. For example, an increase in the mutational rate in infections in the presence of the nucleoside analog ribavirin not only led to an increase in nonviable genomes but also caused a reduction in the total number of viral genomes produced [102]. One of the advantages of the quasispecies nature of poliovirus offspring is that isolates with a selective advantage to new growth conditions may already exist in the population [102]. Studies on mixed infections in PVR Tg21-transgenic mice suggest that random selection may play a role in the selection of which genomic variants within a mixed infection in the gut infect the CNS since virus isolated from the CNS was not always the most neurovirulent [137]. Other experiments showed that increased fidelity of the polymerase reduced viral fitness in the PVR Tg21-transgenic mice [138] or in tissue culture [139]. Andino and colleagues proposed an alternate explanation for selection [140]. They provided evidence that the quasispecies was not just a collection of individual variants but a group of interactive variants and that fitness and selection may occur at the level of the population rather than at the level of individual genomes. In their study, an increase in polymerase fidelity affected viral adaptation and pathogenesis in addition to genome variability. Data supporting the suggestion that minor components can alter the

Polio and Its Epidemiology

phenotype of quasispecies comes from retroviral infections [141] and studies with VSV [142]. A number of studies have shown that single nucleotide misincorporations by the polio RNAprimed RNA polymerase accumulate at a more or less constant rate that can be used as a “molecular clock” to estimate evolutionary time between isolates and to determine whether sequence differences between two polioviruses isolated within a given time interval are consistent with a shared, direct evolutionary pathway between them [41, 42, 143, 144]. In general, the rate of accumulation and fixation of single nucleotide substitutions appears to be similar for all isolates regardless of kind (all three serotypes of wild, vaccine, or vaccine-derived polioviruses), type of polymerase (original intact polymerase or chimeric or complete recombinant from the same serotype, a different serotype or even a group C non-polio enterovirus), or type of infection (transient in immune competent individuals, persistent in immunodeficient patients, or even in the very elderly where waning immunity may play a role in selection) [5, 42, 110, 118, 132, 143, 145– 151]. Moreover, the rate of third codon position synonymous substations appeared to be fairly constant throughout the period of virus excretion in a persistently infected individual [152]. Using molecular observations from full-length genome sequences from viruses isolated during a 10-yearlong outbreak established from a single imported founder virus, Jorba et al. [42] calibrated five clocks based on five different classes of nucleotide substitutions. The constants for total substitutions (Kt), synonymous third position substitutions in coding regions (Ks), synonymous transitions (As), synonymous transversions (Bs), and non-synonymous substitutions (Ka) were 1.03  0.10  102, 1.00  0.08  102, 0.96  0.09 _ 102, 0.10  0.03 _ 102, and 0.03  0.01  102 substitutions/site/year, respectively. The rates were similar whether calculated using linear regression, a maximum likelihood/single-rate dated tip method, and Bayesian inference. The first two constants were mostly controlled by the third. As for saturation, third position synonymous transitions become evident by 10 years and complete saturate within

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65 years, while saturation of synonymous transversions was predicted to be minimal at 20 years and incomplete even at 100 years. This wide variation in calculated time constants depending on the type of substitutions together with differences in the estimated time until all possible changes become saturated provides a flexibility that allows one or more clock to be applied to characterize the range from evolution in outbreaks between very closely related isolates with short intervals between isolations to comparison between much more distantly related polioviruses or related enteroviruses. It is interesting to note that the molecular clocks are fairly constant given that intratypic and intrageneous recombination can result in complete or partial substitution of the polymerase whose intrinsic properties presumably govern the rate of misincorporation. Mutations may increase non-synonymous mutation rates [153], while others decrease them [138]. Multiple recombination events [125, 127] must be ruled out or taken into account when calculating time clocks based on the number of nucleotide differences. The genetic code introduces a bias in the position of observed substitutions. Substitutions in the third position of a codon are least likely to result in an amino acid change, and these synonymous substitutions are by far the most abundantly observed in wild poliovirus, polio vaccine, and VDPV infections [5, 118]. Non-synonymous substitutions that occur in the initial stages of vaccine infections restore replicative fitness and in many cases neurovirulence [4, 154], while those in persistent infections may influence receptor-virus interaction. Three-dimensional requirements also bias the observed distribution of substitutions. Maintenance of stem of the stem-loop structure in the conserved region of the 50 UTR especially within the IRES appears to be one of the major constraints on viability and variability. For example, complimentary paired double substitutions that maintained stem structure were frequently found in the loop V of evolutionarily related environmental isolates [151]; a single nucleotide substitution in a loop of domain V is a dominant determinant of attenuation of neurovirulence and

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growth at elevated temperatures [47]; and paired substitutions deliberately introduced into stem structures of domain V have increased genetic stability of new oral polio vaccine candidate strains [34]. Other examples of three-dimensional effects are mutations that occur at distances from or adjacent to functional sites that influence the viral response to antiviral drugs in the hydrophobic pocket [91], mutations that occur adjacent to amino acids that determine neurovirulence attenuation [155], and mutations that occur at the interfaces between proteins and at the N-terminals of VP1 and VP4 that may affect structural stability and the receptor-induced transitions [52]. Due to the complex nature of polioviral replication and multi-functionality of viral enzymes and viral three-dimensional structures, selective pressures that operate on one structure or function may affect another seemingly unrelated property. One of these apparent paradoxes is the fact that some RNA viruses including poliovirus may diverge antigenically in the absence of immune selection [156]. One of the features that distinguishes polio vaccine evolution during persistent infections in total B-cell-deficient immunodeficient patients from evolution during person-toperson transmission in immune competent but naïve individuals is that isolates from the former but not the latter have high numbers of amino acid substitutions in and around neutralizing antigenic sites [4, 5, 98, 151, 157]. Antibody titers tend to wane in the elderly. The finding of amino acid substitutions at or near neutralizing antigenic sites during infection of the elderly with type 1 monovalent mOPV [145] may suggest that waning immunity may create a situation resembling the early stages in establishment of persistence in immunodeficient patients. Since some of the amino acids and structural organizations are shared by neutralizing antigenic sites and receptor-binding sites, the high mutation rate in neutralizing antigenic sites is more likely the result from selective pressures governing receptor-virus interaction during establishment and maintenance of persistence than on nonhumoral-mediated immune selection or selection by variations in anti-polio antibodies in the IVIG regimens these immunodeficient patients receive

Polio and Its Epidemiology

to compensate for their B-cell deficiencies. This sharing of functions has also been suggested to be one of the reasons why there are only three serotypes of poliovirus [28, 87]. It is commonly accepted that the evolution of a fourth serotype would require receptor switching of a non-polio enterovirus to the use of the PVR, CD155. A somewhat paradoxical alternative for emergence of a fourth serotype may be through antigenic evolution during persistent infections in immunodeficient individuals as a result of selective pressures relating to receptor binding. Consistent with this is the observation that cohorts of immunized individuals who had high titers against vaccine strains had significantly reduced geometric mean titers against highly diverged neurovirulent vaccine-derived viruses that were isolated from environmental samples [151, 158] and individual titers against some of these isolates were 50% of poliovirus isolates had recombinant genomes [123, 176–179]. Supporting this is evidence from environmental surveillance where vaccine viruses with minimal divergence (0.5–1%) in their VP1 sequences had already recombined with polio and non-polio enteroviral genomes in regions encoding nonstructural proteins [180]. The ability to simultaneously reverse multiple mutations by recombination could foil efforts to develop improved oral vaccines. Introducing mutations that decrease the chance for reversal by single nucleotide replacement such as incorporation of polymerases with improved fidelity or a total redesign of the genome of each serotype based on rare codon usage and increasing the number of GC pairs [4, 34, 45, 181, 182] could be bypassed by recombination. Finally, the general consensus is that selective pressure or a higher mutation rate due to local sequence or secondary structure leads to a higher frequency of mutations at certain “hot spots” [135]. However, after reviewing the pattern of changes in substitution frequencies throughout the genome, it may be more accurate to think of the real frequency of substitutions as that observed in the so-called hypervariable region of the 50 UTR which may be under minimal selective pressure, and consider all other regions as “cold or colder spots” with lower observed rates of substitution derived from negative selection driven by the requirement for viability. Virion assembly, maturation, and release in picornaviral infections have been reviewed [27]. The ratio of viral particles to infection particles ranges between 30:1 and 1,000:1. Many of the viral particles are noninfectious due to lethal

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mutations in their genomic RNA and/or incomplete maturation. Some are viral like particles (VLPs), e.g., assembled viral capsids lacking genomic RNA. Polio VLPs are an alternative direction for producing safer vaccines [183]. Poliovirus Infections at the Level of the Individual Host The incidence of poliovirus infections is significantly higher in summer and autumn in temperate zones, becoming less seasonal as the environment becomes more tropical (reviewed in [4]). Improved sanitation and vaccination have reduced natural endemic infections in the very young and together with incomplete vaccine coverage has led to an increasing number of infections in older individuals. There are two major routes of host-to-host transmission. The most common and most efficient is fecal-oral, followed by oral-oral transmission as Dowdle et al. described for the fate of poliovirus in the environment and their review of the infectious dose for transmission in humans [184]. It has been postulated that there has been a shift from the former to the latter route, as the level of community hygiene improved [94]. The infective dose after ingestion of Sabin vaccine strains is approximately 100-fold higher than that for wild poliovirus, 1000 CCID50 compared to 10 CCID50, respectively [4, 184, 185]. Nerve damage in the lower spinal cord results in paralysis of the lower limbs (spinal poliomyelitis), whereas damage in the upper spinal cord and medulla may result in bulbar poliomyelitis and paralysis of breathing [108]. The percent of infections ending in paralytic poliomyelitis is further reduced in highly immunized populations. This ratio of asymptomatic cases to paralytic cases has implications for surveillance strategies based on investigation of all AFP cases. Between these two extremes falls the “minor disease” [108, 186], approximately 5% of infections with wild polio that result in abortive poliomyelitis with fever, fatigue, headache, sore throat, and/or vomiting, and another 1–2% result in non-paralytic poliomyelitis with aseptic meningitis, pain, and muscle spasms. The incubation period is between 7 and 14 days but ranges between 2 and 35 days

Polio and Its Epidemiology

[108]. Virus can be recovered from the throat, blood, and feces by 3–5 days. It was initially thought that viremia was infrequent, but this was based on observations in patients with paralytic poliomyelitis who most likely already had high circulating titers of neutralizing antibodies [108, 187]. However, when observations were made early after exposure, for example, in contacts of cases, a high frequency of viremia was demonstrated, implying that the viremia might play a vital role in the development of paralytic poliomyelitis [186, 188]. This was strengthened by concurrent experiments that demonstrated a protective effect against CNS lesions by antiserum in experimentally infected primates. The genetic basis for neurovirulence of poliovirus isolates is addressed below. Paralytic poliomyelitis, encephalitis, and aseptic meningitis occur after a biphasic infection where viremia in some systemic infections is followed by infection of the CNS [186, 188] (Fig. 5). Studies of virus in the CNS and stools in VAPP patients suggested that the virus that invades the CNS was randomly selected [137]. Acute flaccid paralysis (AFP) is a direct result of destructive replication in motor neurons followed by atrophy of de-enervated muscles. Skeletal muscles are affected when nerves in the anterior horn of the spinal cord are infected, and bulbar paralysis occurs when cranial nerves are infected [5, 189]. The maximum effect on muscles occurs within a few days after the start of symptoms. Muscle recovery can occur when infection only results in temporary loss of nerve function. Residual paralysis may last from months to the life of the infected individual [108]. Poliovirus infections are not the only cause of AFP. Non-polio AFP occurs with an incidence of 1 per 100,000 children for the implication this has for surveillance. Guidelines that help epidemiological investigators distinguish AFP caused by polio from AFP caused by other causes are reviewed in Sutter et al. [5]. Final diagnosis requires laboratory confirmation of a poliovirus infection. Poliovirus infections start as a local infection of cells in the tonsil, intestinal M cells, Peyer’s patch of the ileum, and mesenteric lymph nodes

Polio and Its Epidemiology

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Fecal-Oral Transmission Oral ingestion Tonsils Peyer’s patches

Barrier

Viremia

Sk e

let al

mu

sc

le

CNS Gut associated lymphoid tissue Ailementary tract

CSF

Ax M on m edia al tr ov te an es d sp as by or wh P V t ole re pa cep rti tor cle

