Viral Hepatitis: Current Status and Issues [1st ed.] 978-3-211-82387-3;978-3-7091-4437-4

Table of contents :
Front Matter ....Pages I-X
Introduction (Edouard Kurstak)....Pages 1-12
Front Matter ....Pages 13-13
Hepatitis A virus properties and replication (Edouard Kurstak)....Pages 15-23
Clinical aspects of hepatitis A virus infection (Edouard Kurstak)....Pages 24-27
Pathogenesis of hepatitis A virus infection (Edouard Kurstak)....Pages 28-32
Hepatitis A virus infection: diagnostic tests (Edouard Kurstak)....Pages 33-37
Epidemiology and transmission of hepatitis A virus (Edouard Kurstak)....Pages 38-43
Hepatitis A: therapeutic approaches, prevention and control (Edouard Kurstak)....Pages 44-57
Front Matter ....Pages 59-59
Hepatitis B virus: molecular characteristics and subtypes (Edouard Kurstak)....Pages 61-68
Acute hepatitis B virus infection (Edouard Kurstak)....Pages 69-74
Chronic hepatitis B virus infection (Edouard Kurstak)....Pages 75-82
Hepatitis B virus: epidemiology, transmission and carrier state (Edouard Kurstak)....Pages 83-92
Hepatitis B virus infection: serology and diagnostic techniques (Edouard Kurstak)....Pages 93-104
Immunology of hepatitis B virus infection (Edouard Kurstak)....Pages 105-110
Hepatocellular carcinoma and hepatitis B virus (Edouard Kurstak)....Pages 111-118
Treatment of hepatitis B virus disease (Edouard Kurstak)....Pages 119-127
Control and prevention of hepatitis B virus infection (Edouard Kurstak)....Pages 128-148
Front Matter ....Pages 149-149
Hepatitis D virus: characteristics, replication and infection (Edouard Kurstak)....Pages 151-155
Hepatitis D: epidemiology, prevention and treatment (Edouard Kurstak)....Pages 156-159
Diagnosis of hepatitis D virus infection (Edouard Kurstak)....Pages 160-171
Front Matter ....Pages 173-173
Current nomenclature, viral agents, and clinical aspects of hepatitis C and hepatitis E (Edouard Kurstak)....Pages 175-180
Hepatitis C and hepatitis E viruses: epidemiology and transmission (Edouard Kurstak)....Pages 181-184
Diagnosis of hepatitis C and hepatitis E virus infections (Edouard Kurstak)....Pages 185-193
Hepatitis C and hepatitis E: prevention, prophylaxis and treatment (Edouard Kurstak)....Pages 194-201
Front Matter ....Pages 203-203
Differentiation between viral hepatitis (Edouard Kurstak)....Pages 205-210
Back Matter ....Pages 211-219

Citation preview

Edouard Kurstak

Viral Hepatit is Current Status and Issues

In Collaboration with Christine Kurstak, A. Hossain, and A. Al Tuwaijri

Springer-Verlag Wien GmbH

Prof. Dr. Edouard Kurstak Director of ICVO, Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Canada World Health Organization, Geneva, Switzerland King Fahad National Guard Hospital, Riyadh, Saudi Arabia

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machines or similar means, and storage in data banks. © 1993 Springer-Verlag Wien Originally published by Springer-Verlag/Wien 1993 Printed on acid-free paper Product Liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. The use of registered names, trademarks, 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.

With 26 Figures

ISBN 978-3-211-82387-3 DOI 10.1007/978-3-7091-4437-4

ISBN 978-3-7091-4437-4 (eBook)

Preface Viral hepatitis, especially hepatitis B, continues to kill, despite the availability of safe and effective vaccines. With recent addition of highly immunogenic hepatitis A vaccine, these two viral hepatitis infections could be prevented. A such major achievement is possible only if universal programs of immunization are implemented, as it was the case for eradication of smallpox disease. Nearly 350 million of hepatitis B virus and 100 million of hepatitis C virus infective carriers are estimated worldwide in comparison to 14 millions people infected with human immunodeficiency virus. Adding other types of hepatitis caused by viruses we are facing the major and growing global health problem. In term of patients hospitalized with acute and chronic infections and sequelae, including related hepatocellular carcinoma and high mortality rate, viral hepatitis are an enormous financial burden to the society and unresolved problem to health administrations. With recent avalanche of new research data on hepatitis C virus and the development of highly sensitive molecular genetic diagnostic tools it was timely to publish a monograph on current status and issues regarding all types of hepatitis A, B, C, D and E virus infections. The virological, clinical, epidemiological, diagnostic, therapeutic and preventive aspects pertaining to all viral hepatitis, known up to date, are discussed in this single volume. Some of data presented are very recent breakthroughs of dedicated scientists, who piece by piece clarify the complexity and diversity of hepatitis viruses and diseases. A such volume impose some limitations in choice of subjects and references. Our wish was to have a comprehensive and balanced presentation of different hepatitis viruses and related infections, with emphasis on recent developments regarding vaccines, immunization, therapy and diagnosis of these diseases, which are based on new findings in molecular characterization of viruses, their epidemiology, immunology and pathogenesis. It is our hope that this volume will benefit medical practitioners, microbiologists and infectious diseases specialists, as well as, those involved in health administration worldwide. Special thanks are addressed to the staff of Springer-Verlag for their part in the publication of this volume.

Prof Dr. Edouard Kurstak

President · International Comparative Virology Organization Member · Viral Diseases Panel, World Health Organization

Contents Introduction. Viral hepatitis: current status and issues . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10

Part I. Hepatitis A virus and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

1. Hepatitis A virus properties and replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The nature of the virus particle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Morphology and size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Sedimentation co-efficient and buoyant density . . . . . . . . . . . . . . . . . . . . . . . . C. Reactivity of physical and chemical agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. The virus genome and structural proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Virus genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Virus structural proteins and their function . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The virus replication cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Clinical aspects of hepatitis A virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Clinical features of hepatitis A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Infection incubation period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Asymptomatic and fulminant infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Polyphasic course of infection in children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathogenesis of hepatitis A virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Pathologic features of disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Virologic events of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Infection immunopathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Hepatitis A virus infection: diagnostic tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Immune electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Rapid viral diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Serological identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Radio-immunoassay and enzyme-immunoassay techniques . . . . . . . . . . . . . . . IV. Current trends in molecular biotechniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Epidemiology and transmission of hepatitis A virus . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Epidemiologic characteristics of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Seasonal and geographic variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Age incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Inapparent and chronic reinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Virus incubation period and transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Hepatitis A: therapeutic approaches, prevention and control . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Measures for prevention of virus transmission in hospitals . . . . . . . . . . . . . . . . . .

15 15 15 15 16 17 18 18 20 22 24 24 24 26 26 27 28 28 28 29 30 33 33 33 34 34 35 36 38 38 38 39 40 40 41 44 44 45

VIII

Contents

III. Passive immunoprophylaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. Active immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inactivated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Live attenuated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other strategies for vaccine development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

46 48 48 50 51 52

Part II. Hepatitis B virus and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59

7. Hepatitis B virus: molecular characteristics and subtypes . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The virus particles and genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Virus structure and antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Virus genomic organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Subtypes of infectious virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Acute hepatitis B virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Clinical presentation of acute hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Prodromal phase of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Icteric phase of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Clinical pathology of hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. Different clinical forms of hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inapparent and anicteric forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cholestatic-type form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Fulminant infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Chronic hepatitis B virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Chronic persistent hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Clinical course of infection and diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Prognosis and management of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Chronic active hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Clinical picture of infection and diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Treatment and prognosis of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. Chronic hepatitis B in special high risk patients . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hemodialysis and renal transplant patients . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hemophiliacs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Homosexuals and drug addicts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Interactions between human immunodeficiencyvirus-1, hepatitis D virus and hepatitis B virus infections in chronic carriers ofHBV . . . . . . . . . . . . . . . 10. Hepatitis B virus: epidemiology, transmission and carrier state . . . . . . . . . . . . . . . . . I. Epidemiology and characteristics of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Worldwide prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Virus transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Incubation period and mode of transmission . . . . . . . . . . . . . . . . . . . . . . . . . . i) Transmission by blood transfusion of blood products . . . . . . . . . . . . . . . . ii) Sexual route and transmission through fluids . . . . . . . . . . . . . . . . . . . . . . . iii) Vertical transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv) Horizontal transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Virus carrier state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Definition, duration and prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Markers of virus infectivity and replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Epidemiological aspects of carrier state and management . . . . . . . . . . . . . . . 11. Hepatitis B virus infection: serology and diagnostic techniques . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. General serological aspects of viral infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61 61 61 62 64 68 69 69 69 70 71 71 72 72 73 73 75 75 76 76 76 77 78 79 80 80 81 81 82 83 83 84 84 86 86 86 87 87 89 90 90 90 91 93 93 95

Contents III. Serology of active hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Serology of chronic hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Techniques for virus markers detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Detection of HBsAg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Detection ofanti-HBs ............................................. C. Detection ofHBcAg .............................................. D. Detection ofanti-HBc ............................................. E. Detection ofHBeAg and anti-HBe ................................... F. Detection of hepatitis B virus DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Immunology of hepatitis B virus infection .................................. I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Liver antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Humoral immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Cellular immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Anti-idiotypic responses .............................................. 13. Hepatocellular carcinoma and hepatitis B virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Clinical aspects: PHC/HBVassociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Pathological aspects: PHC/HBV association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Geographical localization and familial incidence: PHC/HBV . . . . . . . . . . . . . . . V. Serological association of HBV markers and PHC . . . . . . . . . . . . . . . . . . . . . . . . . VI. Molecular biology perspectives: HBVand PHC relationship ................. 14. Treatment of hepatitis B virus disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Treatment of acute hepatitis B ......................................... A. Restricted activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Diet ............................................................ C. Drugs .......................................................... II. Therapeutic approaches in chronic hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . i) Suramin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii) Acyclovir (acycloguanosine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii) Adenine arabinoside (vidarabine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv) Thymosins/prostaglandins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Interferons in hepatitis B treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Drugs combination therapy in hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Monoclonal antibody to HBsAg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Interleukin-2 in hepatitis B therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15. Control and prevention of hepatitis B virus infection . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Passive immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III.Active immunization ................................................. A. Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Safety of the vaccines .............................................. C. Dosage and schedules ............................................. D. Administration route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Adverse reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Antibody persistence and booster doses/revaccination . . . . . . . . . . . . . . . . . . IV. Hepatitis B virus vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Polypeptide vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Recombinant DNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Hybrid vaccinia virus vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Vaccines using hybrid particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Chemically synthesized vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Anti-idiotype antibody as a prospective vaccine . . . . . . . . . . . . . . . . . . . . . . . . G. Combined hepatitis A and B virus vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Universal hepatitis B virus vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ................................................................