Blood Brain Barrier

Brain Excretion Paralytic poliomyelitis Meningitis Encephalitis

Polio and Its Epidemiology, Fig. 5 Poliovirus infection and disease in individuals. Poliovirus is transmitted from host to host by a fecal-oral and, to a lesser extent, by oral-oral routes of transmission. Virus first infects cells in the tonsils, Peyer’s patches, and gut-associated lymphoid tissues, and viral progeny is excreted in feces. This phase is followed by a systemic infection during which there is viremia for a short period of time. In some individuals, the virus crosses the blood-brain barrier by entering the CSF, by axonal transport along nerve cells, and possibly from infected white blood cells that enter the brain. These

individuals may develop meningitis, encephalitis, or paralytic poliomyelitis. Destructive viral replication in nerves of the anterior horn of the spinal cord may lead to irreversible atrophy of de-enervated muscles, while bulbar paralysis occurs when cranial nerves are infected. Most (>90%) infections of naïve individuals even with the most neurovirulent strains are asymptomatic, and 5% result in meningitis, encephalitis, and/or transient paralysis. Only 0.1–1% of the infections will result in permanent paralytic poliomyelitis or death

[4, 108]. This replication in the gut results in the excretion of poliovirus during defecation by all individuals with asymptomatic as well as symptomatic infections and is the basis for fecal-oral transmission. It also provides the rationale for supplementary environmental sewage surveillance for poliovirus infections. A review of publications between 1935 and 1995 on excretion of polioviruses by Alexander and associates [190] indicated that in most infections of naïve children, wild polioviruses were excreted for 3–4 weeks with a mean rate of 45% at 28 days, and 25% of the cases were still excreting during the sixth week. In contrast, fewer than 20% excreted vaccine strains after 5 weeks. Excretion of polio ranged from a few days to several months [191]. The highest probability of detecting poliovirus-positive stool samples was reported to be at 14 days after the onset of paralysis [190] and

is the basis of stool sample collection for diagnosis of polio AFP surveillance. The disappearance of poliovirus from sewage samples and from stool samples of immunized children within 6–8 weeks after an immunization campaign [192] or after transition to exclusive immunization with IPV [193] provides additional confirmation for the short duration of excretion. Persistent poliovirus infections are the exception and will be discussed in more detail below. Interestingly, more than one evolutionarily linked lineage of the same serotype may co-circulate in the gut of such persistently infected individuals [118, 146, 151, 152] or in the gut and oropharyngeal mucosa [109, 111]. Excretion and the duration of excretion are dependent on host factors and on vaccination history of the infected individual. Immunization history may start with passive immunization from maternal antibodies. However, maternal

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antibodies have an estimated half-life of approximately 1 month [194]. Based on a comparison between titers in cord blood and at 6 weeks, the half-lives for maternal neutralizing antibodies against types 1, 2, and 3 poliovirus were 30.1 days, 29.2 days, and 34.6 days, respectively [162]. Immunization history obviously also includes polio vaccinations and natural exposure to endemically circulating wild poliovirus and waning immunity in aging cohorts. Skeletal muscle injury, including injury caused from intramuscular injections, increases the likelihood of poliomyelitis in children infected with wild or vaccine poliovirus. Mouse model studies have suggested that in this provocative poliomyelitis, the muscle injury facilitates viral entry to nerve axons and subsequent damage to the motor neurons in the spinal cord [195]. Some individuals who had poliomyelitis develop new muscle pains, hypoventilation, new or increased weakness or fatigue, and paralysis decades later after a period of relative stability. This reappearance of polio-related symptoms is referred to as post-polio syndrome. There is a large body of literature relating to post-polio syndrome that will be left up to the reader to pursue. Suggested starting points include the websites of the Post-Polio Health International (www.postpolio.org), the Mayo Clinic (www.MayoClinic. com), a 1992 paper on the “Epidemiology of the post-polio syndrome” by Ramlow et al. [196], and a 2010 review on the pathophysiology and management of post-polio syndrome by Gonzalez et al. [197]. There is still a debate whether persistent poliovirus or mutated poliovirus contributes to the development of post-polio syndrome [197]. The risk factors include the extent of permanent residual impairment after recovery from the poliovirus infection, an increased recovery after AFP possibly related to the extra stress on compensatory neural pathways and overuse of weakened muscles, the age of onset of the initial illness, and physical activity performed to the point of exhaustion. Natural infections with poliovirus stimulate both humoral and cell-mediated immunity (see [198, 199] and reviews [5, 200]). Neutralizing antibodies appear in exposed individuals around

Polio and Its Epidemiology

the time that paralytic symptoms become evident in the few individuals who develop symptomatic infections [108]. Neutralizing IgG and IgM antibodies are also induced in response to immunization with inactivated polio vaccine. The neutralizing antibodies induced after exposure to live or inactivated poliovirus prevent disease by blocking virus spread to motor neurons of the central nervous system [4]. Once seroconversion occurs after vaccination, individuals are protected from disease for life, although circulating antibody titers may wane late in life and may drop below protective levels against one or more serotypes in some individuals [201]. The epitopes on vaccine-derived and wild poliovirus strains that induce neutralizing antibodies may differ from those on vaccine strains. Neutralizing antibody titers 1:8 against each of the three Sabin OPV serotypes are considered protective; however, higher titers may be needed to compensate for the relatively lower antigenicity of wild and vaccine-derived strains [202]. For example, the highest serum neutralizing antibody titers were recorded from individuals immunized exclusively with OPV or IPV when the live challenge virus was the same as that used in vaccination, slightly lower for the respective heterologous strain, and significantly lower for wild and vaccine-derived strains. Serum from some individuals who had titers of >1:50 against Sabin vaccine strains had titers of 350,000 per year in 1988 to 33 in 2018 (for up to date number, see http://polioeradication.org/ polio-today/polio-now). Difficulties in reducing this further are discussed below. Criteria for quality control for production of polio vaccines introduced by the WHO in 1962 have been updated in relation to newly acquired knowledge about the epidemiology of poliovirus and polio vaccine. One of the major risks associated with the use of live vaccine is that progenies of the vaccine readily accumulate mutations, some of which may reverse attenuation. The highest risk for vaccineassociated paralytic paralysis, VAPP, comes from

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Sabin 3, the vaccine serotype that also has the highest variability across production lots [4]. However, the risk of OPV-associated polio is less than 0.3 per million doses [23] with the risk highest in naïve children receiving their first dose [123]. An additional risk occurs when vaccine strains loose attenuation and continue to pass from person to person resulting in outbreaks of cases [148, 218]. In fact, in 2018, there were 104 cases of paralytic poliomyelitis caused by these circulating VDPVs, in comparison with the 33 cases caused by wild poliovirus strains. The risk of not using oral vaccine for global eradication compared to its use at the current stage in the Global Polio Eradication Initiative remains overwhelmingly in favor of its use [1]. Three incidents nearly derailed early efforts to develop and employ effective vaccines. Firstly, and the most glaring of these, primarily from the point of views of negative publicity for use of polio vaccines, was the “Cutter incident” in 1955 where wild poliovirus was inadequately inactivated probably because of failure to remove clumps that may have sequestered and protected infective vaccine virus and a nonlinear tailing-off of inactivation at low titers [23, 219, 220]. Altogether, more than 400,000 children were inoculated with an inadequately inactivated vaccine batch produced by the cutter vaccine production facility which resulted in 94 cases of poliomyelitis among primary vaccinees, 126 cases in family contacts, and another 40 cases among community contacts and 10 deaths. The publicity caused great concern throughout the world until the cause was discovered and corrective measures applied. Secondly, apparent failure of early vaccines to protect against subsequent infection and paralytic disease due to an initial lack of awareness in the 1950s that there were three non-cross-reacting serotypes of polio has already been mentioned. And thirdly, the contamination of live polio vaccine with SV40 virus, a simian virus, continues to raise concerns about long-term effects from human zoonotic infection with this virus that was shown to cause cancer in mice [23, 221–224]. The SV40 was inadvertently introduced through the use of SV40-infected simian cell cultures in some early vaccine production batches. So far, there is little

Polio and Its Epidemiology

evidence for any contribution to the incidence of tumors in the humans who received SV40. The many vaccination formulation and vaccination schedules that have been employed during the effort to eradicate polio and the rationale for their use have been reviewed in depth [5, 215]. Changes in schedules and formulations mean that in any one region, different cohorts in the total population will have received different vaccine formulations and immunization schedules. This complicates determining duration of protection and interpretation of events. The evolution of vaccination policy in Israel [225–227], a graph showing the history of poliomyelitis in Israel (Fig. 6), and the two disagreeing discussions that were published within the same report on the underlying causes that enabled the last outbreak in Israel in 1988 [228] are a good example of this difficulty. Isolation of poliovirus with attenuated neurovirulence was a prerequisite for the development of oral polio vaccines (OPV; see reviews [4, 5, 217].). Vaccine candidates were either derivatives of neurovirulent or even highly neurovirulent (e.g., Sabin 3) isolates selected for attenuation after passage in primates, primate cell cultures, and/or non-primate cell cultures or starting from isolates with low neurovirulence (e.g., Sabin 2). Neurovirulence refers to the ability of an isolate to cause an infection adversely affecting functions of the CNS, keeping in mind that for any neurovirulent isolate, only 5% of infections cause transitory adverse CNS effects and less than 1% cause permanent paralytic poliomyelitis. The total number of nucleotide differences between vaccine strains and their respective parental strains was found to be 57 nucleotides and 21 amino acids for serotype 1 [229–231], 2 nucleotide differences and 1 amino acid difference for serotype 2 [232, 233], and 10 nucleotide differences and 3 amino acid differences for serotype 3 [234, 235]. Sequence analysis coupled with genetic manipulation has allowed investigators to pinpoint which of these nucleotide differences between vaccine candidates and vaccine strains account for the loss of neurovirulence (Fig. 3c) [135, 236]. “Quantitative determination of the contributions of each substitution is complicated

Polio and Its Epidemiology

a

283

Poliomyelitis in lsrael: number of cases and rate per 100,000 1000

100 Cases

90 IPV!

80

800

1957

70 60

600

50

OPV!

40

1961

IPV plus! booster ! 7y 2006 400

elPV & OPV! 1989

30 Last cases! 1987–88

20

OPV booster! 6–7y1999

Number of cases

Rate per 100,000

Rates

200

10 0 51

b

57

63

69

75

81 Year

87

93

99

05

0

Polio vaccine immunization schedules in lsrael 1957–2010 1957–1960 exclusive use of IPV (mainly local production) varied number of doss (2 to 4 ) varied quantity of antigen per dose initially administered in campaigns then by routine vaccination schedule! estimated coverage 75% to 90% 1961–1963 exclusive use of OPV coverage 95% 1961 mOPV1 1962 add mOPV2 and mOPV3 1963 tOPV3 dose schedule (2,6 and 12 months) 1964–1978 4 doses by (2, 4, 6, 24 mo) coverage 81% to 91% 1979–1981 same as in 1964-78 plus a supplimental mOPV1 once a year for ages 0–2 yrs 1982–1987 three programs a. 4 OPV doses in 12/14 health sub-districts b. 4 OPV doses as in a. plus one dose mOPV1 in selected groups at risk c. exclusive eIPV 3 doses in 2 to 14 health sub-districts at least one dose in combination with DTP. 1989–2005 combined eIPV/OPV 2 doses of eIPV (2 and 4 mo) 2 OPV doses (4 and 6 mo) simultaneous IPVplus OPV at 12–16 mo) 1990 IPV booster added at 6–7 yr 2006–2010 exclusive use of eIPV 4 doses of eIPV (2, 4, 6, 12 mo) plus booster 7 yrs 1957–2010 Immigration of families with children who were vaccinated by different vaccination schedules used at their countries of origin.

Polio and Its Epidemiology, Fig. 6 Prevention of poliomyelitis through universal vaccination and evolving vaccination strategies as illustrated by the history of cases and vaccination schedules in Israel between 1957 and 2019. Figure (a) represents the annual number of cases (blue bars) of laboratory confirmed poliomyelitis cases and the rate per 100,000 children (red line) between 1951 and 2010. The red arrow indicates the last cases of poliomyelitis that occurred during an outbreak in 1987–1988. Israel has been poliomyelitis-free since 1989. Black arrows indicate major

changes in vaccination policy. Previous attack rates of 14.2 and 146.9 per 100,000 in 1949 and 1950, respectively, signaled the transition from endemic to epidemic epidemiology of poliomyelitis in Israel. A full list of vaccination schedules is indicated in (b). (Data presented in (a) was supplied with permission by the Israel Center for Disease Control. The vaccination schedules in (b) were taken from Swartz TA. The Epidemiology of Polio in Israel A Historical Perspective. Tel Aviv: Dyonon Pub. Ltd.; 2008 [225], Shulman et al. [324] and Kaliner et al. [265])

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by several factors: (a) The role of minor determinants of attenuation is difficult to measure, (b) some substitutions have pleiotropic effects on phenotype, (c) some Sabin strain phenotypes require a combination of substitutions, (d) second-site mutations can suppress the attenuated phenotype in various ways, and (e) the outcome of experimental neurovirulence tests may vary with the choice of experimental animals (monkeys versus transgenic mice) or the route of injection (intraspinal versus intracerebral) [4].” The propensity of vaccine to evolve and revert to neurovirulent phenotype is discussed throughout the current review. The safety and effectiveness of live attenuated polio vaccine strains in preventing poliomyelitis were very clearly demonstrated in large clinical studies involving millions of children in the 1950s [20]. A number of factors including vaccination schedules, the presence of maternal antibodies, hygiene, and nutritional status of the individual influence the efficiency of induction of seroconversion by OPV strains. Early studies showed that viral interference between strains in the trivalent vaccine and from concurrent infections with nonpolio enteroviruses also influences vaccination outcome [20]. The take of OPV is negatively influenced by the presence of maternal antibodies. Nonetheless, when infants are fed OPV at birth, 30–60% excrete virus, 20–40% of infants seroconvert, and the subsequent take of OPV is better when a birth dose is given (reviewed in [237, 238]). Multiple doses of OPV are recommended to ensure seroconversion rather than to boost waning immunity [238]. The number of OPV doses that is needed to reach 90–95% seroconversion rates in naïve children is not the same for all populations. For example, three doses will seroconvert 90–95% of naïve children in developing countries, whereas in certain regions within developing countries such as India, the same three doses will only seroconvert a maximum of 60% of vaccinees [238]. Supplemental immunization activities (SIAs) employed sometimes more than once a year are needed to ensure adequate primary vaccination coverage and to fight endemic circulation of wild poliovirus or reintroductions of wild poliovirus. In SIAs, all children in national or

Polio and Its Epidemiology

subnational regions are immunized in national immunization day (NID) and/or subnational immunization day (SNID) campaigns, respectively, with a dose of OPV irrespective of vaccine history. The costs of the additional doses needed to raise seroconversion rates to above 90% significantly raise the cost for effective immunization with OPV and require the coordination and use of many paid and voluntary staff. cVDPVoutbreaks may occur when coverage is much lower than 90% [239]. In fact, in the end, it may actually be easier to immunize three times with IPV (even at current costs) than with the additional number of doses of OPV especially when access to populations is difficult and environmental conditions challenge maintenance of viability of the live vaccine. This counters both the lower cost and difficulty of administration rationales for using OPV instead of IPV. Mass immunization campaigns have rapidly boosted herd immunity [4]. Vaccination formulation must also take into account differences in the efficacy of induction of intestinal immunity by the different vaccine serotypes [206]. For example, type 2 was 100% effective with two doses, whereas types 1 and 3 needed more than three doses. Since the elimination of wild type 2 in 1999 [240], and the significant decrease in the number of endemic regions where wild types 1 and 3 co-circulate, SIAs have increasingly turned to the use of monovalent and divalent OPV. Monovalent OPV vaccines improve seroconversion rates compared with tOPV [241]. In 2015, here was a globally coordinated removal of OPV2 from tOPV vaccines to prevent reemergence of VDPV2 and a switch to bOPV (serotypes 1 and 3 only) [239]. New guidelines for the use of mOPV1, mOPV2, and bOVP (1+3) were issued in 2009 [242]. Routine immunization should include the use of at least one dose of IPV to prevent the accumulation of large cohorts of individuals who are naïve to type 2 poliovirus and who could serve as a reservoir for transmission of neurovirulent type 2 VDPV, as has occurred and is still occurring in Nigeria [148, 239]. Research has been underway to develop a new, safer form of OPV2 (nOPV2) to minimize the

Polio and Its Epidemiology

risk of any resulting cVDPV or VAPP cases since mOPV2 is still planned to be used in any supplementary immunization activities (SIAs) required to control current and potential future outbreaks of poliovirus type 2 after the global switch from the tOPV to the bOPV vaccine. A global consortium of governmental, nongovernmental, academic, and global health organizations and investigators have been working together to develop novel live attenuated OPV type 2 (nOPV2) vaccine candidates, which would be significantly less likely than existing OPV2 to cause VAPP or cVDPV. The continued emergence of new clusters of cases of poliomyelitis after the withdrawal of OPV2 from tOPV from use of mOPV2 in SIAs to control ongoing cVDPV2 outbreaks has provided a strong incentive to develop new candidate strains of OPV2. Two monovalent serotype 2 oral polio vaccine candidates, #1, S2/cre5/S15domV/rec1/ hifi3, and #2, S2/S15domV/CpG40, are currently undergoing clinical trials in humans [45, 46]. Both candidates are genetically modified polioviruses that are genetically stable and hyperattenuated. Both candidates have the S15 modification of domain V of the 50 NCR (Fig. 3d). Intentional genetic modifications result in multiple AU pairs in place of the thermodynamically more stable GC pairs in the stem of stem-loop secondary structure of domain V. Reversion by single nucleotide substitutions would require two successive nucleotide substitutions involving a less stable intermediate stem structure. Candidate #1 has two additional modifications in the 3D polymerase t increase stability of attenuation and translocation of a key replication element, cre, from the 2C coding region to the 50 UTR to inhibit recombination. For Candidate #2, series of synonymous nucleotide substitutions were introduced into the P1 capsid coding region to form GpC dimers to reduce replicative fitness and potentially improve stability of the attenuated phenotype while reducing transmission. Phase 1 trial was conducted in containment facilities for 28 days, since it involved use of live type 2 poliovirus after global containment of PV2 under GAP III. In phase 1 trial, both candidates were immunogenic and had acceptable tolerability. Median neutralizing antibody titers rose 8.0-fold in participants who

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received #1 and 12.7-fold in those who received #2. nOPV2 was detected in stools of all 15 volunteers vaccinated with #1 and most (13 of 15) with #2. Median shedding lasted 23 days with #1 and 12 days with #2. However, the shedding period was longer than the containment period for 7 participants who received #1 and 4 who received #2, and the longest period of shedding was 89 days for #1 and 48 days for #2. Reversion to neurovirulence of virus recovered from the last stools with sufficient titer to test in transgenic mice low absent and sequence analysis demonstrated no loss of attenuation in domain V of the 50 UTR. Of interest is that nasopharyngeal swabs taken on days 0, 3, 7, and 28 were all negative for poliovirus. Phase 2 trials are currently underway since both candidates were safe, stable, and immunogenic. Additional nt substitutions were introduced into domain V of the 50 UTR of OPV3 to successively reduce thermodynamic stability and produce polioviruses that had increased stability and hyperattenuation compared to the S15 candidate #1 isolates described in the previous paragraph [34]. These have been designated S17, S18, and S19, respectively (Fig. 3d). Decreasing thermostability was correlated with increasing temperature sensitivity. The next step was to prepare five additional “new oral vaccine-like strains” by substituting the P1 capsid sequence of OPV3 with the P1 capsid sequences of OPV1, OPV3, IPV1 (Mahoney), IPV2 (MEF-1), and IPV3 (Sauket) strains. A second set of six new oral vaccine-like strains was designed to include an amino acid substitution in nonstructural viral protein 2A that allowed polioviruses with this substitution to produce much higher yields when poliovirus is grown in Vero cells. The S19 variants maintained their extreme stability and hyperattenuation upon passage in Vero, Hep2, and MRC-5 cells. None of the S18 and S19 OPV3 constructs were neurovirulent. Thus, if an S19 were to revert to an S18 strain, it would still be safe. It took longer for cultures infected with S19 variants to reach full CPE than unmodified polioviruses with the same capsid genome; however, CPE was complete by 24–48 h. The Containment Advisory Group, CAG, after reviewing data from

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these studies has concluded that the S19 series of polioviruses with and without the amino acid substitution optimizing growth in Vero cells can be considered for use outside of the containment requirements of Annex 2 and 3 of GAP III for IPV production, rat neutralization IPV potency assays, human serum neutralization tests for poliovirus antibody titer determination, and potency testing for human immunoglobulin lot control and release (up-to-date CAG reports can be downloaded from http://polioeradication.org/ tools-and-library/policy-reports/advisory-reports/ containment-advisory-group/). The use of inactivated wild poliovirus strains was an alternative approach to vaccination against polio (reviewed in [215]). Salk developed an inactivated polio vaccine, IPV, using neurovirulent strains of the three serotypes of poliovirus. IPV was successfully tested by a placebo-controlled trial in over 400,000 children and in unblinded observations on another 1,000,000 children before certification for use in the mid-1950s [17, 18, 243]. A relatively higher difficulty in production, greater production costs, higher difficulty in administration, and the initial belief that only live vaccine would efficiently evoke intestinal immunity led to the choice of OPV for most routine national vaccination programs [165]. Countries are currently switching to vaccination with IPV in combination with OPV or more often in place of OPV because of improvements in manufacture that have increased effectiveness and reduced the cost difference between a dose of OPV and IPV, and the paradoxical success of OPV in reducing poliomyelitis as an epidemic disease in most countries [215], and its relative safety record, specifically no VAPP cases and no emergence of cVDPVs that behave like wild polioviruses [180, 244, 245]. Emergence of cVDPVs must be prevented in order to attain final success for poliomyelitis eradication. Early studies on genetic and antigenic variation such as a study of Sauket strains, the type 3 used in the production of IPV [246], were instrumental in the establishment of rigorous standards for seed stocks for vaccine production. The original IPV formulation has since been improved. This enhanced IPV, eIPV, has a higher immunogenicity

Polio and Its Epidemiology

and protective efficacy than IPV [208]. A number of factors contributed to this improvement. These included new production protocols, new tissue culture techniques including a microcarrierbased technology, and a more optimal balanced formulation of the three serotypes. It can be administered alone or can be combined with other vaccines such as DTP. The immunogenicity of eIPV was at least as good as that of OPV, and there was good long-term immunity [208]. Subdermal administration of fractional doses of IPV was one of the approaches tried in the early 1950s [17]. Subsequently, seroconversion rates from fractional doses were shown to be adequate but somewhat lower than for full-dose intramuscular injection fractional doses [162, 163, 247]. Ironically, recent shortages in availability of IPV during transition from tOPV to bOPV [248] and safety issues during vaccine production have restimulated interest in use of fractional doses and new clinical trials for fractional doses, with and without use of adjuvants, and possible use of needle-free devices (non-inclusive example of recent references: [162, 163, 249–252]). Substitution of Sabin strains or the new candidate OPV strains for IPV strains in a new inactivated polio vaccine (sIPV) to provide safe, affordable next-generation inactivated poliovirus vaccines is another alternative that is currently being explored [80, 82, 248, 251, 253– 255]. Chumakov et al. [82] suggested four possible solutions to the problem of lower stability and immunogenicity of Sabin OPV strains when inactivated to be used for IPV: (1) increase antigen content to a level that would ensure adequate seroconversion (disadvantages: it requires growing greater quantities of virus made more difficult since yields of Sabin strains are lower than wild-type viruses; increases costs); (2) stimulate immunogenicity with adjuvants; (3) explore use of alternative inactivating agents that do not damage antigens as much as formaldehyde; and (4) make IPV from new, genetically stable, nonpathogenic, hyperattenuated engineered poliovirus strains with antigenic structures identical to the currently used wild-type strains [34, 256]. One of the intended uses of the S19 domain V poliovirus constructs discussed above for live

Polio and Its Epidemiology

vaccines is to use them for safely producing IPV and sIPV [34]. Antiviral drugs offer a promising complimentary or alternative approach to the use of vaccine to control poliovirus infections especially for persistent infections in immunodeficient individuals, during the final stages of eradication, and for posteradication reemergence [165]. Presumably, these drugs may also work to control severe infections by non-polio enteroviruses or have been chosen because they have been shown to do so. Drugs with unique virus-specific targets such as capsid proteins, the hydrophobic pocket, the RNAprimed RNA polymerase, protease inhibitors, protein 3A inhibitors, nucleoside analogs, proteinase 2c inhibitors, and compounds with unknown mechanisms of action have been reviewed [257]. There is still a long way to go to find truly effective universal anti-polio or antienteroviral drugs; thus, only a few examples will be provided. Pocket factor drugs such as WIN 51711 51711 [113], isoflavines [91, 155], pleconaril [258], and capsid inhibitor V-037 also called pocapavir [259, 260] prevent viral entry by interfering with receptor binding or by preventing conformational changes needed for viral capsid uncoating. One of the difficulties in developing pocket factor drugs comes from the quasispecies nature of enteroviral infections, where mutants may rapidly emerge [91, 99] or there may be viral isolates already present in the quasispecies that have mutations in the capsid that may either render the isolate resistant or even dependent on the drug for growth. Furthermore, these resistance mutations may not even have to be at the drug binding site (see [91]). Ribavirin is a drug that normally interferes with mRNA capping. While enterovirus mRNA is uncapped, the polio polymerase can incorporate ribavirin into both negative- and positive-strand progeny RNA molecules increasing mutagenesis above the catastrophe limit causing a decrease in the reproductive capacity of the viruses [102, 136] (see discussion on 3Dpol polymerase). Passive immunization has also been tested as a means of preventing polio. Administration of immunoglobulin shortly after exposure to polio