IX 96 97 99 99 100 101 101 102 103 105 105 106 107 108 110 111 111 112 112 114 115 116 119 119 119 120 120 120 121 122 122 123 123 125 126 127 128 128 129 130 130 130 131 131 131 131 132 133 133 133 134 135 135 136 136 137

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Part ill. Hepatitis D virus and disease

149

16. Hepatitis D virus: characteristics, replication and infection . . . . . . . . . . . . . . . . . . . . I. Virus characteristics and replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Clinical course of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17. Hepatitis D: epidemiology, prevention and treatment . . . . . . . . . . . . . . . . . . . . . . . . . I. Infection incubation period and mode of transmission . . . . . . . . . . . . . . . . . . . . . II. Epidemiology and virus distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill. Prevention and treatment of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18. Diagnosis of hepatitis D virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Serological identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill. Diagnostic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Radio-immunoassays (RIA) ........................................ B. Enzyme-immunoassays (EIA) ....................................... C. Immunofluorescence and immunoperoxidase techniques . . . . . . . . . . . . . . . D. Immunoblot techniques ........................................... E. Molecular hybridization assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

151 151 153 156 156 156 158 160 160 161 163 163 163 165 166 167 168

Part IV. Hepatitis C virus, hepatitis E virus and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 19. Current nomenclature, viral agents, and clinical aspects of hepatitis C and hepatitis E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Current nomenclature and viral agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nomenclature: hepatitis C virus and hepatitis E virus . . . . . . . . . . . . . . . . . . . II. Clinical aspects of hepatitis C and hepatitis E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hepatitis C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hepatitis E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20. Hepatitis C and hepatitis E viruses: epidemiology and transmission . . . . . . . . . . . . . I. Epidemiology and distribution of infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Incubation period and mode of transmission ............................. 21. Diagnosis of hepatitis C and hepatitis E virus infections . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Antibody assays and seroprevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Detection of hepatitis C virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Diagnosis of hepatitis E virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Hepatitis C and hepatitis E: prevention, prophylaxis and treatment . . . . . . . . . . . . . I. Prevention and prophylaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Treatment of hepatitis C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

175 175 175 175 178 178 180 181 181 183 185 185 186 190 192 194 194 195 197

Part V. Different forms of viral hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 23. Differentiation between viral hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Hepatitis viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Clinical features of viral hepatitis ....................................... IV. Epidemiology of viral hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Diagnosis of viral hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205 205 205 207 208 208 209

Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

Introduction Viral hepatitis: current status and issues Hepatitis is generally defined as a inflammation of the liver and may be due to a variety of causes as drugs, toxins and viruses. The most common causative agent of acute hepatitis is viral in nature. In recent years, significant advances in the understanding of viral hepatitis have occurred with the elucidation of the viral structure, molecular events during infection and pathogenic mechanisms (Krugman, 1985; Zuckerman, 1988; Hollinger et al., 1991; McLachlan, 1991; Kurstak, 1992). Since the suggestion of the viral etiology of hepatitis in the context of acute yellow atrophy of liver, knowledge of the nature and diversity of viruses causing hepatitis in humans has substantially increased. The agents of hepatitis currently known include at least five different viruses: hepatitis A virus (HAV)-causing classic short-incubation infections or epidemic hepatitis; hepatitis B virus (HBV)-long-incubation serum hepatitis and a possible variant of hepatitis B virus (HBV-2); hepatitis D virus (HDV); and one or more still not entirely defined viruses responsible for non-A, nonB hepatitis parenterally transmitted and recently classified as hepatitis C virus (HCV) as well as hepatitis E virus (HEV), an epidemic form of enterically transmitted non-A, non-B hepatitis. The recent development of an antibody test for the hepatitis C virus (antiHCV) (Choo et al., 1989; Kuo et al., 1989) has led to rapid advances in our knowledge of the epidemiology and possible oncogenicity of this virus. HCV is now known to be the chief cause of transfusion-associated non-A, non-B hepatitis, but the prevalence ofHCV among blood donors and the frequency of transmission by blood transfusion remains to be unequivocally determined. It appears, based on this test, that approximately 90% of post-transfusion hepatitis cases are caused by HCV (Esteban et al., 1990). A recent study byMcHutchson etal. (1991) indicates that anti-hepatitis Cvirusantibodywas more often detected in intravenous (IV) drug users (89%) and after transfusion (71 %) in chronic infection in comparison to 61 % (IV drug abusers) and 33% in acute transfusion-associated hepatitis. There is also the theoretical possibility of yet another parenteral non-A, non-Bagent (hepatitis F and

2

Introduction

beyond) in view of the fact that a large proportion of presumed HCV-positive patients show negativity for HCV even on supplementary testing. Viral hepatitis, particularly hepatitis B virus infection, remains a major public health problem worldwide (Dindzans et al., 1985; Hollinger et al., 1991; Ghendon, 1992). Approximately 2 billion people appear to have been infected by hepatitis B virus and of these 350 million people are chronically infected (World Health Organization Press Release, 1992). Acute viral hepatitis a generalized infection with inflammation of the liver, has been noted for its variable clinical picture with presentations as asymptomatic or subclinical infection, mild gastro-intestinal symptoms and the anicteric form of the disease, acute illness with jaundice, severe prolonged jaundice or acute fulminant hepatitis. Hepatitis B virus disease and co-infection with the hepatitis D virus and infection with the parenterally transmitted hepatitis C virus (non-A, non-B hepatitis) may be associated with a persistent carrier state and these forms of infection may possibly progress to chronic liver disease which may be severe. In fact, HBV carriers have a risk of developing primary liver cancer that is 12 to 300 times higher than others (Melnick, 1992). The molecular mechanisms underlying hepatitis B virus persistence in individuals positive to hepatitis B surface antigen (HBsAg) remain unresolved. Data from a recent study however suggest that mutations accumulating during the natural course of hepatitis B virus infection may be a mechanism underlying viral persistence in HBsAg individuals, presumably through escape from immune surveillance (Blum et al., 1991). Any emergence of an escape mutant functionally lacking the a determinant would be of major concern since the antigenic subtypes of HBV had been generally known to have contained the a determinant, the antibody to which confer protection against all HBV subtypes. In the child analyzed in detail by Carman et al. (1990) recently, the presence ofanti-HBs apparently did not confer protection against infection with the mutant strain of HBV. Another HBV variant has been described in which a novel core antigen resulting from a series of mutations in the precore and core regions of the genome is associated with the failure of development of antibody to hepatitis B core antigen (anti-HBc) (Bhat et al., 1990). Despite the fact that HBV is highly evolved, it still has the capacity to produce genetic variation with an obvious clinical relevance (Carman and Thomas, 1992). It has been estimated that at a single nucleotide position ofHBV, 1-3 x 10-5 mutations occur in an infected person each year (Okamoto et al., 1987). The recently published work ofKosaka et al. (1991) helps confirm and infact extends previous evidence that HBeAg-defective HBV causes a form of hepatitis B with severe pathogenicity. The clinical importance of HBV mutants remains to be determined. There appears to be conflicting reports about the response to interferon therapy in chronic HBeAg-negative HBV DNA-positive hepatitis B associated with the precore mutant. The precore mutant strain ofHBV lends to a particularly severe form of chronic hepatitis (Moriyama et al., 1991). In the case of hepatitis C virus infection, no clear relation has yet been established between HCV replication and severity of the disease. A recent

Viral hepatitis: current status and issues

3

study by Pozzato et al. (1991) has shown however, that the detection of Japanese-type HCV-RNA in the serum may seem to be associated with the presence of more serious liver disease. Recently, evidence of vertical transmission ofHCV has been provided (Degos et al., 1991; Thaler et al., 1991). The findings suggest that perinatal infection may initiate a silent process or more perhaps a chronic carrier state. Fong et al. (1991) in an interesting study assessed the prevalence and clinical significance of antibodies to HCV among a cohort of 148 patients with chronic hepatitis B virus infection and suggested that patients with chronic hepatitis B with anti- hepatitis C virus antibodies were more likely to be cirrhotic. The hepatitis C virus appears to suppress hepatitis B virus replication and cause more severe disease in patients with chronic HBV infection. In the acute phase of hepatitis D infection, occurring either by coinfection with HBV or by superinfection of a chronic HBV carrier, synthesis of HBV proteins appears to be significantly inhibited but for persistent HDV infection, a subliminal or latent infection of the helper virus is necessary (Rizzetto, 1984). Recently published results of a study by Smedile et al. ( 1991) seem to suggest that active hepatitis B virus replication represents an important, previously unrecognized, determinant of severe liver damage in patients with chronic hepatitis D virus infection. Since hepatitis B virus provides the means for hepatitis D virus replication and release from infected cells, active hepatitis B virus multiplication favoring the spread of hepatitis D virus from cell to cell may increase the pathogenetic potential of the HDV defective agent. A trigger compassing specific helper T cells and antibodies to the asialoglycoprotein receptor for autoimmune chronic hepatitis type 1 has now been identified (Vento et al., 1991). Chronic hepatitis B virus infection affects at least 5-7 percent of the world's population and the main cause of cirrhosis and possibly hepatocellular carcinoma. The World Health Organization has listed hepatitis B virus disease as the ninth major cause of death worldwide, close behind the chronic pulmonary disease. According to Hoofnagle (1990) these estimates suggest chronic hepatitis B virus (HBV) infection as the most important chronic infection affecting humans. Evidence now exists of an etiological association between hepatitis B virus and primary hepatocellular carcinoma (PHC), one of the ten most common malignant tumors worldwide (Hsu et al., 1989). Hepatocellular carcinoma and cirrhosis appear to be the major sources of morbidity and mortality in Southeast Asia and Africa and to a lesser extent in North America and Europe. HBV has been infact noted to be the most important determinant of PHC in almost all countries in the world, with a risk 10 to 20 times higher for HBsAg carriers as compared with non-carriers. However, a striking difference between males and females in PHC incidence exists despite the similarity in HBsAg carrier prevalence. Although sex hormones have been suggested as an explanation for the gender discrepancy, difference in life-style habits such as cigarette smoking and alcohol drinking have been suggested as cofactors. A recent study by Chen et al. (1991) has shown a significant association between the hepatocellular carcinoma and carrier status of HBsAg and HBeAg with odds ratio of 16. 7 and 56.5 respec-