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may reduce the incidence or severity of paralytic disease although its general use is not practical due to the short time during which it is effective [261]. Intravenous preparations of immunoglobulin prepared from human populations exposed to enteroviral infections have however helped to decrease chronic meningoencephalitis infections by these enteroviruses in agammaglobulinemic patients [262]. Regular intravenous treatment may help prevent poliomyelitis in immunodeficient individuals but may not prevent virus replication and shedding [98]. Passive immunization with immunoglobulin or human milk rich in antipolio IgA together with another antiviral pleconaril may have helped to resolve at least one persistent poliovirus infection [168]. However, efforts to cure another persistent poliovirus infection with human milk and ribavirin, or other antiviral treatments, did not prove successful [99], and this individual has continued to excrete highly diverged vaccine-derived poliovirus for more than 30 years [98, 263]. Anecdotally, shedding of intestinal mucosa associated with a superinfection with Shigella sonnei may have helped to cure another persistent excretor [98]. Poliovirus Infections in Populations Poliovirus infections in populations have been the subject of many reviews over the years. The older reviews are still of interest not only because of the information they review but because they also provide a picture of policies and knowledge available at the time. The following paragraphs will concentrate on those aspects of poliovirus infections in populations that impact the most on the endgame strategy of poliomyelitis eradication. The discussion will start with a brief overview of the changing nature of the epidemiology of poliovirus infections. This will be followed by a description of the Global Polio Eradication Initiative and will end with a discussion of the three main problems that have led to a delay in its realization, namely, “failure to vaccinate,” “vaccine failure,” and “vaccine-derived polioviruses.” When reading this section which will highlight some of the current problems and their solutions, the reader must keep in mind the overwhelming success of the Global Polio Eradication Initiation

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to date: a major reduction in the number of endemic countries where polio is still transmitted from 126 to 2; a decrease in the number of annual cases by >99% that prevented lifelong paralysis in more than five million children between 1988 and 2005; eradication of one wild poliovirus serotype, type 2, in 1999; the probable eradication of a second wild serotype, type 3, in 2012, and elimination of the majority of wild lineages throughout the world. The nature of poliovirus infections in populations has gone through a number of phases. Before the appearance of outbreaks of poliomyelitis starting in the nineteenth century, poliovirus circulated endemically. Infections occurred in the very young and conferred lifelong immunity against reinfections with the same serotype. The epidemics became more frequent and grew in size, and infections included older children and adults who were not naturally immunized. Vaccination has drastically reduced the number of people who have been exposed to natural infection with wild polioviral strains. There also appears to be a shift in person-to-person transmission routes. Oral-oral transmission has increased in importance, while fecal-oral transmission decreased as a result of improved hygiene [94]. Control of poliomyelitis requires breaking all chains of wild poliovirus transmission by immunizing all children with three doses of polio vaccine or at least enough children so that herd immunity protects the remaining population. The percent of the population that needs to be immunized for establishment of herd immunity against wild poliovirus is >85% for developed countries and >90–95% in tropical developing countries. In 2010, there were still three groups of countries: four “endemic countries,” Afghanistan, India, Nigeria, and Pakistan, where the transmission of wild viruses has not yet been completely halted; “polio-free countries” where vaccination has broken all endemic chains of wild poliovirus circulation, and there have been no cases other than VAPP within the last 3 or more years; and “importation countries” that were formerly polio-free, but where there are poliomyelitis cases caused by wild poliovirus imported from one of the “endemic countries” and where there may be local transmission of the imported wild

Polio and Its Epidemiology

poliovirus. Between 2002 and 2006, there were 26 importation countries, 7 with viruses imported from India and 19 with viruses imported from Nigeria [5]. There were 21 importation countries in 2009 and 13 in 2010. These included ongoing outbreaks in Tajikistan and the Russian Federation and apparently expanding outbreaks in Angola and the DRC. The former represents the first cases from wild poliovirus in the European Region since regional eradication was declared in 2006, and the latter had the potential to spread to polio-free countries in Africa and other regions. All wild type 1 poliovirus cases that have been reported since 2018 occurred either in Pakistan or Afghanistan, the only countries where WPV has continued to circulate endemically. Countries using IPV exclusively in routine vaccination programs and that have vaccine coverage in excess of 90% are not necessarily protected against transmission of wild or VPDV strains. The penetration and sustained transmission of WPV1 in Israel in 2013–2014 in the absence of AFP cases is one example [264–268]. The list of importation countries is updated on a weekly and monthly basis and can be accessed at the web page of the Global Polio Eradication Initiative, www.polioeradication.org. Transmission routes are dependent on population movements. One or more outbreak founders may be introduced by infected persons coming from an endemic or importation country or by returning travelers from such countries. One event with very high risk for spreading poliovirus from one country to another is the Hajj in Saudi Arabia. To counter this threat, it is now mandatory for foreign pilgrims coming on Hajj and Umrah to be vaccinated for communicable diseases including polio, especially pilgrims with young children arriving from countries with polio cases. The children must have received a dose of OPV 6 weeks before their arrival and another upon arrival. Smallpox was eliminated as a circulating human pathogen in 1977 after an 11-year extensive vaccination and surveillance program [269]. Only two sources of smallpox virus have been reserved for research purposes, one in the United States and one in Russia. Final eradication will be achieved when these last two remaining,

Polio and Its Epidemiology

contained sources of smallpox virus are finally destroyed. Proposing a similar approach in 1988, the World Health Assembly set a goal of eradicating poliomyelitis by the year 2000 (resolution WHA41.28). A group of experts at the global, regional, and country level set the criteria and conduct the process of certification of eradication [270]. These experts must be independent from the vaccine administration and polio laboratories. Global polio eradication, first targeted for completion by 2000, was limited to the eradication of all wild polioviruses with the caveat that “the occurrence of clinical cases of poliomyelitis caused by other enteroviruses, including attenuated polio vaccine viruses, does not invalidate the achievement of wild poliovirus eradication” (report of the first meeting of the Global Commission for the Certification of the Eradication of Poliomyelitis, Geneva, World Health Organization, 1995. WHO document WHO/EPI/GEN/ 95.6). The aim was that OPV vaccination would cease within a few years after eradication of poliomyelitis from wild polio, and industrialized nations would save not only the large sums of money needed for the maintenance and rehabilitation of individuals with paralytic poliomyelitis but the costs of vaccine and vaccine administration as well [271–274]. In order to easily eradicate an infectious agent, (a) the agent should replicate in a single host with no intermediate vector, alternative reservoir species, or carrier state; (b) vaccines and/or antiinfectious agents must be available to break chains of transmission whether directly between susceptible organisms or after exposure to the infectious agent in the environment; (c) if transmission involves environmental exposure, there must be a finite and relatively short survival time for the infectious agents in the environment; (d) all or the majority of infections should be clinically apparent with unique symptoms; and (e) there must be an easy and cost-effective surveillance system for detection of the infectious agent, for identifying infected individuals, and for determining the efficacy of treatments in individuals and populations [4, 275]. Deviations from some of these requirements for eradication have made eradication of polio more difficult from the

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start [94]. In particular, most (>95%) poliovirus infections are clinically asymptomatic, while symptoms associated with the few infections that result in poliomyelitis are not unique to poliovirus infections, and although effective vaccines were available, there is no easy and cost-effective method to determine the effectiveness of vaccination in vaccinated individuals. This makes surveillance and the identification and isolation of infected individuals much more difficult. When the GPEI resolution was passed in 1988 and even as late as 1996, it was stated [276] that there was no indication of chronic excretors. However, persistent infections do exist and have emerged as one of the difficulties in achieving eradication, and there is evidence of at least two instances where VDPVs circulated [277, 278]. Differences between smallpox and polio have made it relatively more difficult to achieve polio eradication. For example, live polio vaccine is made from three temperature-sensitive serotypes of poliovirus because there were three non-cross-reacting poliovirus serotypes, whereas the vaccine for smallpox was a single more stable unrelated bovine virus. This has complicated vaccine formulation and administration. In addition, since the vaccine contains live poliovirus, there are also a number of more severe safety issues concerning pre- and post-eradication vaccine production compared with the smallpox vaccine. Specific eradication strategies for polio included (a) high routine immunization with at least three doses of vaccine for all children and an additional birth dose in countries where polio has remained endemic; (b) SIAs, either national or subnational immunization campaigns targeting children under 5 years of age; (c) surveillance, primarily investigation of all cases of AFP with an increase in the number of supplementary surveillance programs such as sewage surveillance and enterovirus surveillance as the endgame of eradication approaches; and (d) house-to-house mopping-up immunization campaigns to block final chains of wild polio transmission [1, 214, 271]. The WHO requires genomic sequencing of all isolates of potential interest to the Global Polio Eradication Initiative. An isolate is of interest when results from either standard immunological

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or molecular tests conducted by accredited laboratories using standard methods (see next section) indicate that the isolate has behaved differently than standard Sabin strains of the corresponding serotype. A Global Polio Laboratory Network, GPLN, was established to monitor poliovirus infections throughout the world using standardized methods, cell lines, reagents, and reporting methods [279– 283]. These standard methods (WHO/EPI.CDS/ POLIO/90.1) have been reviewed and revised and improved as knowledge about the epidemiology of polio expands and as new analytical methods become available. There are two classes of methods, recommended methods and approved methods. Recommended methods are those that have been tested and validated and for which the WHO maintains cell lines, reagents, equipment, and supplies for distribution to laboratories of the GPLN network. Accepted methods are alternative methods that have been validated and demonstrated to also provide acceptable results and can be used instead of the recommended method, but methods for which the WHO is not obligated to provide support in the form of cell lines, reagents, equipment, and supplies. Revisions have included even the flow charts or “algorithms” for culturing viruses, identifying polioviruses, and characterizing the serotype (typic differentiation) and determining wild, vaccine, or vaccine-derived virus within specific time frames (intratypic differentiation). The current fourth version of the Polio Laboratory Manual (WHO/IVB/04.10) was adopted in 2004. An updated version is scheduled for completion by 2020. The three levels of laboratories, national and subnational laboratories, regional reference laboratories, and global specialized laboratories, are certified each year through on-site visits, after accurate testing and timely reporting of a minimum number of relevant assays, and by results from proficiency tests. The requirements for certification, quality assurance, and safety and the responsibilities of the three types of laboratory are spelled out in the Polio Laboratory Manual (WHO/IVB/04.10). By the end of 2009, the GPLN consisted of 146 laboratories of which 139 were fully accredited by the WHO and another 5 in the process of accreditation

Polio and Its Epidemiology

(WHO 16th Informal Consultation of the Global Polio Laboratory Network, September 2010, Geneva, Switzerland). The polio laboratories are coordinated on a regional basis by regional laboratory coordinators who report to the global WHO polio coordinator in Geneva. Identification tasks such as intratypic differentiation and sequence analysis originally assigned to the more specialized labs are now being certified for use in national and regional reference labs as expertise increases and methods – especially molecular methods – are simplified. In fact, today, it is more correct to refer to three levels of laboratories based on their ability in increasing order of difficulty to isolate viruses (viral isolation or VI laboratories), conduct intratypic differentiation (viral isolation and intratypic differentiation or VII laboratories), and conduct genomic sequencing (viral isolation, intratypic differentiation, and sequencing or VIIS laboratories). This trend was driven by the increasing difficulty and costs of shipping material that may contain live infectious wild polioviruses between the different levels of laboratories, the need for decreasing the time between isolation and final notification of characterization of the poliovirus isolates, and easy access to new technologies. Rapid identification is especially critical for eradication efforts in endemic regions, for identifying introductions of polioviruses to polio-free regions from these endemic regions or emergence of VDPVs and for identifying and controlling outbreaks when they occur. The global specialized laboratories continue to maintain additional testing and analytical abilities. GPLN laboratories work in close coordination with epidemiologists and medical staff in the investigation of all cases of AFP and/or supplementary enterovirus surveillance and with municipal employees and epidemiologists where supplemental environmental surveillance is utilized to screen for poliovirus presence and circulation. In late 2010, a commercially available method for preparing non-infective viral RNA suitable for molecular analysis based on automatic nucleic acid extraction, immobilization, and storage on Flinders Technology Associates (FTA) filter papers was being evaluated to increase safety