4

Introduction

tively, for carriers ofHBsAg alone and for carrier of both HBsAg and HBeAg. A dose-response relationship between cigarette smoking and PHC was found. Also a significant association with PHC was also observed for the habitual alcohol consumer with an odd ratio of 3.4. Serum alpha fetoprotein (AFP) determinations are useful in the diagnosis of advanced PHC. To achieve an improvement in the diagnostic usefulness of AFP, Lee et al. (1991) recently investigated the different specificities and predictive values positive (PVPs) of serum AFP and their results suggest that regular serum AFP determination may be more useful in HBsAg- patients with chronic liver disease for the early diagnosis of PHC than in HBsAg+ patients. In a very interesting study Arville et al. (1991) very recently investigated the regulation of insulin-like growth factor II gene expression and their results imply a role for this insulin-like growth factor II in the pathogenesis of cirrhotic/non-cirrhotic chronic liver disease to hepatocellular carcinoma where there is evidence of hepatitis B virus involvement. The status ofHBV DNA in tumor and non-tumorous liver tissues in carrier children with PHC was investigated by Chang et al. (1991). The PHC tissues from five of the eight children showed integration of hepatitis B virus DNA into host cellular DNA sequences. Using subgenomic fragments of the HBV genome as probes, the X gene fragment and the surface antigen gene fragment were found to be the most conserved sequences. The authors believe that the single-site integration of HBV DNA in childhood primary hepatocellular carcinoma may hit the critical region, resulting in insertional mutagenesis and early development of PHC. HBxAg and HBsAg could be expressed in the absence ofHBV replication, from viral nucleic acid integrated into host chromosomes, and are linked to PHC development (Feitelson, 1992). It has long been suspected that non-A, non-B hepatitis infection may occasionally lead to PHC and the development of an assay for circulating antibodies against hepatitis C virus has indeed confirmed that HCV is frequently involved in chronic liver disease. A recent publication has highlighted the indication that HCV infection does have an interactive role in the origin of PHC (Kaklamani et al., 1991). Hepatitis A, B and D viruses have already been characterized and infections due to these viruses can now be readily differentiated by sensitive and specific laboratory tests. Recently, a method to amplify target DNA sequences (polymerase chain reaction-PCR-method) has been described. The principle of this method involves the amplification of DNA sequences exponentially by repeating cycles of denaturation of DNA, annealing of synthetic oligonucleotide flanking primers to the target DNA and extension of these primers by polymerase reaction. If the reaction efficiency is 100%, DNA can be amplified to 2N times the original DNA after N cycles. This amplification method has now been applied towards the detection and sequencing of the HBV gene and HBV DNA was reportedly detected at the level of one virion and its sequence determinable by direct sequencing in a few days (Yokosuka et al., 1991). An HBV PCR DNA typing procedure based on restriction endonuclease analysis of PCR products has been utilized in strain analysis ofHBV (Shih et al., 1991). HBV DNA sequence polymorphisms involving short sequences

Viral hepatitis: current status and issues

5

in polyvariable regions of the HBV genome has recently been used in the studies ofHBV transmission (Lin et al., 1991). The genome of HBV is known to consist of four open-reading frames, each of which is believed to encode one or more viral gene productions. The fourth open-reading frame encodes hepatitis Bx antigen (HBxAg) polypeptides. Recent work in renal dialysis patients who became transistently or persistently positive for HBsAg has shown that antibody directed against the viral polymerase (anti-pol) is the earliest marker of HBV infection, that HBxAg in the serum correlates with other markers of viral replication and the appearance of antibodies against the HBx.Ag (anti-HBx) correlated with a decrease in the amount of detectable virus in the serum. A recent study (Horiike et al., 1991) of these newly characterized HBV markers including the most recent anti-pol in sera and livers from Japanese patients infected with HBV suggests that HBx.Ag may be common in the liver among patients with chronic hepatitis independent of HBV replication markers but closely correlated with liver aminotransferase indicative of its importance in the pathogenesis of chronic infection. The detection of anti-pol in many samples positive for HBeAg and HBV DNA and less often in serum samples without markers ofHBV replication appears to suggest that this marker could reflect ongoing viral replication in the liver. Although hepatitis D virus (HDV) infection can now be detected by a wide range of methods, including immunobloting and cDNA probes, the routine laboratory detection of HDV markers is generally carried out by serological assays involving either enzyme immunoassays (EIAs) or radioimmunoassay using commercially available reagents. One or more of three serological markers are used: hepatitis D virus antigen (HDAg) for acute infection; anti-HDV antibody (anti-HDV) for past infection, epidemiologically based investigations and chronic infection; and immunoglobulin M (IgM) anti-HDV for the 'window' period between the appearance ofHDAg and IgM anti-HDV and for indicating chronic HDV infection at a high titer. In up to 78% of coinfections, HDAg and IgM anti-HDV appear to be detectable concurrently. Commercial EIAs for detection of hepatitis D antigen and antihepatitis D virus (HDV) and IgM anti-HDV antibodies have been recently evaluated by Shattock and Morris (1991) and have noted a substantial improvement over previously evaluated assays in sensitivity and specificity of commercial assays for anti-HDV detection, and the sensitivities of IgM antiHDV assays are also appear to be comparable. However, these still remain major differences in sensitivity among some assays for HDAg detection. The clinical significance of two forms oflgM antibody to hepatitis D virus (HDV) has been elegantly assessed in a recently published article by Jardi et al. (1991). The high molecular weight IgM form (19 S) was predominantly detected in acute hepatitis D virus cases, whereas the low molecular weight (7 S) form was found in chronic hepatitis D virus cases. During their investigation of the serological profile of these two forms of IgM antibody the authors found that in the acute stage of the disease, the 19 S form was predominent whereas 6 months later, a predominance of 7-8 S IgM was observed. The results of these investigations thus suggest that IgM antibody

6

Introduction

anti-HDV are different in acute and chronic HDV infection and their detection only appears to be of value in the differentiation of an acute infection from a chronic infection but not to differentiate a hepatitis HDV-HBV coinfection from hepatitis D virus superinfection in the acute stage of the disease. The etiological agent of blood-borne non-A, non-B hepatitis recently identified and called the hepatitis C virus (HCV) is a flavivirus- like with a linear single-stranded, positive polarity RNA genome approximately 10 kb long. Molecular cloning and expression techniques have revealed the existence of a single open-reading frame in the genome from which a polypeptide (CI00-3) consisting of 363 viral amino acids has been synthesized in yeast. Since the discovery of nucleotide sequences of the HCV genome an enzymelinked immunosorbent assay (ELISA) was developed for blood-donor screening in order to prevent post-transfusion non-A, non-B hepatitis. However, only 17%-25% of HCV CIOO ELISA-positive blood products are known to transmit HCV (Garson et al., 1990). Most of the HCV ClOO ELISA-positive samples may therefore represent nonspecific ELISA reactivity. Although the recombinant immunoblot assay with the 5-1-1 and CIOO antigens (CIOO RIBA) improved specificity (Ebeling et al., 1990) this assay reportedly does not give independent confirmation but might merely represent a supplementary test. Moreover, the CIOO RIBA/ClOO ELISA combination even has been noted to be less sensitive than the CIOO ELISA alone. HCV infection confirmation can now be obtained by amplification of HCV sequences with the polymerase chain reaction (PCR) (Garson et al., 1990; Weiner et al., 1990) but the main hurdle appears to be the non-availability of such technique for routine purposes. A new second generation RIBA ( 4-RIBA) has recently been developed by Chiron Corporation in which two additional HCV recombinant antigens have been added to the Cl 00 RIBA. One antigen is from the non-structural NS3 region (C33c), and the other is an HCV-coreassociated antigen (C22). Both antigens have been expressed in yeast. This new four-antigen recombinant immunoblot assay has recently been evaluated by Van der Poel (1991) for testing stored serum samples of blood donors and recipients and compared with results from PCR analysis of fresh plasma samples in donors and recipients from the original study. The results of this study validates the 4-RIBA as a candidate confirmation test to discriminate between infective and non-infective HCV Cl 00 ELISA positive blood donors. Marcellin etal. (1991) have also used this test in French patients with chronic non-A, non-B hepatitis and their results confirm that in these patients the new Chiron RIBA for anti-HCV is more sensitive than the first generation ELISA. Another upshot of this study has been that HCV is indeed responsible for practically almost all cases of non-A, non-B hepatitis in France. Detection of hepatitis C virus RNA has been advocated recently to provide a useful indication in the study ofHCV infection. Hosada et al. (1991) has reported a study in which PCR was used to develop an assay for detecting HCV RNA in the liver tissues and the presence ofHCV RNA was investigated in the liver of Japanese patients with chronic liver diseases as well as its association with the presence of anti-HCV antibody in serum. Direct sequenc-

Viral hepatitis: current status and issues

7

ing of amplified complementary DNA enabled partial sequencing of the HCV genome in patients testing positive for HCV RNA in the liver. In yet another report for the detection ofHCV RNA, Cha et al. (1991) have used a signature nucleotide sequence ofHCV, believed to be well conserved among viral isolates throughout the world. Based on this signature sequence for HCV, the viral RNA agsay was developed uging cDNA synthesis with reverse transcriptase, followed by the polymerase chain reaction. The putative 5 'untranslated ( 5PUT) PCR primers thus used were found to be superior to the NS-3 primers in sensitivity and specificity. The utility ofNS-5 as a new marker for detection of previously unrecognized HCV infection had been demonstrated recently (Lesniewski et al., 1992). The clinical significance of the presence or absence ofHCV RNA in samples from patients however remains unclear at the present time. Although the development of the immunoassay for the detection of the IgG antibody to HCV (IgG anti-HCV) based on the solid-phase immobilization of Cl 00-3 recombinant HCV antigen allowed the proper classification of non-A, non-B hepatitis patients, the appearance of IgG anti-HCV could be delayed for up to one year after HCV infection and its presence does not imply ongoing viral replication. Also, anti-HCV testing cannot be used as a prognostic marker because the IgG titer does not correlated with the cause of HCV infection. Quiroga et al. (1991) has infact developed a single modification of the immunoassay for anti-HCV that invariably permits the detection of IgM specific anti-HCV which usually precedes the detection oflgG antibody and can reflect the presence of ongoing viral replication. The study of the clinical significance oflgM anti-HCV in acute and chronic hepatitis C showed that IgM antibody to HCV persists after acute infection in patients contracting chronic hepatitis C. Therefore, testing for this antibody (IgM) may be useful in early identification of patients for antiviral therapy. Epidemics of viral hepatitis in which specific serological markers for hepatitis A and B cannot be detected are thought to be caused by enterically transmitted non-A, non-B hepatitis agents. Once such agent, hepatitis E virus (HEV) exists as 27-34 nm particles; it is believed to be transmitted in primates causing hepatic injury with characteristic rises in serum aminotransferase activity and excretion of virus in the stool. The HEV genome has been assessed to be a single positive strand RNA of about 7.6kb. The recent examination (Ray et al., 1991) of stool samples from patients affected during the large epidemic of hepatitis in north India for the presence of hepatitis E virus genome by means of reverse transcription - polymerase chain reaction strongly suggests that HEV indeed was the causative agent for this large epidemic. Effective preventive immunization for hepatitis B has been developed but treatment for both acute and chronic viral hepatitis is still far from satisfactory (Alexander and Williams, 1988; Hoofnagle and Di Bisceglie, 1988; Mazella et al., 1988; Caselmann et al., 1989; Henrietta et al., 1989). For now, the treatment with interferon (IFN) alpha offers the best hope. It has been proven to be the most successful agent to date and results in HBeAg seroconversion and loss of serum HBV DNA in 30% to 50% of