Polio and Its Epidemiology

and drastically reduce costs of shipping material between laboratories (Summary and Recommendations of the 16th Informal Consultation of the Global Polio Laboratory Network, 2010, Geneva). Using this technology, viable virus could be reconstituted from the RNA after it is transfected into eukaryotic cells. Intratypic differentiation (determination of the serotype of an isolate) was based on results from one immunological ELISA test [284] and one molecular-based test, either probe hybridization [285], diagnostic RT-PCR [286], RT-PCR and RFLP [287], real-time RT-PCR [288], or microarray-based systems [119]. At the 16th Informal Consultation of the Global Polio Laboratory Network, 2010, Geneva, the recommendation for ITD testing from ITD-accredited laboratories was modified to one of the following three options: (a) two real-time RT-PCR procedures, one for ITD and one for detecting vaccinederived poliovirus and sending all non-Sabin-like viruses or Sabin-like viruses with non-Sabin-like VP1 to higher-level labs for full-length sequencing and molecular analysis of VP1, (b) one validated ITD method (ITD, or molecular) and shipment of all viruses to higher-level labs for full-length sequencing and molecular analysis of VP1, or (c) on-site full-length VP1 sequencing of all isolates or referral of all virus isolates to higher-level labs for full-length sequencing and molecular analysis of VP1. Results are confirmed by sequence analysis of the entire VP1 capsid gene. Molecular analysis of the sequences of the genomic RNA encoding the VP1 capsid gene is in fact the definitive method to determine whether an isolate is a vaccine strain, a VDPV, or a wild isolate. Sequence analysis of the VP1, all four capsid genes (e.g., P1), and even the entire genome infers evolutionary relatedness to other isolates in endemic or external reservoirs. The methodology and results from such analyses that help trace the origin of viruses founding outbreaks have been clearly presented in reviews by Kew et al. [143] and Sutter et al. [5]. Sequence data is kept in databases maintained by the specialized laboratories of the Polio Laboratory Network, such as the CDC in Atlanta, GA, Pasteur Institute in Paris, and the HTL in Finland. Phylogenetic

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comparisons of sequences from new isolates, routinely provided by the CDC, indicate evolutionary relationships to previously isolated polioviruses from the same region and trace importations to or from external reservoirs. Important epidemiological information can be obtained from this phylogenetic analysis. For example, a significant gap between a new sequence and all other known sequences indicates a gap in surveillance, while an importation implies that there are cases or silent circulation of related viruses in the region containing the reservoir that it is most closely related to. Knowledge obtained about time clocks [42] for nucleotide substitutions allow investigators to infer whether nucleotide differences between two isolates are consistent with local transmission or represent separate introductions (e.g., see Manor et al. [144]). Three major types of surveillance are currently available for detecting poliovirus infections by WHO-accredited GPLN laboratories: (i) the gold standard, acute flaccid paralysis (AFP) surveillance; (ii) supplementary enterovirus surveillance (EVS); and (iii) supplementary environmental surveillance (EnvS). Integration of multiple surveillance programs that are performed in parallel improves reliability and strengthens interpretation of data when conducting risk analysis [289]. The currently recommended standard method for poliomyelitis surveillance is based on the isolation and molecular and serological analysis of all viruses from all cases of acute flaccid paralysis, AFP, to rule in or rule out polioviral etiology [290]. The definition of an outbreak varies depending on whether endogenous poliovirus transmission has remained unbroken or the area has been found to be polio-free. In the latter, given the goal of eradication and the number of asymptomatic infections, a single AFP case can be considered to be an outbreak. The previous paragraphs describe what is needed for timely high-quality testing of all poliovirus isolates from cases and from other sources such as environmental surveillance. Much time, effort, and money has been spent on maintaining and improving lab quality assurance and performance of laboratories in the Global Laboratory Network. However, two factors outside of the control of the

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laboratories strongly influence the final result. The first is sample collection and the second is the conditions under which the sample is stored and shipped to the first processing lab. The most appropriate sample with the highest probability of detecting poliovirus is a stool sample of approximately 5 g. For AFP cases, two stool samples (not rectal swabs) should be collected 1–2 days apart within 14 days of onset of paralysis. This is based on a review of studies measuring the timing of viral excretion in infected individuals [190] and the fact that detectable viral excretion is sometimes intermittent. Standardized tissue culture conditions using limited passages of poliovirus-sensitive L20B, HEp2C, and RD cells provided by specialized laboratories of the Global Polio Laboratory Network are used according to standard operating procedures to isolate polioviruses from clinical samples [290]. An amended algorithm for isolating polioviruses designed to reduce the workload and the time between receipt of sample and identification of viruses of interest has been successfully evaluated in a number of national poliovirus laboratories (WHO 16th Informal Consultation of the Global Polio Laboratory Network, September 2010, Geneva, Switzerland). Standard typing and intratypic differentiation assays are based on results from serological assays and molecular assays as described above with final characterization based on the full-length sequence of VP1. Additional regions such as the 50 UTR, the entire P1 region encoding all four capsid proteins, the 3D polymerase, or the entire genome may be sequenced for higher resolution and to determine whether and to what extent genomic recombination has occurred. Molecular data from any polioviral isolates recovered from the stool samples provides information about the serotype of the isolate or isolates and may be sufficient to differentiate between VAPP, persistent VDPV, and circulating VDPV or wild polioviruses. The different time clocks for single nucleotide substitutions [42] and unique recombination patterns are important tools for these analyses. Timely AFP surveillance also provides the necessary critical information about the temporal and geographic distribution of the isolates for efficient and economical infection and outbreak

Polio and Its Epidemiology

response. The rationale for AFP surveillance is based on the observation that AFP from all nonpolioviral causes occurs with an incidence of 1 per 100,000 in children up to the age of 15. When all AFP cases are investigated and the AFP incidence is within the range for non-polio causes, the absence of poliovirus in the stool samples from any AFP case is considered to indicate absence of circulating poliovirus in the region under surveillance. A surveillance area is considered to be wild poliovirus-free when adequate AFP surveillance levels for greater than 3 years indicate absence of wild poliovirus, and the entire WHO-designated regions are considered to be free from endogenous wild polioviruses when all countries within that region are wild poliovirus-free. Sequence analysis of poliovirus isolates, as was performed for isolates from Mumbai, India, can confirmed cessation of local chains of transmission and/or the reintroduction of viral lineages still circulating elsewhere [291]. Wild poliovirus-positive AFP cases in previously polio-free areas or WHO regions can occur. Molecular analysis then reveals the most likely external reservoir from which the virus was transmitted [143]. Three examples of country-to-country transmission which have seriously impacted the eradication initiative are the spread of wild polioviruses to >21 polio-free countries [292] as consequences of the temporary cessation of vaccination in Nigeria starting in 2003; the spread of wild polio into the European Region in 2010 [293] enabled by low vaccine coverage in Tajikistan; and the spread of WPV1 from Pakistan to Egypt [294], Israel where there was sustained transmission in the absence of AFP cases [295], and Syria where there were at least 37 AFP cases [296, 297]. Most countries employ AFP surveillance. However, not all are able to reach the required incidence of AFP investigations. Some of these countries supplement AFP surveillance with enteroviral surveillance and/or environmental surveillance. Enteroviral surveillance, EVS, is the systematic identification of the enterovirus genotypes in all clinical infections in general or more specifically from all cases with associated meningitis and encephalitis, symptoms that may appear more

Polio and Its Epidemiology

frequently than AFP in patients with poliovirus infections (approximately 5% of poliovirus infections compared to 0.5–1% for AFP). In some countries, enteroviral surveillance and/or environmental surveillance is used exclusively. “Guidelines for enterovirus surveillance (EVS) for polioviruses have been established [298]. EVS for poliovirus infections targets the 5% of unvaccinated individuals who develop less severe symptoms after infection by polioviruses [298]. According to WHO Guidelines [298], four types of information need to be available for an effective EVS Program. The same information can be used to evaluate the sensitivity and specificity of an EVS program for detection of poliovirus infections. The types of information include (i) a clear case definition for inclusion in the surveillance program; (ii) the proportion of the total population that is under surveillance by the system; (iii) the proportion of patients with specific clinical illness that are actually tested for enteroviruses; and (iv) information on diagnostic procedures, e.g., the types of clinical samples, the laboratory procedures used to test these specimens, and the quality management programs employed by those laboratories during each diagnostic test and in general” [299]. Countries in the WHO European Region used different inclusion criteria for EVS that included investigation of one or more of the following clinical presentations: acute gastroenteritis infections; Guillain-Barré syndrome cases; meningitis and encephalitis; and respiratory infections; and/or in some cases (in alphabetical order): acute hemorrhagic conjunctivitis only or all conjunctivitis; AFP (but not as part of an AFP surveillance program); ataxia; cases associated with outbreaks, but not sporadic cases of meningitis, respiratory illness, or acute gastroenteritis (AGE); cases with EV-like symptoms; cerebral edema; diseases of blood circulation; enterovirus positive blood (cases with EV or EV(+) RNA isolated from blood); enterovirus positive CSF (cases with EV or EV(+) RNA isolated from CSF); exanthema; febrile diseases, sepsis, and severe infections in neonates; febrile rash; febrile syndrome (nonspecific fevers); handfoot-and-mouth disease (HFMD); heart diseases; hepatitis; immune deficiencies; laryngotracheitis;

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low birth weight; mental anorexia; metabolic diseases; mobility disorders; muscle diseases; myocarditis; myositis; n. facialis; neurologic manifestations of acute onset identified by syndrome surveillance in refugee camps; neurological symptoms; neuropathy; pancreatitis; paralysis; paraplegia; paresis; pericarditis; residual stools from microbiology labs (“convenient samples”); seizures; sepsis; septicemia; sudden death; vacuities; and vesicle fluid [299]. The absence of uniform inclusion criteria makes comparisons between programs difficult. A number of different sampling techniques have been used to obtain environmental samples including grab sampling during peak sewage usage, trapping with silicates or gauze, and automatic composite sampling of sewage aliquots at given time intervals over a 24-h period (Guidelines for environmental surveillance of poliovirus circulation, WHO/V&B/03.03 [300] and Environmental Surveillance Guidelines Working Draft, March 2015 [301]). All sample storage and shipment must be at low temperatures (4–8  C) to maintain viability since currently certified tests require an amplification step in tissue culture. The FTA technology trial referred to above may eliminate the need for maintaining low temperatures. The usefulness of L20B cells to isolate polio isolates in the presence of high titers of non-polio enteroviruses [291] has already been mentioned. It is still important to characterize the viruses that grow, since L20B cells can also support growth of some other human and bovine enteroviruses, as well as less well-characterized viruses [94]. Additional steps involving molecular screening and/or growth at elevated temperatures have enabled investigators to more easily identify and characterize wild and vaccine-derived polio in the presence of high titers of vaccine viruses [41, 144, 285, 286, 288, 302]. Selective growth of nonvaccine poliovirus at elevated temperatures [303] is based on a relative small decrease of titer for these viruses compared to a much higher reduction in yield for vaccine strains at elevated temperatures. The main molecular determinant responsible for this difference is a single nucleotide change in loop V of the 50 UTR which can

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revert or be modified by other changes; hence, some polioviruses of interest may escape detection and some minimally diverged vaccine virus may be included among the selected isolates. Confidence that polioviruses isolated by environmental surveillance reflect circulating viruses comes from the high sequence homology between environmental samples and isolates from cases [193, 304, 305]. One of the major contributions of environmental surveillance reviewed by Hovi et al. [193] is that it can be used to establish the presence and/or circulation of wild or vaccinederived polioviruses before the appearance of AFP cases [144, 291, 304, 306, 307]. Environmental surveillance has also revealed the presence of presumptive primary vaccinees excreting OPV in Switzerland where vaccination is by exclusive use of IPV [94] and wild type 2 poliovirus shed in 2017 as a result of infection after accidental exposure to WPV2 in a vaccine production facility [308]. Different methods for analyzing environmental samples are also currently employed in different laboratories, since unlike AFP surveillance [300, 301], there is as of yet no single standard recommended method. The probability of detecting poliovirus in environmental samples [307] depends on the duration and amount of poliovirus excreted by one or more infected individuals, the effect of physical and mechanical factors on the dilution and survival of poliovirus in the sewage system (reviewed in [184]), and the frequency of collection and laboratory processing of the environmental samples [300]. A model based on these factors [307] predicted that environmental surveillance could outperform AFP surveillance for small outbreaks as well as detect circulation before the appearance of cases. The location of the sampling site relative to the excretor and the number of excretors (Fig. 7) also determines the probability of detection [193]. In general, polioviruses can be quantitatively recovered from the environment [309– 313]. Decreasing this distance between the excretor and the sample collection site is more effective in increasing the probability of detection and less labor-intensive than increasing the sampling frequency [151]. Environmental