8

Introduction

patients (Perrilo et al., 1990). The response appears to fairly impressive since it is two to four times the rate of spontaneous seroconversion (Perrilo et al., 1990). Additionally, it has recently been shown by the sensitive polymerase chain reaction that most patients with chronic hepatitis B responded to the IFN-alpha therapy with loss ofHBeAg and improved serum aminotransferases status was found. Consequently, they were clear ofHBsAg and all evidence of residual HBV replication (Korenman et al., 1991). It remains, however, that more than half the patients in the overt replicative phase of HBV treatment who presumably do not respond to interferon treatment. Recently, Lau et al,(1991) have examined the expression and regulation of IFNalpha receptor on peripheral monocuclear cells and mononuclear subsets in chronic HBV patients to determine whether these could possibly account for the varied response to INF-alpha therapy. Their data indicate that INF-alpha receptors appear to be expressed and regulated normally in chronic HBV infection and that the variable response to IFN-alpha therapy is not due to a variation in interferon-alpha receptor. Despite the considerable progress achieved in the treatment of patients affected by chronic hepatitis B with recombinant IFN-alpha, the therapy shown to be effective in a considerable proportion of these patients and the licensing of IFN-alpha in many parts of the world other than the United States, the role of interferon in treating children appears to be less clear. In a recent study however, Ruiz-Moreno et al. (1991) assessed interferon treatment in children with chronic hepatitis Bin a randomized controlled trial of a 6-months course of interferon-alpha in 36 children with this disease. The findings indicate that a 6-months course ofIFN-alpha is effective in inducing a serological, biochemical and histological remission of disease in approximately 50% of children with chronic hepatitis B. Recently interest has been focussing on thymosin alpha, an agent other than IFN-alpha, as viable alternative for those who do not respond to interferon treatment. Thymosins are hormonelike polypeptides produced by thymus epithelial cells and the thymosin-alpha is believed to stimulate in vivo production of interferon-alpha. Thymosin alpha may eventually prove to be as effective as IFN-alpha and has the advantage of being relatively free of significant side effects (Mutchnick et al., 1991). At the present time, little is however known about the mechanism of action and clinical effect of thymus derived agents as thymosin alpha in HBV infection. Hepatitis C virus (HCV) has been shown to be the most important etiologic agent of chronic non-A, non-B hepatitis. Furthermore, prospective studies have shown that chronic liver disease would develop in about half of the patients receiving multiple transfusions in the industrialized world, of whom about 20% will progess to liver cirrhosis. Interferon alpha appears to be the only treatment for hepatitis C virus infection that has been evaluated thoroughly and almost exclusively for chronic non-A, non-B hepatitis patients. Recently, however, a prospective controlled trial of short-term interferon treatmentfor the acute form has been carried out (Omata et al., 1991) in which serum HCV RNA became undetectable in 10 or 11 treated patients which seems to suggest that natural interferon beta prevents the progression

Viral hepatitis: current status and issues

9

of acute non-A, non-B hepatitis to chronicity by eradicating HCV. A study by Chayama et al. (1991) indicates that the detection of hepatitis C virus RNA may indeed be a useful marker of viral replication in chronic hepatitis C and further suggest that interferon should be readministered to patients who turn HCV RNA negative on treatment but again exhibit the marker of viral replication on cessation of therapy. This direct test for HCV through HCV RNA helps provide a firm biological basis for therapy of this disease and may allow for more intelligent use of antiviral agents. Recently also, oral ribavirin therapy for chronic hepatitis C virus infection has been evaluated in a pilot study including ten patients (Reichard et al., 1991). Treatment was instituted with oral ribavirin 1000-2000mg per day in two devided doses for 12 weeks. Since significant decreases in aminotransferase, after the 12 weeks treatment was achieved, it was concluded that ribavirin with mild side-effects, full reversible after cessation of therapy may infact be the first drug to offer a potentially effective oral treatment for chronic hepatitis C. A need however exists for its further evaluation on controlled trials possibly in combination with interferon alpha. Eradication of hepatitis B virus infection will only be possible in the foreseeable future if prenatal transmission can be abolished, vaccine nonresponse nullified and carrier state eliminated. Due to available HBV vaccines more than 100 million people have been vaccinated against hepatitis B with an outstanding degree of safety and efficacy. The clinical trials with available recombinant HBV vaccines demonstrated their protective efficacy, immunogenicity and safety (Andre and Safary, 1988). The HBV vaccine infact could be considered the first vaccine against a major human cancer as HBV chronic infection and primary hepatocellular carcinoma links were established. The price of the HBV vaccine in developing countries has fallen to less than US$ 2 for a full course of immunization thus making HBV vaccination possible to children in some developing countries (World Health Organization Press Release, 1992). Tron et al. ( 1989) have recently studied the safety and immunogenicity of different doses of a recombinant (rHBV) vaccine containing S and pre-S2 sequences produced in mamalian cells in comparison to those of a plasma-derived HBV vaccine. The striking feature of this rHBV vaccine was an early high production of antibodies to pre-S2 which may constitute an advantage in prevention of HBV infection. For vaccination against HBV two different immunization schedules are currently in use depending on the manufacturer: three vaccinations given at 0, 1 and 6 months as recommended by Merck Sharp & Dohme and Smith Kline Beecham and four vaccinations at months 0, 1, 2 and 12 was also suggested. Vaccination at 0, 1and12 months is believed to lead to significantly higher anti-HBs levels than those achieved after conventional vaccination at 0, 1and6 months with the same vaccine Uilg et al., 1988). Based on the results of a recent detailed study it is suggested by Jilg et al. ( 1989) that for achieving a higher anti-HBs concentration guaranteering a long-lasting persistence, vaccination at months 0, 1 and 12 had been advocated to be preferable. For individuals at high risk ofHBV infection vaccination at months 0, 1, 2 and 12 might be a useful consideration for obtaining an optimal early

10

Introduction

seroconversion as well as long-term protection. Recently Coursaget et al. (1991) has suggested scheduling ofrevaccination against hepatitis B virus. They applied a random effects regression model to data from 118 Senegalese infants given three injections of HBV vaccine about 6 weeks apart and a booster injection at 13 months and showed that revaccination can be scheduled on the basis of an anti-HBs titre recinded at the time of the booster dose. The titre at booster, according to these authors is no less accurate in predicting future titre than I-month post-booster titre. This scheduling of HBV vaccine boosters has been pointed out by others (Prince, 1991) to be a cumbersome approach for developing countries where the need for HBV vaccine appears to be the greatest and may infact pose a major hindrance towards underlying a national HBV vaccine immunization programme. Coursaget et al: ( 1991) assume that booster will be necessary for HBV but no evidence exists at the present time for such a conclusion. At a time when the incidence of hepatitis A virus infection is increasing in several industrialized countries, the first hepatitis A vaccine, inactivated with formalin has now been licensed. The vaccine is highly immunogenic inducing a protective immune response after one or two doses (Editorial, 1992). It could be predicted that in the near future the manufacturers will produce combined vaccines permitting to immunize against several infectious diseases with a limited number of injections. Association of hepatitis B virus vaccine with other vaccines, like yellow fever virus, DTP, DT poliovirus, measles vaccine, among other, has been found to be compatible and without adverse effects (Ajjan, 1991). Such combined vaccines are to be expected for future immunization programs and strategies. However, the key issues in the future is to produce highly immunogenic specific and safe vaccines, including for viral hepatitis using an expression vector. This approach was already experienced using vaccinia virus and recently adenovirus expression vector systems. The canine adenovirus as a recombinant non-entirely replicating expression vector and non-pathogenic will be a candidate of choice for human vaccine production.

References Ajjan N (1991) Vaccines. Pasteur Merieux SV, Lyon, 1-188 Alexander GJM, Williams R (1988) AmJ Med 85 (Suppl 2A): 143-146 Andre FE, Safary A (1988) In: Zuckerman AJ (ed) Viral hepatitis and liver disease. Alan R Liss, New York, pp 1025-1030 Aiville CN, Nouri-Aria KT,Johnson P, Williams R (1991) Hepatology 13: 310--315 Bhat RA, Ulrich PP, Vyas GN (1990) Hepatology 11: 271-276 Blum HE, Liang TJ, Galun E, Wands JR (1991) Hepatology 14: 56-62 Carman WF, Zanetti AR, Karayiannis P, WatersJ, Manzillo G, Tanzi E, Zuckerman AJ, Thomas HC (1990) Lancet 336: 325-329 Carman WF, Thomas C (1992) Gastroenterology 102 (2): 711-719 Caselman \J, EisenburgJ, Hofschneider PM, Kishy Rl (1989) Gastroenterology 96: 449-455 Cha TA, Kolberg], Irvine B, Stempien M, Beall E, Yano M, Choo QL, Houghton M, Kuo G, Han JH, Urdea MS (1991) J Clin Microbiol 29: 2528-2534