Polio and Its Epidemiology

surveillance is resource- and labor-intensive and may require large capacity high-speed centrifuges that are not commonly present in most national poliovirus laboratories [309]. It requires judicious choice of potential target populations, a competent laboratory, a plan for routine surveillance and reporting, and the cooperation of municipal authorities. The WHO has recommended principles for selecting sites, sampling strategies, and interpretation of results [300, 301]. Lengthy periods of poliovirus-free monitoring are needed to confirm that poliovirus transmission has stopped since even the most comprehensive surveillance covers only subgroups of the entire population of potential excretors [314]. Many poliovirus-positive environmental samples contain one or at best a few polioviruses of interest indicating that the surveillance is operating at the lower limits of detection. Thus, while negative findings cannot rule out the presence of polioviruses at levels below detection, they gain significance when they are part of a long sequence of negative results from frequent routine surveillance at the given site. A positive finding of a wild poliovirus or a VDPV can trigger a response ranging from public announcements to remind individuals scheduled for routine vaccinations to be vaccinated in time in areas with high vaccine coverage to scheduling NIDS or SNIDS in regions where immunization coverage is below that required for establishing herd immunity. Sequence analysis can differentiate between multiple importations and local circulation when more than one poliovirus is isolated within a short interval of time [144]. Detection of “orphan polioviruses” or viruses that are not closely linked to previously sequenced isolates indicates gaps or suboptimal surveillance. The length of the gap is roughly proportional to a nucleotide substitution rate of 1% per year in the gene encoding VP1 [42]. Orphan viruses [133, 147, 245] were the earliest identified isolates in most cVDPV outbreaks. This is in contrast to the situation in Nigeria where intensive AFP surveillance triggered by the circulating wild polioviruses also revealed the initial stages in circulation of multiple lineages of predominantly type 2 cVDPVs [148] and during follow-up of

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Polio and Its Epidemiology, Fig. 7 Population-based environmental surveillance for poliovirus. The figure is a schematic representation of a network of sewage drainage pipes leading to a sewage treatment plant starting from individual homes, schools, or places of work or entertainment (thinnest lines) and converging into larger and larger trunk lines (thicker lines) until entering the treatment plant. There is an in-line automatic sampler at the inlet to the treatment plant (green arrow). Portable automatic samplers like those illustrated in the photograph (black arrow) can be lowered into sites at branch points to determine which of the branches contain virus detected by downstream

sampling sites. Yellow squares represent virus excreted by a single infected individual living at the periphery of the system (large yellow square). Large red circles represent the situation where more than a single individual is infected and the viruses that they excrete are represented by the smaller red circles. The incrementally increasing black numbers in the circles represent upstream the order in which the portable samplers can be placed at major branch points to approach and determine the location of the single excretor. (Adapted from Hovi et al. [193] and Shulman et al. [409])

mOPV2 SIAS [239]. The presence of type 2 cVDPVs, iVDPVs, and aVDPVs is of concern since despite elimination of transmission of wild type 2 poliovirus in 1999, neurovirulent serotype 2 poliovirus is still among us [167, 171, 239]. In 2017, a limited trial of a new artificial genetic variant of OPV2, nOPV2, was conducted under containment conditions in a GAP III compliant contained village for a time interval thought to be sufficient for cessation of post-vaccination

shedding. At the end of this time, five participants, for whom the end of nOPV2 shedding was not proven, returned to the Netherlands where one participant continued shedding nOPV2 for another 24 days. In this case, no nOPV2 was recovered from a single environmental surveillance sample collected from a site downstream of the shedders’ home. A number of countries that switched from OPV or combinations of OPV and IPV to exclusive use

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of IPV conducted environmental surveillance for OPV after the transition (reviewed in [193]). The OPV rapidly disappeared from the environmental samples, but imported OPV-like isolates have been isolated from time to time presumably imported from OPV-using countries [315]. One of the important milestones toward achieving eradication is the containment of all potential sources of the pathogen. In 1999, a process for containing all laboratory stocks of wild poliovirus was initiated by the World Health Assembly entitled Global action plan for the laboratory containment of wild polioviruses or GAP 1 (WHO/V&B/99.32). A revised plan, GAP II, included two phases: (1) the identification in all facilities of all known poliovirus stocks and any material that could potentially contain live wild poliovirus, for example, any stool specimens that were collected at times when poliovirus was endemic, and (2) the containment of these stocks by destroying them, rendering them noninfectious, or transferring them to a minimal number of laboratories certified by the WHO as poliovirus essential facilities (PEFs) having appropriate BSL3/polio biosafety facilities and justification to work with wild viruses. All other non-PEF laboratories would be prohibited from using or storing the proscribed poliovirus containing materials. GAP III is the WHO Global Action Plan to minimize poliovirus facilityassociated risk after type-specific eradication of wild polioviruses and sequential cessation of oral polio vaccine use (GAP III) [174]. The first of the eradication milestones was reached in September 2015 when WPV2 was declared eradicated [239]. A draft of the next version, GAP III, extends containment of wild polio to now also include containment of OPV/Sabin strains and concentrates on minimizing risks associated from facilities that work with polioviruses and vaccine production and storage facilities after eradication of wild poliovirus transmission and cessation of OPV vaccination. Declaration of eradication of WPV2 triggered phase 2 of GAP III [174]. Pathways of exposure from these facilities and assessment of the risks from a literature review have been calculated [184]. Gap III has three annexes that are relevant for the

Polio and Its Epidemiology

biosafety and containment of polioviruses in PEFs and non-PEFs. Annex 2 deals with biorisk management standard for essential poliovirus facilities holding WPV materials, e.g., PEFs. Annexes 2 and 3 of GAP III apply to poliovirusessential facilities; Annex 6 applies to facilities that may encounter poliovirus materials in the course of their activities, but will not store those materials. Annex 3 deals with biorisk management standard for essential poliovirus facilities holding only OPV/Sabin; and Annex 6 deals with safe handling of new samples potentially containing poliovirus material in nonessential laboratories, e.g., non-PEFs. After risk analysis, a goal was set to reduce the number of such facilities globally to 1,000 [210]. One month after the last vaccination, they were challenged with an additional dose of OPV. Up to 60% of children excreted at least one OPV serotype between 1 and 3 weeks, the upper range of similar studies reviewed in that report. There was no evidence of transmission to siblings or mothers of these children, most likely because of good hygiene [210]. These rates were comparable to other similar studies [210]. Hygiene and high coverage probably also contributed to the fact that there was also no evidence for OPV circulation in IPVvaccinated populations in the United States living adjacent to OPV-vaccinated populations in Mexico [325].

Polio and Its Epidemiology

A third example of vaccine failure occurs when the vaccine itself is defective. The example here is the Cutter incident discussed above where children were vaccinated with incompletely inactivated, neurovirulent IPV [23, 219, 220]. Emergence of vaccine-derived polioviruses (VDPVs). A number of comprehensive reviews on vaccine-derived polioviruses have been published [4, 98, 118, 147, 157, 326–332]. As described above, vaccine-derived polioviruses evolve either during person-to-person transmission (cVDPVs) or during relatively rare [333], persistent infections in immunodeficient patients (iVDPVs) (see [4, 5, 147, 157]). By 2010, reports indicated 12 cVDPV outbreaks [148]. Using the definition of outbreak in the context of eradication (i.e., even a single case), the number of outbreak is even higher. For example, in 2009, 21 cases due to cVDPVs were found in four countries in addition to 153 in Nigeria and a case in Guinea traced back to Nigeria [334], and the cases in Nigeria represent emergence of multiple independent lineages [148]. The current numbers and geolocations of cVDPV cases in the world can be found on the GPEI website using the URL: http://polioera dication.org/polio-today/polio-now/ Most outbreaks caused by cVDPVs have been caused by a single lineage that spread rapidly through a susceptible cohort within the general population. Most outbreaks were only discovered after silent circulation of the VDPVs for more than 1 year or more, which indicates gaps in surveillance. By the time such outbreaks became evident, the genomes of the isolates had usually recombined with those of the progeny of other vaccine serotypes or closely related non-polio enteroviruses. When mOPV is introduced or reintroduced into a population with cohorts of naïve individuals as in Nigeria, adequate surveillance revealed that in early stages of reemergence, more than one independent lineage may emerge [148, 239]. There is also a potential for recombination of cVDPVs with wild-type viruses in areas where both co-circulate as shown from retrospective molecular analysis of isolates during endemic circulation of wild polioviruses [124]. Luckily, from the point of view of eradication, cVDPV outbreaks resemble outbreaks of poliomyelitis from

Polio and Its Epidemiology

wild polioviruses introduced into polio-free areas, and their chains of transmission can be broken by similar vaccination responses [148, 316]. In certain circumstances, poliovirus can establish persistent infection in immunodeficient individuals. The types of immune deficiencies of known chronic excretors have been reviewed [4, 5, 98, 147, 218, 331, 335–337]. The genomes of the Sabin strains are unstable [157], and reversions of nucleotide changes that attenuate neurovirulence appear even among the progeny virus excreted by primary OPV vaccinees. These reversions are believed to improve the replicative fitness of the isolates [166] and are responsible for the rarity of vaccine-associated paralytic poliomyelitis cases (VAPP; 1 per 500,000–1,000,000 vaccinations of naïve infants and 7,000 times higher for some immunodeficient individuals) and cVDPV outbreaks [4, 147]. Thus, it is not surprising that reversion of attenuation also occurs at an early stage in chronic excretors [98]. During 4 months of observation of long-term excretion in a healthy child [98], type 1 virus diverged by 1.1% and evolved toward full reversion to wildtype phenotypic properties similar to the Mahoney parent of the Sabin 1 strain. It is less obvious why these isolates so quickly predominate in the quasispecies of persistently infected immunodeficient individuals. The process by which persistence is established and maintained may present selection through bottlenecks within a single individual that is similar to the bottleneck by which only a single progeny or a subpopulation of the quasispecies is passed onto the next host in person-to-person transmission among immune competent hosts. Selection by passage through bottlenecks was also suggested to explain evolution of wild poliovirus during long-term expression [338]. Amino acid changes in capsid proteins were thought to allow polio to establish persistence in cells of the CNS [339]. Examination of the genomes of iVDPV isolates differentiates them from cVDPVs in that significantly fewer heterotypic recombinations occur [5, 110, 340] and intrageneous recombination appears to be largely absent [5, 147]. Interestingly, more than one highly divergent lineage may be recovered from a single stool sample from persistently

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infected individuals [4, 110] or from the gut and oropharyngeal mucosa [109, 111]. This suggests that persistence and evolution occur in separate sites although intratypic recombination indicates that some mixed infections in a single cell must occur. Only a single serotype was detected in most (30 of 33) long-term excretors identified between 1962 and 2006 [5] and by 2018 was the case for 132 of 139 reported cases of long-term excretors of poliovirus (Shulman, unpublished). Molecular analysis of phylogenetically related highly diverged (>10%) aVDPVs isolated from sewage in Finland, Slovakia, and Israel reveals a pattern of amino acid substitutions in or near neutralizing antigenic epitopes and lack of intrageneous recombination that resembles the pattern of changes in iVDPVs and is qualitatively different from evolutionary changes in cVDPVs [151, 315, 340]. This pattern and the extended periods of time over which phylogenetically related polioviruses have been isolated from the same sewage systems and surveillance sites within those systems strongly suggest that replication of the related viruses has taken place in one immunodeficient host or a very limited number of individuals in contact with such a host. Routine monthly sewage surveillance of catchment areas representing 35–40% of the population in Israel intermittently and repeatedly revealed the presence of highly diverged type 2 VDPVs 2 between 1998 and 2012. Phylogenetic analysis indicated that the isolates came from two different and unrelated persistent infections [289]. Isolates from one of these aVDPV2 epidemiological events were isolated intermittently between 1988 and 2012 and the second between 2001 and 2011. In additional, highly diverged isolates of aVDPV1 have been found in sewage collected in Haifa between 2002 and 2019. The situation in Finland is particularly interesting and unusual, since evidence suggests that the infected individual is simultaneously and persistently infected with three highly diverged VDPV serotypes [315]. Most mutations in iVDPVs and aVDPVs are synonymous and are observed in third position codons. These synonymous substitutions occur at similar rates to those for poliovirus during person-to-person transmission [42, 143, 151,

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152, 340]. Non-synonymous amino acid substitutions affect antigenicity, neurovirulence, receptorbinding motifs, hydrophobic pockets, and drug sensitivity. The prevalence of aVDPV excretors is unknown, but additional countries with excretors of aVDPVs are being reported as environmental surveillance is introduced into more and more regions [315, 329, 341–344]. Between January 2017 and June 2018, 28 aVDPVs were isolated from 7 countries, compared with 11 countries in the previous year [239]. Most (82%) were from mOPV responses to cVDPv2 outbreaks. Proposals to regularly monitor cVDPVs through increasing the number of laboratories that employ routine environmental surveillance are being adapted [193, 345–347]. It is important to determine the exact nature of the immune status of these types of persistent excretors since it may be different than that for identified persistently infected individuals who were primary B-cell immunodeficient individuals [5, 147] [331]. Types of primary B-cell deficiencies included AGG, CVID, HGG, HLA DR, ICF syndrome, MHC II, PID, SCID, and XLA. It is unfortunate that the individuals infected with and excreting these aVDPVs remain unidentified and will most likely remain so for a long time. The most frustrating attempt to locate such an excretor occurred in Slovakia where moving sampling sites progressively upstream successively restricted the excretor to a population of 500 individuals before detection ceased [193]. There is no consensus on the extent that persistent VDPV infections may affect the realization of eradication [180, 214, 319, 336, 348– 351]. Determining the number of unidentified persistent infections is becoming more urgent as eradication of wild polio approaches (WHO 16th Informal Consultation of the Global Polio Laboratory Network, September 22–23, 2010, Geneva, Switzerland, WHO/HQ. Summary of Discussions and Recommendations). Some researchers believe that VDPVs may pose an insurmountable problem [157, 170], while others feel that the problem is less severe [4, 5, 169]. Molecular epidemiological analysis has indicated that highly diverged neurovirulent aVDPVs isolated from