References

11

Chang MH, Chen PJ, ChenJY, Lai MY, Hsu HC, Lian DC, Liu YG, Chen DS (1991) Hepatology 13: 316-320 Chayama K, Saitoh S, Arase Y, Ikeda K, Matsumoto T, Sakai Y, Kabayashi M, Unakami M, Morinaga T, Kumada H (1991) Hepatology 13: 1040-1043 Chen CJ, Liang KY, Chang AS, Chang YC, Lu SN, Liaw YF, Chang WY, Sheen MC, Lin TM (1991) Hepatology 13: 398-406 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M (1989) Science 244: 359-362 Coursaget P, Yvonnet B, Gilks WR, Wang CC, Day NE, ChermJP, Diop-Maar I (1991) Lancet 337: 1180-1183 Degas F, Maisonneuve P, Thiers V, Noel L, Edinger S, Brechot C, BenhamouJP (1991) Lancet 338: 758 Dindzans YJ, Cai MY, Levy GA (1985) Med North Am 21: 2770-2779 Ebeling F, Naukkorinen R, LeikolaJ (1990) Lancet 335: 982-983 Editorial (1992) Lancet 339 (8803): 1198-1199 Esteban JI, Gonzalez A, Hernandez JM, Viladomiu L, Sanchez C, Lopez-TalaveraJCL, Lucea D, Martin-Vega C, Vidal X, Esteban R, Guardia] (1990) N Engl] Med 323: 1107-1110 Feitelson M (1992) Clin Microbiolog Rev 5 (3): 275-301 Fong TL, Di Bisceglie AM, Waggoner JG, Banka SM, HoofnagleJH (1991) Hepatology 14: 64-67 GarsonJA, Tedder RS, Briggs M, Tuke P, GlazebrookJA, Trute A, Parker 0 (1990) Lancet 335: 1419-1422 Ghendon Y (1992) In: Kurstak E (ed) Control of virus diseases, 2nd edn. Marcel Dekker, New York HenriettaJH, Lelie PN, Wong VCW, Kuchns MC, Reesink HK (1989) Lancet i (8635): 406--409 Hollinger FB, Lemon SM, Margolis H (eds) ( 1991) Viral hepatitis and liver disease. Williams and Wilkins, Baltimore Hoofnagle JH, Alter HI ( 1984) In: Vyas GN, DienstagJL, Hoofnagle JH (eds) Viral hepatitis and liver disease. Grune & Stratton, Orlando, pp 97-113 HoofnagleJH, Di Bisceglie AM (1988) In: Zuckerman AJ (ed) Viral hepatitis and liver disease. Alan R Liss, New York, pp 823-830 Hoofnagle.JH (1990) N Engl] Med 323: 337-339 Horiike N, Blumberg BS, Feitelson MA (1991) J Infect Dis 164: 1104--1112 Hosada K, Yokosuka 0, Omata M, Kato N, Omito M (1991) Gastroenterology 101: 766-771 Hsu HC, Wu TT, Sheu JC, Wu CY, Chiou TJ, Lee CS, Chen DS (1989) Hepatology9(5): 747-750 Jardi R, But! M, Rodriguez F, Garcia-Lafuente A, Sjogren MH, Esteban R, Guardia] (1991) Hepatology 14: 25-28 Jilg W, Schmidt M, Deinhardt F (1988) J Infect Dis 157: 1267-1269 Jilg W, Schmidt M, Deinhardt F (1989) J Infect Dis 160: 766--769 Kaklamani E, Trichopoulos D, Tzonou A, Zavitsanos X, Koumantaki Y, Hatzakis A, Hsieh CC, Hatziyannis S (1991) JAMA 265: 1974--1976 KorenmanJ, Baker B, Waggoner J, EverhartJE, Di Bisceglie AM, HoofnagleJN (1991) Ann Intern Med 114: 629-634 Kosaka Y, Takase K, Kojima M, Shimizu M, Inoue K, Yoshiba M, Tanaka S, Akahane Y, Okamoto H, Tsuda F, Miyakawa Y, Mayumi M (1991) Gastroenterology 100: 1087-1094 Krugman S (1985) In: Gerety RJ (ed) Hepatitis B. Academic Press, Orlando, pp 1-4 Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, DienstagJL, Alter .M.J, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee WS, Kuo C, BagerK, Shuster JR, Overby LR, Bradley DW, Houghton M (1989) Science 244: 362-364 Kurstak E ( 1992) Control of virus diseases, 2nd edn. Marcel Dekker, New York, pp 1-448 Lau JYN, Sheron N, Morris AG, Bumford AB, Alexander GJM, Williams R ( 1991) Hepa to logy 13: 1035-1039 Lee HS, Chung YH, Kim CY (1991) Hepatology 14: 68-72 Lesniewski RR, Desai SM, Johnson RG, Nelson LR, Schlouder GG, Sant CL, Mushahwar IK (1992) Proc European HCV Council Group Meeting, Venice, July 1992 Lin I~J, Lai CL, Lauder IJ, Wu PC, Lau TK, Fong MW (1991) J Infect Dis 164: 284-288

12

Introduction

McHutchsonJC, Kuo G, Houghton M, Chao QL, Redeker AG (1991) Gastroenterology 101: 1117-1119 Marcellin P, Martinott-Peignoux M, Bayer N, Pouteau M, Almont P, Erlinger S, BenhamouJP (1991) Lancet 337: 551-552 Mazella G, Rizzetto M, Amed MA, Quiritela G, Rosina F, Barbara L, Roda E (1988) Amj Med 85 (Suppl 2A): 141-142 McLachlan A (1991) Molecular biology of the hepatitis B virus. CRC Press, Boca Raton, 1-312 MelnickJL (1992) J Virol Dis 1: 6-14 Moriyama K, Nakajima E, Hohjoh H, Asayama R, Okoch K (1991) Lancet 337: 125 Mutchnick MG, Appelman HD, Chung HT, Aragona E, Gupta TP, Cummings GD, WaggonerJG (1991) Hepatology 14: 409-415 Okamoto H, Imai M, Kametani M, Nakamura T, Mayumi M (1987) Jpnj Exp Med 57: 231:236 Omata M, Yokosuka 0, Takano S, Kato N, Rosada S, Imazeki F, Tadu M, Ito Y, Ohto M (1991) Lancet338:914-915 Perrillo RP, Schiff ER, Davis GL, Bodenheimer HC, Lindsay K, Payne], DienstagJL (1990) N Engl Med 323: 295-301 Pozzato G, Moretti M, Franzin F, Croce LS, Tiribelli C, Masayu T, Kaneko S, Unoura M, Kobayashi K (1991) Lancet 338: 509 Prince AM (1991) Lancet 338: 61 QuirogaJA, Campillo ML, Catillo I, Bartolome], Porres JC, Carreno V (1991) Hepatology 14: 38-43 Ray R, Aggarwal R, Salunke PN, Mehrotra NN, Talwar GP (1991) Lancet 338: 783-784 Reichard 0, Anderson], Schvarcz R, Weiland 0 (1991) Lancet 337: 1058-1061 Rizzetto M (1984) Hepatology 3: 729-737 Ruiz-Moreno M, Rua MJ, Molina], Moraleda G, Moreno A, Garcia-Aguado J, Carreno V ( 1991) Hepatology 13: 1035-1039 Shattock AG, Morris MC (1991) J Clin Microbiol 29: 1873-1878 ShihJWK, Chueung LC, Alter HJ, Lee LM, Gu HR (1991) J Clin Microbiol 29: 1640-1644 Smedile A, Rosina F, Saracco G, Chiaberge E, Lattore V, Fabiano A, Brunetto MR, Venne G, Rizzetto M, Bonino F (1991) Hepatology 13: 413-416 Thaler MM, Park CK, Landers DV, Wara DW, Houghton M, Wauters GV, Sweet RL, Han JH (1991) Lancet 338: 17 Tron F, Degos F, Brechot C, Courouce AM, Goudeau A, Marie FN, Adamowicz P, Saliou P, Laplanche A, BenhamouJP, Girard M (1989) J Infect Dis 160: 199-204 Van der Poel CL, Cuypers HTM, Reesink HW, Weiner AJ, Quan S, Di Nello R, Van BovenJJP, Winkel I, Mulder-Folkerts D, Excel-Verlers PJ, Schaasberg W, Leentvaar-Cuypers A, Polito A, Houghton M, Lelie PM (1991) Lancet 337: 317-319 Vento S, Garofano T, Perri G, Dold L, Conda E, Basetti D (1991) Lancet 337: 1183-1187 Weiner AJ, Kuo G, Bradley DW (1990) Lancet 335: 1-3 World Health Organization (1992) Press Release WH0/12, February 21 YokosukaO, OmataM, HosadaK, TadaM, Ehata T, Ohto M (1991) Gastroenterology 100: 175181 Zuckerman AJ (ed) (1988) Viral hepatitis and liver disease. Alan R Liss, New York, pp 1-1136

Part I Hepatitis A virus and disease

1 Hepatitis A virus properties and replication I. Introduction About 19 years have elapsed since the causative agent of hepatitis A was identified by immune electron microscopy in feces of infected individuals by Feinstone et al. (1973) and about a decade since the hepatitis A virus (HAV) particles purified from such fecal samples shown to bear similarity in various properties with the picornaviruses. The picornaviruses contitute a family of small, stable, RNA viruses that includes well known and widely investigated agents as poliovirus, rhinovirus, encephalomyocarditis virus and foot-andmouth disease virus (Siegl, 1988). The conclusions based on analogy with established properties of known picornaviruses as poliovirus have proved invaluable in the analysis of the hepatitis A virus. Current evidence is indicative of HAVas distinct picornavirus with several as yet unrecognized properties (Stapleton et al., 1991a; Vento et al., 1991). This chapter deals with the current status of properties and replication of HAV, a virus thus far known to be a genetically and antigenically stable one with a single identifiable serotype.

II. The nature of the virus particle

A. Morphology and size Small spherical virus-like particles with antigenic properties of hepatitis A virus (HAV) were identified byFeinstone etal. (1973) in the feces of patients suffering from hepatitis A. Electron microscopy revealed a diameter of27 nm for the virus particles (Fig. 1; courtesy of Dr. G. Siegl). Similar particles were subsequently detected in varied clinical samples of human origin (Locarnini et al., 1975, 1978; Siegl and Frosner, 1978) and also from infected cell cultures (Provost and Hilleman, 1979; Kojima et al., 1981). These observations confirmed the mature infectious hepatitis A virion as a spherical nonenveloped particle measuring 27 nm in diameter (Figs. 1 and 2).

Hepatitis A virus properties and replication

16

Fig. 1. Electron micrograph of hepatitis A virus (HAV) particles of 27 nm in diameter

Copsid

RNA Fig. 2. Schematic representation of hepatitis A virus (HAV) particle components

Even though electron micrographs have repeatedly revealed the presence of both full and empty particles and related to the complete virion and its empty capsid, information on the detailed morphology of HAVis lacking. The HAV capsid by the superimposition technique appears to be composed of 32 distinct capsomeres (Siegl, 1982).

B. Sedimentation co-efficient and buoyant density Buoyant density centrifugations of HAV particles purified from human and primate fecal and liver specimens have led to a great variety of density values ranging from 1.24 to 1.50 g/ml (Feinstone et al., 1974; Provost et al., 1975;

The nature of the virus particle

17

Rakela et al., 1976; Siegl and Frosner, 1978). This variation was attributable in part to the purification procedures which failed to disintegrate lipoprotein-HAV complexes and insufficient accuracy in the determination of density fractions. The main fraction of the particles does however band at approximately 1.34 g/ml. Infectivity of both types of particles have been demonstrated in marmosets (Bradley et al., 1975). Compared to the major component of infectious HAV, the latter type of particles are of distinctly lower specific infectivity (Siegl, 1988). Replication in vitro of HAVin cell cultures with the simultaneous radioactive labelling of viral nucleic acid and protein components enabled the unequivocal determination of the buoyant and sedimentation characteristics of mature HAV particles. In clinical specimens the mature hepatitis A virion bands around a density of l.34g/ml similar to enteroviruses of the picornavirus family. Likewise HAV sediments at about 160 S. Centrifiguration studies using poliovirus type 2 (l.34g/ml, 160 S) as internal marker has however revealed HAV to have a slightly lower buoyant density (l.34g/ml) and sedimenting more slowly (156 S) (Siegl et al., 1981; Lemon et al., 1985). Recently, Ruchti et al. (1991) have identified and characterized incomplete hepatitis A virus particles generated during persistent infection. A major portion of the viral antigen was found to be associated with noninfectious, empty particles banding at l.305g/ml and l.20g/ml CsCl and sedimented in sucrose gradients at 76S and 59S. Empty HAV particles were similar to those of poliovirus with respect their physical stability and had the characteristic capsid protein content (VPO, VPl and VP3).

C. Reactivity ofphysical and chemical agents The resistance of HAV to various physical and chemical agents have been frequently overestimated. However, reliable data to date indicate that HAV equals or exceeds all known picornaviruses in stability and may very well be among the most resistant human viral pathogens (Siegl, 1984). The stability favors its effective distribution via fecal material and contaminated food under both endemic and epidemic conditions (Siegl, 1988). Stability of HAV may be linked to its amino acid composition, the size, and possibly, arrangement of the capsid proteins. HAVin feces or serum samples have been shown to remain infective after exposure to 56°C during 60 minutes (Krugman et al., 1970). The stability at elevated temperatures is considered a criterion of value in the classification of the virus and thus the resistance ofHAVand poliovirus type 2, the prototypical enterovirus have been compared (Siegl, 1984). Under strictly controlled experimental conditions it was found that the temperature at which 50% of poliovirus particles became disintegrated during heating at pH 7 for 10 min (T50,10 = 43°C) differed significantly from the one characteristic for HAV (T50 ,10 = 61 °C). In the presence oflM MgCl known to stabilize enteroviruses, shifting of T 50,10 to 61°C and 81°C were observed for poliovirus and HAV,

18

Hepatitis A virus properties and replication

respectively. Thus, the thermal stability ofHAV is substantially greater than that of other picomaviruses. lnfectivity is reduced only 100-fold when the virus is held at room temperature for four weeks (Siegl, 1984). HAV has been found to be stable at pH3 as are enteroviruses (Provost et al., 1975; Siegl, 1984). The absence of lipids from the compact HAV particle is indicated by the high buoyant density in CsCl and this is consistant with the resistance of HAV to treatment with 20% ethyl ether at 4°C/24h (Provost et al., 1975). Purification schemes for HAVin the past several years have included extraction of virus suspensions with Freon (Trichlorotri-fluoroethane) up to concentrations of 50% without noticeable loss in infectivity or antigenicity and similar results have been obtained with chloroform (Siegl and Frosner, 1978; Locarnini et al., 1979). The early recognition of the importance of the fecal-oral route in the spread of hepatitis A virus prompted the investigation of disinfectants in inactivation of HAVin feces. Chlorine in large amounts was found to destroy the infectivity of HAVin crude fecal extracts. In a report of the World Health Organization (McCollum & Zuckerman, 1981) free chlorine (hypochlorous acid) at a concentration of I mg/liter was indicated to be enough to inactivate HAVin 30 min. Peterson et al. ( 1983) found enhanced resistance of HAV to chlorination as compared to common picornaviruses. Provost et al. ( 1975) found complete inactivation of purified HAV during incubation with formalin at a final dilution of I: 4000 at 37°C/71 h. Purified HAV in phosphate buffered saline is readily inactivated by irradiation with UV light at I.I W within a minute (Provost et al., 1975) and likewise the inactivation kinetics of HAV during irradiation with Co60 has been reported to resemble closely those characteristic ofpoliovirus (Frosner, 1982).

III. The vims genome and structural proteins A. Virus genome Subsequent to the initial identification of hepatitis A virus in human feces studies with indirect methods suggested the genome of the HAV to consist of RNA. These included: a) the detection ofviral particles and antigen in the cytoplasm of infected marmoset and chimpanzee hepatocytes both by electron microscopy and by staining with labeled antibodies (Provost et al., 1975; Murphy et al., 1978; Shimizu et al., 1978); b) observation of concentrated purified HAV particles staining orange-red with acridine orange, like single-stranded RNA and DNA (Provost et al., - 1975); c) enhanced reduction of virus infectivity by digestion of virus particles with pancreatic RNase at 60°C/lh in comparison to the degree ofinactivation by heat treatment alone (Provost et al., 1975);

The virus genome and structural proteins

19

d) significant alteration in the sedimentat ion characterist ics of the virus particles after pretreatme nt of HAVat pH 10, 4 °C/ lh and incubation with RNase but not with DNase (Bradley et al., 1978). Electron microscopic and ultracentrif ugal sedimentat ion studies of nucleic acid molecules released from highly purified HAV samples provided confirmation of the earlier conclusions . Linear, single-stran ded molecules which like poliovirus, unlike parvovirus and phage X-174 single-stran ded DNA, could be hydrolized by incubation of virus particles at pH 12.9 following treatment of HAV samples with 4M urea and 90% formamide. Two m ain species with molecular weights of 1.9 x 106 and 1.3 x 106 were detected (Siegl and Frosner, 1978). These molecules were subsequent ly shown to sediment at 33 S under non-denatu ring conditions in sucrose gradients and were selectively abolished in nucleic acid samples pretreated with RNase. Hence the conclusion that the genome of hepatitis A virus is a linear, single-stran ded RNA molecule (Fig. 3, courtesy of Dr. G. Siegl) with positive polarity and a molecular weight of 2.25 x 106 . The single-stran ded RNA genome of HAV is approximat ely 7500 nucleotides long and has a polyadenyli c acid tail at its 3' end (Bradley et al., 1978; Siegl et al., 1981; Ticehurst et al., 1983). Complemen tary DNA transcripts of RNA from several HAV strains have been subjected to molecular cloning

Fig. 3. Linear, single-stranded RNA mole cule of h e patitis A virus (HAV) o btained under denaturating conditions

20

Hepatitis A virus properties and replication

(Ticehurst et al., 1983; 1988; Linemeyer et al., 1985) and the complete nucleotide sequence of one viral strain has been reported (Najarian et al., 1985). A study on the cloning and sequencing of RNA from an HAV isolate have been indicated to show the molecule consists of a total of 7478 nucleotides and like the genome of a typical picornavirus it can be functionally divided into i) a 5 '-terminal untranslated region composed of 733 nucleotides, ii) a single long open reading frame (ORF) of 6681 nucleotides encoding a polyprotein of approximately 250kDa. Viral structural proteins are generated by post-translational proteolytic processing of this polyprotein and, iii) a short 64 nucleotides long, untranslated segment at the 3 '-end of the molecule (Cohen et al., 1987; Winokur et al., 1991). On the basis of analysis of molecular sequences Ticehurst et al. (1988) demonstrated that HAV has characteristic genomic organization of a picornavirus. However, they concluded that HAV sequences are clearly distinct from those of enteroviruses and other picornaviruses. The nt sequence of HAVis not homologous with other picornaviruses and HAV nucleotide and aa sequences are distinct when compared with enteroviruses. Ticehurst et al. (1988) suggested that HAV classified as enterovirus type 72 within the family of Picornaviridae should be reclassified.

B. Virus structural proteins and their function The polypeptide composition of the hepatitis A virus capsid have been determined on virus particles purified from human stool (Coulepis et al., 1978; 1982; Tratschin et al., 1981) with the viral polypeptides either radioiodinated prior to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or staining with Coomassie blue. Subsequently, labeling ofHAV structural proteins have been carried out during the process of HAV replication in cell culture (Siegl et al., 1981). The capsid of a typical mature picornavirus particle is known to consist of 60 copies with at least three major structural polypeptides VPl, VP2 and VP3 ranging in size from 33,000 to 22,000 daltons and they closely match the six of the structural polypeptides VPl, VP2 and VP3 of the corresponding picornavirus particle (Cooper et al., 1978; Coulepis et al., 1982). A fourth polypeptide (VP4) with a molecular weight of about 2500 is probably also present (Baroudy et al., 1985). Together these polypeptides form a tight protein shell ( capsid) which contains the viral RNA. Furthermore, a 40,000 MW protein have also been identified (Tratschin etal., 1981; Siegletal., 1981) which may represent the equivalent of the precursor polypeptide (VPO) of picornaviruses. This polypeptide is present in empty viral capsids as well as in the provirion of the mature picornavirus and during maturation of the virus particle it is cleaved into VP2 and VP4 (Putnak and Phillips, 1981).

21

The vims genome and structural proteins

Most of the HAV proteins have been tentatively mapped to their corresponding site in the genome by using similarity of HAVamino-acid sequences with amino-acid sequences from other picornaviruses. A total of 11 proteins in three distinct regions Pl-P3 have been included. The location ofVPl and VP3 have been determined by sequencing the amino-acids at the aminotermini of these HAV proteins (Lin em eyer et al., 1985). The location of HAV VP2 was confirmed by raising antibody to a synthetic peptide corresponding to this protein. Antibody to the peptide reacted with VP2 on a Western blot (Wheeler et al., 1986). HAVVP2 is smaller than VP3 whereas in most other picornaviruses VP2 is larger than VP3. The assembly of capsid proteins into subviral or virion structures may be necessary for the generation of HAV epitopes which efficiently elicit neutralizing antibodies (Winokur et al., 1991). Consistent with the low overall homology between the nucleotide sequences ofHAV and members of the entero, rhino, cardio and aphtoviruses, homology in amino-acid sequences corresponding proteins is barely evident. Most extensive homology was detected among proteins required for RNA replication or cleavage of the polypeptide precursor. At best, however, it amounted to 29% of amino-acid identity (Siegl, 1988). All the information on the structure of the hepatitis A virus genome in relation to the protein cleavage sites are summarized in Fig. 4.

5'

HAV RNA (7 .5 Kb)

0.73Kb

6.2

7.4

VPg-~~1~1-·8~~1+.6~~~2.~2~~-3~i1~3~i-7-:~1~~~~5~l-+l+t~~5~.9~~~~1-po:(A) AUG + UGA poly protein TRANSLATION Region protein - - - - P1 - - - • -

P2 -

name

VP4

VP2

VP3

VP 1

2A

28

D c:==:J C=:J C==:J i:::=:::J D

Length (amino acids)