Polio and Its Epidemiology

sewage in Finland, Slovakia, and Israel [151, 315, 352] resemble the molecular epidemiology of poliovirus isolates excreted over time by identified excretors of iVDPVs, and the exact nature of the immune status that has presumably enabled infection to persist in these aVDPV excretors remains unknown. The time course of excretions, the rate of nucleotide substitutions in virus isolated from identified persistent excretors, and genomic recombination patterns have been consistent with the establishment of persistence and evolution of the virus in these individuals rather than transmission of an iVDPV. Highly diverged iVDPVs (as opposed to cVDPVs and less diverged iVDPVs) have not been found during routine AFP surveillance of cases of immune competent individuals [4]. Clear indication that iVDPV-like aVDPVs are transmissible comes from a study of silent transmission in infected children in an undervaccinated community in Minnesota [278] where 8 of 23 infants had evidence of type 1 poliovirus of VDPV infection and from Spain [277] where 3 of 7 family contacts excreted VDPV2, one of whom excreted VDPV 2 for 216 or more days. While this absence of documented transmissibility of very highly diverged VDPVs is encouraging, it may only be circumstantial, since most of the highly diverged neurovirulent aVDPVs have been found in the environment of countries with high vaccine coverage and good hygiene barriers that have also prevented circulation and appearance of wild poliovirus cases even after neurovirulent wild polio was introduced from external reservoirs [144]. An alternative explanation, suggested above, is that amino acid substitutions in neutralizing antigenic epitopes/receptorbinding residues in iVDPVs and iVDPV-like aVDPVs help establish and maintain persistence in specific microenvironments containing different combination or densities of CD155 and other secondary poliovirus receptors, and that this specialization renders it more difficult for the iVDPVs to transmit to other individuals. These same changes might affect/reduce transmission via the oral-oral route in communities where there is high vaccine coverage and good hygiene. If true, this might significantly reduce the threat to

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eradication, despite the highly neurovirulent nature of these isolates in animal model systems and the decreased geometric mean neutralizing antibody titers against some of these excreted iVDPV and aVDPV isolates in the general public in the highly immunized communities where these isolates are found [151, 315]. It must also be taken into account that identified and unknown excretors are free to travel to communities with poor vaccine coverage and substandard hygiene where the fecal-oral transmission route is more important and continuous person-to-person transmission easier to maintain.

Future Directions: The Endgame Stage of Eradication and Sustainability Afterward This section will start with a brief overview of current accomplishments to provide a suitable background for the discussion of future directions and sustainability. One of the best online sources for keeping up to date on eradication can be found at www.polioeradication.org. One of the most important problems that must be successfully dealt with is to answer the question: “How can current achievements and eradication be sustained once the endgame has been concluded?” Ideally, a major public health undertaking such as eradication requires a cost-benefit analysis, sufficient funds at the beginning, the means for achievement at hand, and the political and social will to carry the process through to the end. Delays and problems with fundraising, especially when they occur during the endgame, may derail the entire effort [1]. New unanticipated scientific problems may appear which further delay polio eradication. After all, awareness of (1) the potential problems from cVDPVs in communities with low vaccine coverage and (2) the presence of chronic excretors of VDPV only became evident as the endgame of eradication approached and the global burden of cases had decreased by >99% and/or when appropriate analytic tools to document and confirm VDPV became widely available [4, 353]. There has been a >99% overall reduction in the number of cases since adoption of the Global Polio Eradication Initiative in 1988 [334]. For up-

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to-date number, see http://polioeradication.org/ polio-today/polio-now. The Region of the Americas (AMR) was the first region to be certified to be free from all three serotypes of indigenous wild polioviruses in 1994 [354, 355]. Subsequently, the Western Pacific Region (WPR) in 2000 [356], the European Region (EUR) in 2002 [357], and the Southeast Asian (SEA) Region in 2014 were also certified free from all three serotypes of indigenous wild polioviruses. The African Region could be certified wild poliovirus-free at the end of 2019 or beginning of 2020 assuming that there are no new cases of AFP due to WPV. This will leave the Eastern Mediterranean Region (EMR) as the only region where endemic transmission of wild type 1 poliovirus still exists (specifically in Pakistan and Afghanistan). The last case anywhere in the world from wild type 2 polio occurred in India in 1999 [240, 358], and wild type 2 poliovirus was declared eradicated in September 2015 [239]. The last reported AFP case due to wild poliovirus type 3 occurred in Nigeria in 2012 [359]. A declaration of eradication of wild type 3 poliovirus is therefore imminent. These successes have been due to the dynamic nature of the eradication program where vaccination strategies have been adapted in response to specific problems and to changing conditions emerging as the endgame approached [1]. A number of decisions affecting sustainability were made in the last few years. Some were based on incomplete knowledge, because of the long lead time needed between planning facilities and final production of regulatory agency-approved products. For example, it was clear that fractional doses of IPV significantly reduce costs; however, it was not known whether or not they were less effective than full doses. Since 2004, there have been a number of clinical studies of effectiveness of fractional doses and use of adjuvants and on alternative methodologies for vaccinating individuals that are helping to fill in informational gaps. To date, there has been insufficient time to gather data on kinetics of waning. Questions concerning sufficient antigenicity of Sabin IPV have been partially answered. Differences in antigenic structure of inactivated polio vaccines made from

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Sabin live attenuated and wild-type poliovirus strains effected equivalency of vaccine potency assays used to measure the D-antigen content of the vaccines [255]. This required development of new international standard, IS 12/104, for sIPV [80, 81]. (1) Financial Considerations for Sustainability: Financial considerations are one determinant for sustainability of the Global Polio Eradication Initiative. The financial requirements for the transition period are complicated and have been set forth by the WHO (WHO Global Polio Eradication Initiative – Programme of Work 2009 and financial resource requirements 2009–2013 WHO/POLIO/09.02). The bottom line is that alternatives to OPV must be affordable [316]. We need to succeed in reaching the endgame before financial resources dry up, and we need financial reserves to maintain success during the years following eradication. The accumulated costs for the vaccination program exceeded 4.5 billion US dollars by 2004. The WHO Strategic Advisory Group of Experts on Immunization (SAGE) has developed a series of Polio Eradication and Endgame Strategic Plans (PEESPs). The PEESP 2013–2019 added another $7 billion [360], and the current PEESP for 2019–2023 will add up to another $4.4 billion [361]. A growing annual gap between financial needs and funds on hand is of concern [362]. This cost is offset by a $40 to $50 billion benefit between 1988 and 2035 estimated by Duintjer et al. for successful eradication [363], most importantly is the reduction in lifelong human suffering. National governments (listed by alphabetical order: Australia, Austria, Belgium, Canada, Denmark, Finland, Germany, Ireland, Italy, Japan, Luxembourg, the Netherlands, Norway, the United Kingdom, and the United States) have provided a significant portion of the necessary funding. NGOs (WHO, UNICEF, Rotary International, the Bill & Melinda Gates Foundation, and the International Red Cross and Red Crescent societies), the World Bank, and corporate partners (Aventis Pasteur, De Beers) have also made significant contributions toward purchase of vaccines and for applied and basic polio research. In addition to paid professional staff, more than ten million volunteers

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have assisted in the global vaccination program. Their knowledge of local practices and beliefs has provided a significant asset to the GPEI [1]. Reports that deal in depth with the economics and practicality of universal replacement of OPV with IPV include (a) Global Post-eradication IPV Supply and Demand Assessment: Integrated Findings, March 2009; (b) The supply landscape and economics of IPV-containing combination vaccines: Key findings, May 2010, both commissioned by the Bill & Melinda Gates Foundation and prepared by Oliver Wyman, Inc.; and (c) Improving the affordability of inactivated poliovirus vaccines (IPV) for use in low- and middle-income countries – An economic analysis of strategies to reduce the cost of routine IPV immunization, April 20, 2010, prepared for PATH by Hickling, Jones, and Nundy. The second report [364] presents a thorough review of the current options and risks for new vaccines and vaccine formulations for achieving and maintaining eradication that existed in 2010. New generations of inactivated polio vaccines are in the process of being developed and clinically tested, and results will effect endgame and for post-eradication planning. One of the goals of eradication was to reach a stage where vaccination could be discontinued, as was the case for smallpox vaccinations after eradication of smallpox [274, 365–367]. The estimated annual savings would be enormous and could be used to fund other global health initiatives. Widespread vaccination will continue at least during the 3-year period between the last case due to wild poliovirus and certification that wild poliovirus transmission has been interrupted globally. In fact, global vaccination should be continued for much longer. One model [368] predicted that after 3 years, there would only be a 95% certainty that all silent circulation had in fact ceased and the probability after 5 years ranged between 0.1% and 1%. A more recent model predicted a very high probability of reemergence within 10 years after eradication by VDPVs or accidental release of virus from vaccine production facilities, a polio laboratory, or bioterror [369]. Both models imply that vaccination will need to continue for at least 10 years after

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eradication. [Note: A special issue of the journal Risk Analysis (volume 26, Issue 6, 2006) was devoted to risks associated with polioviruses before, during, and after eradication of wild poliovirus.] Some of the projected financial benefits from discontinuing use of polio vaccines may not be achieved since there are some experts who feel that polio vaccination may have to be continued indefinitely [82, 170, 370], at least in countries where aVDPVs continue to be isolated from the environment and possibly in countries with vaccine production facilities (see also discussion on GAP III containment). Current contingency plans for use of OPV in response to reemergence need to be revised based on the data on circulation of live vaccine strains after temporary and/or partial cessation of vaccination [160] and from information from clinical trials on genetic stability, transmissibility, and the relative ability of heterotypic enteroviruses to recombine with nOPVs. An indirect financial advantage is that the organizational capabilities experience expertise and facilities including laboratories in the Global Polio Laboratory Network may be employed to solve other health-related problems (for example, in Nigeria, some staff in the Centers for Disease Control and Prevention will use their technical experience with polio to improve measles surveillance, routine measles vaccination coverage, and measles outbreak investigation and response in high-risk areas [371]). (2) Reagents and Methods Needed for Sustainability: A second pillar for sustainability of the GPEI depends on having appropriate reagents (vaccines, antivirals, etc.) and methods (vaccination options and strategies) to prevent poliovirus infections and break chains of transmission if and when they occur. Risk assessment and appropriate emergency response measures to control reemergence must be in place. “The ideal vaccine choice for the stockpile should be effective in any outbreak scenario, protect all vaccinees with one dose, spread to and protect the unvaccinated population, and have no detrimental effect” [372]. mOPVs are more effective when administered alone than when administered with other serotypes (tOPV and bOPV) most likely because of

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increased titers, decreased interference, and longer replication times. Ironically, this may lead to increased numbers of nucleotide substitutions and may increase the potential for cVDPV outbreaks [145]. Supporting this is the emergence of significantly higher numbers of type 1 viruses with increased antigenic divergence from Sabin 1 after a birth dose of mOPV1 and a second exposure to Sabin 1 [153]. “Although OPV has been the mainstay of the eradication program, its continued use is ironically incompatible with the eradication of paralytic disease (since) vaccinederived viruses consistently emerge as a consequence of the inherent genetic instability of poliovirus [165].” “Eradication of vaccine” suggested in 1997 [272, 273] has become recommended policy on condition that provisions of GAP III for safety in vaccine production and polio laboratories are met [373, 374]. A model describing the impact of cVDPVs on eradication indicated that the probability of eradicating polio with continuous use of OPV was not very likely [375]. This is born out by the emergence of new outbreaks of cVDPV2s as a result of response to ongoing cVDPV2 outbreaks with mOPV2 after global withdrawal of OPV2 from tOPV [239]. These findings also indicate that we do not yet have the best tools for ending cVDPV2 outbreaks. While mOPV might be the most effective in rapidly controlling an outbreak and spreading and protecting unvaccinated individuals [372], plans that preferably do not require use of OPV adjacent to areas with high concentrations of unvaccinated individuals would be better in the long run [160, 376]. [The reader is referred to the website of poliovirus eradication (www.polioeradication. org) for the latest information on past, current, and future vaccination policies.] Initial evidence from ongoing clinical trials with the more stable new OPV strains are encouraging and indicate that the nOPVs are effective substitutes for current vaccine strains, yet significantly reduce the risk for emergence of new epidemiological foci of VDPVs. The three main problems that have delayed eradication originally scheduled for 2000 and now hoped for by 2020 have been discussed. Currently available vaccines can overcome