Function

23

222

248

300

'------..,,...---~; Viral capsld proteins

- - - - P3 - - -

2C

3A

38 3C

C==:J D D CJ

30

~-~

J ... [ x ·-

189

107

335

74

?Transcrlptase

Protease

23

219

489

RNA-polymerase

Protease

-structural - I + - - - - - Non-Structural - - - - •

Fig. 4. The hepatitis A virus (HAV) genome alongwith the protein cleavage sites

22

Hepatitis A virus properties and replication

IV. The virus replication cycle The genomic organi~ation of hepatitis A virus is similar to that of poliovirus and thus a replication scheme resembling picornaviruses. After oral inoculation with HAV, viral replication occurs in the liver with viremia and shedding of the virus in the stool (Klugman et al., 1962). The replication cycle ofHAV in the liver has not been studied extensively with many replicative steps inferred from studies on poliovirus and other picornaviruses. HAV is presumed to enter the hepatocyte after attachment to a viral receptor on the plasma membrane (Cohen, 1989). Following the uncoating of the RNA, host ribosomes bind to the viral RNA with the formation of polysomes. HAV RNA is translated to yield a polyprotein cleaved to yield the capsid proteins (Pl region) and non-structural protein (P2 and P3 regions). The genome plus strand is copied by the viral RNA polymerase to produce a replicative intermediate consisting of both plus and minus strand RNA. The minus strand RNA serves as a template for producing additional plus strand RNA which is used for translation into proteins and for assembly into mature virions (Cohen, 1989). Copies of the viral capsid assemble to form the protein shell which envelopes the plus strand RNA followed by assembly of HAV on cytoplasmic membranes (Shimizu et al., 1978). HAV particles may infect adjacent hepatocytes or the vesicles containing HAV may be released from the hepatocyte into the bile canaliculi. HAV may be released from the vesicles when in contact with bile acids in the canaliculi (Cohen, 1989). A recent study by Anderson et al. (1988) of single-cycle growth kinetics of a HAV strain, HM-175 suggest that uncoating of virus particles delays the initiation of HAV replication of approximately l 2h after which the replication of viral RNA may limit the growth rate and yield of infectious HAV. In monolayers of the BS-C-1 continuous line of African green monkey kidney cells ShavrinaAsher et al. (1988) demonstrated using immunoperoxidase staining that RAV-related antigen was detected 18 hours post-infection and that it increased thereafter. A feature that distinguishes HAV in the cell systems from most other picornaviruses is the resistance of replication to reduce or to block synthesis of many picornaviruses. Included among a total of twenty-five antivirals tested are guanidine, amantine, rhodamine and methyl quercitin (3-MQ) (Siegl and Eggers, 1982; Widell et al., 1986; de Chastonay and Sieg!, 1988). All were found to be ineffective in non-cytotoxic concentrations. This oversation points to the existence of basic differences in replication between HAVand well known picornaviruses. This difference may according to Siegl (1988) offer the possibility of a very specific antiviral prevention or therapeutic treatment of hepatitis A. The cell receptor for several picornaviruses is the major determinant of cell and tissue susceptibility (Crowell and Landau, 1983). Little is known about HAV interactions with specific cell receptors. In a study Stapleton et al. (1991 b) demonstrated that HAV has a calciumdependent receptor on cultured permissive cell lines and that RGD peptides (arginine-glycine-aspartic acid) were unable to interfere with HAV attachment to cells. The calcium-dependent specific attachment of four experi-

The virus replication cycle

23

mented HAV antigenic variant strains showed identical attachment properties with neutralization susceptible strains, suggesting that the immunodominant neutralization antigenic site ofHAV is not directly involved in cell attachment. The identification of HAV receptor is important for understanding of this virus tissue tropism and replication sites as well as in antiviral therapy and in vaccines design.

2 Clinical aspects of hepatitis A virus infection I. Introduction Although clinically recognized acute hepatitis, a generalized infection with inflammation of liver in epidemics, had been known since the time of Hippocrates, it was only after the development of specific serologic tests for the markers of hepatitis A virus (HAV) that it has been possible to obtain a differentiation ofHAV cases with certainty from those caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) or hepatitis E virus (HEV) (non-A, non-B hepatitis) (Tabor, 1984). Serologic test has enabled the recognition of asymptomatic cases of HAV infection and the symptoms characteristic of serologic specific hepatitis A. Various clinical aspects of hepatitis A virus infection are discussed in this chapter.

II. Clinical features of hepatitis A The characteristics, symptoms and signs of hepatitis A virus (HAV) infection may often be indistinguishable from those of hepatitis B virus or hepatitis C virus and hepatitis E virus (non-A, non-B hepatitis) infections. The symptoms of disease are usually non-specific. In individual cases the infecting viral agent cannot even be possibly distinguished by clinical features alone (Dindzans et al., 1985). However, groups of serologically confirmed viral hepatitis cases exhibit distinct clinical patterns. Most cases usually begin with malaise, fatigue and loss of appetite. Nausea and vomitting may begin within several days or simultaneously with the onset of other symptoms. Right upper quadrant abdominal pain may also occur. Jaundice is recognizable and mild icterus may pass unrecognized early in the course of illness if not seen by a physician. Dark urine, light-colored stools or icteric skin usually prompts medical attention. Jaundice may be present at about the time of onset of fatigue and loss of appetite or may develop 1-7 days later. Additional signs often present include hepatomegaly and splenomegaly.

Clinical features of hepatitis A

25

Of primary importance is the recognition of certain aspects of the presentation of a patient with hepatitis A which may permit a provisional diagnosis of HAY infection. Considering the environmental or epidemiological aspects a patient's infection may be attributed to HAV if it follows ingestion of water contaminated with sewage or following close contact with a person known to have an HAVinfection. The subsequent transmission ofacute hepatitis to other close contacts from I to 6 weeks after onset would be suggestive of hepatitis A. Onset of hepatitis A occurs without the prodrome rash and arthralgia characteristic of I 0% of hepatitis B (Tabor, 1988). Fever is the one sign or symptom which occurs primarily in some acute infections. Fever has not been reported in non-A, non-B hepatitis and is known to occur during hepatitis B only in those few fulminant cases rapidly developing coma (Tabor et al., 1976). In hepatitis A fever is a common occurrence rarely becoming higher that 39°C (I 02°F). The fever may be accompanied by chills. Diarrhea occasionally accompanies hepatitis A. Headache may also occur during hepatitis A and is not generally associated with other forms of hepatitis. Myalgia and pharyngitis have also been reported. Age exerts a striking influence on the frequency with which the clinical signs and symptoms of hepatitis accompany primary infection with HAV. Young children, especially under the age of two years, when infected with HAV infrequently have symptoms recognized as being related to hepatitis. About 4% to 6% only of the young children suspected of transmitting HAV acquired at day-care centers to older family members, have had symptoms characteristic of hepatitis (Benenson et al., 1980; Hadler et al., 1980). On the other hand, overt hepatitis develops in the majority of adults infected with HAV (Lednar et al., 1985). In the individual case, the signs and symptoms of hepatitis A virus infection appear to be indistinguishable from those due to other types of viral hepatitis. However, diarrhea has been reported to occur in about 20% of adult patient and perhaps 60% of infected children with the explanation for this still uncertain but does not raise the question of viral replication within the gut (Lemon, 1985). It is not possible to make a definitive diagnosis without the use of serological markers. Laboratory findings in hepatitis A virus infection are usually indistinguishable from those of other forms of viral hepatitis. Although levels of serum aspartate aminotranferase and alanine aminotransferase activity (AST, ALT) usually rise abruptly and reach relatively high levels. These findings are not limited to hepatitis A nor are they typical of every case of hepatitis A. Peak levels of AST and ALT range from 40 to 1790 IU /liter, but are usually greater than 500 IU /liter. Peak serum total bilirubin levels range from I.I to 8.9 mg/dl (Sampliner et al., 1984). The lower range of these values includes the normal range. Occasionally, extreme elevations of aminotransferase and bilirubin levels may be found in hepatitis A virus infection (Tabor, 1988). Abnormalities of coagulation are rare. They are however, usually unaccompanied by clinical evidence of bleeding (Humphrey et al., 1979) and are probably due to the inability of the severely infected liver to manufacture clotting factors.

26

Clinical aspects of hepatitis A virus infection

III. Infection incubation period The incubation period of hepatitis A virus infection is between three and five weeks with a mean of 28 days. Subclinical and anicteric infections are common and although the disease has, in general, a low mortality, adult patients may be incapacitated for many weeks. Death occurs in less than 1 % of hepatitis A cases admitted to hospital. Chronic infection with HAV has never been observed. Usually, hepatitis A is a self-limiting disease with recovery occurring within two to three months. Rarely, late amino-transferase elevations with or without symptoms may occur at five to nine months after onset of HAV infection (Jacobson, 1985). Villarejos et al.(1988) has reported baffling experience of the detection of persistence and reinfection of hepatitis A in Costa Rica. In six family contacts, aged 4, 4, 5, 7, 9 and 10 years, respectively, whose sera were repeatedly negative for both IgM and IgG antiHAV, specific coproantibodies were detected repeatedly at consistently high levels up to 4 weeks before onset of clinical hepatitis A virus infection. Then, these were replaced by high levels of HAV excretion, lasting 1-3 weeks while the serum showed increasing titers ofigM anti-HAV. Eventually, coproantibodies reappeared, attaining higher levels than initially, and IgG anti-HAV gradually replaced the IgM antibodies, conferring lasting immunity. The authors interpret their results as evidence of two separate infections with hepatitis A virus, the first without causing illness, presumably limited to the intestinal lumen, with neither systemic involvement none, consequently, detectable antibody response, and the second occurring as a regular systemic infection with clinical signs and symptoms and a full-blow serologic immune response. They postulate the existence of strain variants that replicate in the intestinal lumen but do not have the potential to penetrate the gut and reach the blood stream or even perhaps the result of infection with very low doses of virus. It should be stressed that the role of intestinal immunity remains uncertain in hepatitis A. In a recent study Stapleton et al. (1991 b) suggested that intestinal immunity does not play a significant role in protection against hepatitis A virus infection. In contrast, secretary IgA plays an important role in intestinal resistance to many enterovirus infections. The oral poliovirus vaccine induction of secretary IgA conferring protection against reinfection is one of examples.

IV. Asymptomatic and fulminant infections Asymptomatic cases of HAV infection serologically documented are rather common. Such infections appear to be predominant among children under 2 years of age (84%) and are more common than among children more than 3years ofage (ages 3 and 4, 50%; age 5 and older, 20%) (Hadler etal., 1980). Furthermore, about one-fifth of healthy adolescents in the United States have shown serologic evidence of previous hepatitis A (anti-HAV) without ever having had clinical symptoms of hepatitis (Tabor et al., 1979). The

Polyphasic course of infection in children

27

prevalence of asymptomatic infections is reported to be lowest among adults (Benenson et al., 1980). It is assumed that asymptomatic HAV infections may often be the source of epidemic of hepatitis A. However, these infections would not result in early public health measures such as isolation of the index case and administration of immune globulin to close contacts (Tabor, 1984). Fulminant hepatitis, characterized by rapid onset of liver failure and coma can result from infection with hepatitis B virus (HBV) alone or with superimposed hepatitis D virus and hepatitis C virus (non-A, non-B hepatitis), but rarely associated with HAV infection. Evidence of acute hepatitis A occurs in about only 10% of cases (Dindzans et al., 1985). Of the eleven reported cases of fulminant hepatitis A, six were reported by Rakela et al. (1978). Serologic evidence of active or fairly recent HAV infections were documented in 14% of the cases of fulminant non-B hepatitis. The clinical presentation in fulminant HAV infection does not appear to be significantly different from other cases of symptomatic acute HAV infection (Rakela et al., 1978; Mathiesen et al., 1979; Mathiesen, 1981). In documented cases, incubation periods ranged from 3 to 4 weeks and symptoms at presentation included jaundice, dark urine, nausea, abdominal pain, headache and thirst.