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“failure to vaccinate,” provided that enough doses of vaccines are made available, that there is the political will to use them, and that natural or manmade disasters do not prevent reaching the children for vaccination. Alternative vaccines and/or vaccine delivery systems need to be developed to deal with “vaccine failure.” Low population immunity remains the main known risk factor for the emergence and spread of cVDPVs [316, 326]. Their use should be continued during, and especially after, the transition to a wild poliovirus-free world to maintain high coverage and to avoid the buildup of large susceptible populations during the time when there is the highest risk for reemergence of OPV strains [160, 184, 369, 375, 377]. Preliminary results from newly approved monovalent and bivalent oral polio vaccines and clinical studies using fractional doses of IPV indicate that there may soon be better solutions for “vaccine failure.” The problem of “vaccine-derived viruses” can be divided into two parts. The extent of the problem of cVDPVs was highlighted in 2017 when the annual number of AFP cases from VDPVs (96) for the first time exceeded the number of cases caused by wild poliovirus (22) [378]. The spread of cVDPVs can be interrupted using the same methods with current and soon new vaccines as were used to stop transmission of wild poliovirus since cVDPVs behave like wild virus [148, 316]. There is currently no universal solution to the problem of prolonged or persistent excretors [99, 168], although antivirals under development and in various stages of clinical testing may help. As long as shedding persists (perhaps as long as some of these individuals remain alive), containment of all polioviruses will be incomplete and high vaccination coverage may need to be maintained. It is easy to say that current GPEI plans to coordinate global cessation of the use of all strains of OPV will prevent VAPP [274] and emergence of new cVDPVs; however, the example of the global withdrawal of OPV2 and activation of phase 2 of GAP III have shown that the actual process is quite complicated and associated with difficulties and risks. The GPEI must be constantly on guard against transmission of wild poliovirus and VDPVs

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across political borders into polio-free areas. Between January 2009 and June 2010, the Global Polio Laboratory Network analyzed 258,000 fecal specimens from 130,000 AFP cases for the presence of poliovirus, and between January 2009 and September 2010, it detected introductions of wild poliovirus into 23 previously “polio-free countries,” countries where indigenous polio transmission had been interrupted. Nineteen were in the African Region and included seven countries (Burkina Faso, Benin, Chad, Côte d’Ivoire, Mauritania, Niger, and Togo) where the outbreak isolates were related to previous importations into those countries as was the case for Sudan in the Eastern Mediterranean Region. One of the more serious setbacks for eradication was the introduction of wild poliovirus into Tajikistan from Uttar Pradesh in India marking the first outbreak in the European Region since it was certified poliovirus-free in 2002 [293]. The large outbreak (>450 confirmed cases) ensued spread into the Russian Federation and resulted in an immediate tenfold increase in the amount of samples that needed to be processed by the polio laboratories in the region. In all of these importation countries and/or regions, large-scale coordinated SIAs were conducted. The spread of wild poliovirus to poliovirus-free countries from Nigeria and India via Tajikistan illustrates the need to maintain high population immunity until all transmission of wild virus has ceased. A number of recent polio events have involved emergence of multiple lineages of VDPVs (as in Nigeria) and transmission of VDPVs across borders (documented at www.polioeradication.org.). The increasing frequency of isolation of aVDPVs, due in part to environmental increased surveillance and better tools for identification of VDPVs, reinforces the need to discontinue use of OPV globally in a coordinated effort or staged manner (see [370] for a review and discussion of key issues that have affected and will affect the GPEI, including safety for volunteers in areas of strife, the low efficiency of OPV to induce herd immunity in certain settings, the requirement for maintaining high coverage even after eradication, the inherent mutability of OPV, problems for establishing

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the safety and efficacy and costs of new vaccines, new vaccine formulations, and scaling up alternatives to OPV). Endgame vaccination strategies have been reviewed [4, 165, 214, 370, 376] and include (1) indefinite use of OPV; (2) cessation of all polio immunization; (3) transition to use of IPV, by synchronous coordinated cessation of all use of OPV with (a) limited use of IPVor (b) replacement of all OPV with IPV; (4) country-by-country cessation of OPV use with options (3a) or (3b); (5) sequential removal of Sabin strains from OPV, as eradication proceeds (note: this has been done for OPV2 but will most likely not be done for OPV3, when WPV3 is declared eradicated); (6) development of new vaccines; and (7) indefinite use of IPV or new vaccines. The synchronous cessation of OPV has several problems particularly if inexpensive alternatives are not in place when it occurs, since this vacuum may result in large populations of naïve individuals, in whom polio could reemerge, after periods of silent circulation, with high force and rapid spread. Such a scenario also does not address the potential risks from unidentified chronic excretors. A gradual shift to IPV or killed OPV (sIPV) may avoid some of the programmatic disadvantages that coordinating a synchronous shift would have on vaccination programs and vaccination production facilities. It also potentially provides a longer window for industry to increase production, integrate information from current fractional vaccine dosage and alternative routes of administration trials, and overcome problems of biocontainment and antigenicity associated with optional use of sIPV (using classic OPVor newly developed nOPVs) as a substitute for the wild strains used in IPV production. One of the problems observed during the switch from tOPV to bOPV was a recommendation to add a single dose of IPV before or at the time of switch to bOPV [379] was that there was not enough vaccine (IPV and/or bOPV) on hand and production delays led to under-vaccination of some birth cohorts in some countries or introduction of a single IPV dose rather than multiple IPV doses [380–382]. The saying “May you live in interesting times,” often attributed to an ancient Chinese proverb or

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curse, appears appropriate for describing the current status in the quest to eradicate wild polioviruses. Eradication, which is tantalizingly close, will require substantial changes in vaccination policy and practice [160]. We have witnessed the disruptions that were caused in routine vaccination programs and in all facilities in every country including PEFs and non-PEFs within the global polio laboratory network when WPV2 was declared eradicated and phase 2 of GAP III put into operation for containment of material containing or potentially containing PV2 [381, 383–396]. Eradication of WPV3 will be declared in the very near future. Rather than repeat this major disruption, containment considerations may be deferred until WPV1 is also eradicated. (3) Regulatory Problems Impacting on Sustainability: The WHO and UNICEF consult informally on a regular basis with vaccine manufacturers to discuss the implications and practicality of vaccine policy decisions. Summaries of these informal consultations are available on the Internet using variations of a search for “WHO/UNICEF Informal Consultation with IPV and OPV Manufacturers” or more recently “WHO/UNICEF Consultation with OPV/IPV Manufacturers and National Authorities for Containment of Polio Vaccine Producing Countries.” For example, the 3rd WHO/UNICEF Informal Consultation with IPV and OPV Manufacturers (2003) included a discussion of post-eradication needs and biocontainment requirements; the 5th (2006) included updated information on progress of the GPEI and OPV cessation strategies; the 16th meeting (2017) discussed current supplies and vaccine requirements (2018–2020), biocontainment, and new QC tests for measuring potency and consistency of Sabin IPV (sIPV); and the 17th meeting (2018) discussed progress in eradication, biocontainment, establishing an international standard for new OPV vaccines (such as sIPV), and clinical trials with new vaccination protocols (such as use of fractional doses of IPV) and sIPV technology transfer. “Although OPV has been the mainstay of the eradication program, its continued use is ironically incompatible with the eradication of paralytic disease (since) vaccine-derived viruses

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consistently emerge as a consequence of the inherent genetic instability of poliovirus” [165]. “Eradication of vaccine” suggested in 1997 [272, 273] has become recommended policy on condition that provisions of GAP III for safety in vaccine production and polio laboratories are met [373, 374]. Withdrawal of vaccine components, changing vaccine formulations, and introduction of new vaccines cause regulatory problems that need to be overcome [397]. Complications involve testing and regulatory approval of new products or formulations are not trivial (see discussion on regulations and standardization of IPV and IPV combination vaccines [398] and views of vaccine producers [399, 400]). The good news is that when a “new” polio vaccine, type 1 mOPV, was needed, it was produced by two companies and licensed in three countries in a relatively short time, 6 months [185, 401, 402]. (Quotation marks have been placed around the word “new” since in actuality millions of monovalent doses of each serotype had been used until introduction of tOPV, at which time licenses for mOPVs were left to expire [403]). Licensing of mOPVs was also aided by the fact that monovalent batches were produced and safety tested before being combined to produce tOPV and only qualified tOPV producers were approached to provide mOPVs [402]. Rapid licensing of bOPV on January 10, 2010, followed release of efficacy results in June 2009 (issue 6, Polio Pipeline, summer 2010). Licensing of new OPV strains may be expedited as well, although there is an additional regulatory hurdle that must be overcome that earlier vaccine strains didn’t face, namely, the new vaccine strains are genetically modified organisms (GMOs). (4) Poliovirus Surveillance and Sustainability: Another important pillar of sustainability discussed above is the requirement for sensitive surveillance programs. As the endpoint of eradication of wild poliovirus is approached, the number of cases of poliomyelitis will decrease, while the number of silent infections may increase as a result of high vaccine coverage. Most of the cVDPVs in outbreaks circulated silently for months or years (VP1 divergence >2%) before detection in AFP cases. Therefore, it is imperative that surveillance be improved and expanded to

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high-risk regions to detect silent circulation of VDPVs as early as possible. Paradoxically, lack of AFP cases may lead to a decreased use of polio vaccine with concomitant increase in the number of naïve individuals enabling easier spread in the case that poliovirus reemerges by accident or deliberately. Under these conditions, supplemental surveillance programs such as enterovirus surveillance and environmental surveillance will become an even more important tool for providing geographical information for designing NIDS, SNIDS, and final mopping-up campaigns for eradication and for monitoring for posteradication reemergence. (See the discussion above on the contribution of environmental surveillance to countries where cVDPVs and WPVs circulate and for countries where vaccine coverage is high and polioviruses might circulate for long periods of time in the absence of cases.)

Concluding Statement In conclusion, the means to identify the presence of poliovirus infections, prevent the disease, and contain the spread of virus transmission when it emerges are already in hand. Safer and more costeffective measures are in the pipeline that include schedule reduction, fractional doses, adjuvant use, optimizing of processing, Sabin or modified Sabin IPV, and noninfectious IPV (non-inclusive list of references: [17, 162–164, 208, 247–252, 323, 364]. Sustainability for achieving eradication will depend on vaccine policy decisions made today and in the recent past, on the length of time it takes to eliminate all wild poliovirus transmissions, on political will and advocacy, on the motivation of volunteers and the level of local community involvement and program-related fatigue, and on the absence of further complications from bioerror, bioterror, or mother nature [404]. The overriding factor will probably be the availability of financial resources [185]. Limited resources mean competition between routine immunization and eradication efforts during endgame. For example, the predicted 1.3 billion USD funding gap in June 2010 forced a reprioritization of planned activities (Global Polio Eradication Initiative Monthly Situation Report, June 2010, www.polioeracdication.

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org.) “Even if there are no competing health needs, it is unlikely that immunization could be maintained indefinitely against a non-existent disease at a level that is sufficient to prevent vaccinederived viruses evolving to cause epidemics” [214]. Programmatic setbacks such as those associated with failure to vaccinate (Nigeria and Tajikistan), vaccine failure (northern India), the frequency of repeated vaccination campaigns, or post-eradication reemergence must not be allowed to derail the current momentum and lead to program-related fatigue [1]. Detailed planning must be made for any post-eradication outbreaks (see Jenkins and Modlin [372] and Tebbins et al. [376]), and provisions to implement them including stockpiling must be in place. Finally, some problems with sustainability will be associated with management rather than scientific problems [1, 405]. It is well worth reading “The pathogenesis of poliomyelitis: what we don’t know” by Nathanson [406] and “Gaps in scientific knowledge for the post-eradication world” by Minor [407].

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Polio and measles are the second and third human pathogens for which there is an ongoing global program for eradication. Polio eradication has reached the endgame. Measles eradication is currently running into trouble from increasing numbers of individuals who refuse vaccination. Smallpox successfully completed the endgame and is now in the stage of post-eradication sustainability with bioterror as the main threat to sustainability of eradication. This chapter will describe some of the difficulties with completing the endgame of polio eradication and then in sustaining post-polio eradication. More than the usual number of items are included in the Glossary to make it easier for the reader to follow the progress of eradication as it unfolds in the large number of official documents that deal with a global eradication program and which contain copious numbers of professional acronyms.

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