V. Polyphasic course of infection in children There have been some reports on course of acute hepatitis in patients positive for IgM antibodies to hepatitis A virus (anti-HAV) ( Gruer et al., 1982; Cobden and James, 1985; Hollinger et al., 1991). It is yet to be clarified whether this pattern is attributable to HAV or to superimposed non-A, nonB agents (hepatitis C virus). Chiriaco et al. (1986) has reported similar clinical behavior in 23 children aged three to eleven year for acute IgM antiHAV positive hepatitis representing about 20% of all pediatric cases of hepatitis A observed during the same period. None of the patients had a history of parenteral exposure to possible sources of viral hepatitis infection and during the acute phase all were reported to be negative for hepatitis B surface antigen, IgM antibody to hepatitis B core antigen, and antibodies to cytomegalovirus and to Epstein-Barr virus. The authors suggest that the polyphasic course of hepatitis, frequently observed in their children in a highly endemic area for viral hepatitis, could be due to simultaneous infection with HAV and with non-A, non-B agents (HCV), transmitted by the same, likely fecal-oral route but with different incubation periods. Alternatively, the hepatitis relapse could have been due to an unknown agent that requires a helper effect by the HAV, in a way similar to that of the hepatitis D virus with respect to hepatitis B virus (Raimondo et al., 1982). However, the long interval between the initial and subsequent alanine aminotransferase (ALT) peaks seen in some of their cases, combined with the fact that four patients had become negative for IgM anti-HAVon relapse, seem to favor the hypothesis of an independent agent linked to HAV only in relation to the route of transmission.

3 Pathogenesis of hepatitis A virus infection I. Introduction Although hepatitis-like illnesses have been known to physicians since antiquity and recognized as an infectious disease with potential for occurrence in large outbreaks (epidemic jaundice) for about a century, it was not until the 1940s that transmission studies in human and epidemiologic observations provided evidence for the existence of the presently recognized two distinct forms of viral hepatitis, A and B. Unlike that of hepatitis B, research on hepatitis A progressed slowly until the 1970s when hepatitis A virus could be visualized and immunologic assays to detect virus antigen and antibody were effectively developed, thus could HAV be identified unequivocally as the causative agent of sporadic and epidemic cases of hepatitis. Among the hepatitis viruses, hepatitis A virus is uniquely and primarily transmitted by the fecal-oral route and thus possesses an unequaled potential for epidemic spread. Although relatively benign and never progressing into chronic hepatitis infection, the hepatitis A virus (HAV) nevertheless remain an important cause of morbidity and occasional mortality (Lemon, 1985; Hoffman, 1991). In Shanghai, China, over 1 million people were infected by HAV in a major outbreak (Yang et al., 1988). Some aspects of the pathogenesis of hepatitis A virus disease are discussed below.

II. Pathologic features of disease Despite the centuries-old recognition of viral hepatitis as a distinct entity, detailed understanding of the clinicopathologic feature of acute hepatitis A and the hosts' immune response has taken place only in the past decade subsequent to the development of methodology for visualization of HAV and detection of viral antigen and antibody. The morphologic lesions of acute hepatitis A are typical of the wellknown changes observed in all types of viral hepatitis. During the period of elevated serum aminotransferase activity, the histologic features include

Virologic events of infection

29

parenchymal cell necrosis and histiocytic periportal inflammation. The reticulin framework of the liver is usually well preserved except in some cases of massive and submassive necrosis. The pattern of changes in the liver is essentially similar in viral hepatitis A and B and consists of marked focal activation of sinusoidal lining cells. Accumulations of lymphocytes in histiocytes within the parenchyma, often replacing hepatocytes lost by necrosis, mild diffuse hepatocytic changes with occasional coagulative necrosis in the form of acidophilic bodies, and focal regeneration and portal inflammatory reaction with alteration of bile ductules. The lesions in hepatitis A develop earlier and the duration of morphologic changes is shorter, while the lesions in hepatitis B longer on, fluctuate and regress slowly. There is also some difference in the distribution of the lesions. In hepatitis A, the localization of parenchymal changes is predominantly periportal, whereas in hepatitis B the lesions are diffuse and tend to accentuate around the hepatic vein tributaries (Zuckerman, 1988). Whether hepatocellular necrosis in hepatitis A is limited to the periphery of the liver lobule remains unresolved. In marmosets with experimental HAV infection, areas of focal necrosis span the entire lobule and extend to the centrilobular zone (Deinhardt et al., 1967; Holmes et al., 1969; Popper et al., 1980), whereas the morphologic features of hepatitis A in chimpanzees are known to spare the centrilobular zone (Dienstag et al., 1975). Studies of histologic changes in acute hepatitis A in humans have also yielded conflicting results. Peripheral localization of focal necrosis in single percutaneous liver biopsy specimens from prisoner volunteers with mild acute hepatitis A and from cases of hepatitis A injapan have been noted in similarity to that found in chimpanzees (Boggs et al., 1970; Popper et al., 1980; Abe et al., 1982). In contrast, considerable centrilobular focal liver cell necrosis have been observed in 16 of 17 liver biopsy specimens from serologically confirmed cases of hepatitis A with atypical clinical features at older age and unexpectedly prolonged jaundice (Teixier et al., 1982). Evidence was provided that cytotoxic T cells capable oflysing HAV infected target cells develop in course of infection (Vallbracht et al., 1986). The hepatitis A virus-specific killing by liver-infiltrating T lymphocytes in man (Vallbracht et al., 1989) support the hypothesis that liver cell injury in acute HAV infection is mediated by HAV-specific CDS+ T lymphocytes and not by a cytopathic effect of the virus itself. In general, in humans the changes in liver morphology are known to last approximately 2-4 weeks and in almost all cases resolve without distortion of lobular architecture or evidence of chronic liver disease (Friedman and Dienstag, 1984; Lau et al., 1991).

III. Virologic events of infection Although the pathogenesis of hepatitis A virus infection remains largely unknown, some understanding has been forthcoming from the limited number of experimental studies involving human beings (Krugman et al., 1960; Boggs et al., 1970) and non-human primate species (Dienstag et al.,

30

Pathogenesis of hepatitis A virus infection

1975; Deinhardt et al., 1975; LeDuc et al., 1983). These studies have confirmed an incubation period of 28 days, generally shorter than that of hepatitis B or non-A, non-B (hepatitis C virus) infection, had have extended the possibility of occurrence of HAV infection by both percutaneous and oral routes. A short period ofviremia is known to precede the onset of hepatitis disease and the fecal shedding maximal during the late incubation period either just before of shortly after the onset of symptoms of liver disease. Unlike hepatitis B or post-transfusion hepatitis C (non-A, non-B hepatitis), HAV yet remains unrecognized as causing chronic or persistent infection. HAV antigen during early infection in chimpanzees has been identified by immunofluorescence in the cytoplasm of as many as 5% to 10% hepatocytes (Mathiesen et al., 1977) and substantially greater proportions ofhepatocytes containing viral antigen in intravenously inoculated marmosets (Mathiesen et al., 1978). Furthermore, the presence of virus particles within cytoplasmic particles has been revealed by electron microscopy (Schulman et al., 1976; Shimizu, 1982). Generally, fecal shedding of virus is temporarily correlated with the appearance of antigen in the liver but the antigen may however, be found in the liver before its appearance in the feces and may persist throughout the period of liver enzyme elevations. Later in the infection the antigen is known to be localized to only a few hepatocytes and Kupffer cells. Antigen has also been detected by immunofluorescence in abdominal lymph nodes, the spleen, kidney where it follows a distribution consistent with the deposition of immune complexes along the glomecular basement membrane (Mathiesen et al., 1978). The question of whether or not HAV initially multiplies within a primary target cell in or near the intestinal epithelium remains unclear. The route of inoculation does not appear to exert greater influence on the length of the incubation period or the amount of virus shed in the feces of experimentally infected primates. Perhaps the length of the incubation period may be more directly related to the titer of the challenge inoculum (Purcell et al., 1984). Negative observations of the searches for HAV antigen in the intestinal epithelium of primates during the early and late stages of infection in the presence of antigen in the feces (Mathiesen et al., 1977, 1978, 1980; Krawczynski et al., 1981) coupled with the finding of viral particles in the bile during acute infection (Schulman et al., 1976; Shimizu, 1982) have paved the way to the current hypothesis that HAV found in feces is derived from the hepatocyte and reaches the intestine through the bile ducts.

IV. Infection immunopathogenesis The humoral immune response to HAV is intense and serum antibody to HAV appears early during the acute illness usually coinciding with acute hepatocellular necrosis and occasionally even before the onset of clinical symptoms (Dienstag et al., 1975; Frosner et al., 1977; Mathiesen et al., 1978). Anti-HAVantibodies are dectectable in serum during viremia documented by immunologic testing and infectivity in marmosets (Mathiesen et al., 1978)

Infection immunopathogenesis

31

and thus suggestive of being non-neutralizing. Titers of serum anti-HAV continue to rise after acute illness and reach peak levels approximately 2-3 months later, then gradually decline. However, years after infection relatively high anti-HAV antibody titers are detectable and anti-HAV can thus persist indefinitely (Frosner et al., 1977). Antibody to HAV that appears during acute illness is predominantly of the IgM class (Bradley et al., 1977) and correlates temporarily with the characteristically high serum IgM levels detectable in patients with hepatitis A (Zhuang et al., 1982; Zuckerman, 1988; Lau et al., 1991). However, the concentration of IgM anti-HAV represents only a small fraction of the total serum IgM and is unlikely to account entirely for the elevated serum IgM in this disease. Increases in IgM may be due to specific antiviral antibody (Lemon et al., 1980) as well as other antigens not related to HAV. The mechanism responsible for non-specific immunoglobulin elevation is unknown. Specific antibody, however, is almost always present by the time of onset of symptoms of hepatitis A virus infection (Locarnini et al., 1977; Rakela et al., 1978; Lemon et al., 1980). This initial antibody response involves IgM and probably also IgG and IgA antibodies since 7S antibodies may be present as early as two days after the onset of illness (Locarnini et al., 1977; Duermeyer et al., 1979; Lemon et al., 1980). Although the IgM anti-HAV response is typically short-lived, sensitive antibody-capture immunoassays have demonstrated its persistence for 6 to 12 months after hepatitis A virus infection (Lemon et al., 1980). Serum neutralizing activity against the virus appears in parallel fashion with antibody detected by immunoassay and may be present three to five days before the onset of symptoms in humans (Lemon and Binn, 1983a). Viral antigen continues to be shed in feces after the development of serum neutralizing antibody (Lemon and Binn, 1983 b) and in some patients have been found to persist well into the second week of illness (Rakela et al., 1977). Despite the fact that both IgG and IgM antibodies possess neutralizing activity against the virus (Lemon et al., 1980) some acute-phase serum samples containing IgM anti-RAV has also been found to contain infectious virus (Purcell et al., 1984). This may perhaps be attributed to the presence in serum of lipid-associated virions (Provost et al., 1975). Unlike anti-HAV IgM, IgG anti-HAV develops more gradually and reaches high levels during convalescence. Serum IgG anti-HAV persists for long periods after infection, perhaps for life. Although IgA antibody has been detected in fecal specimens collected during convalescence from acute hepatitis A (Yoshizawa et al., 1980) its role in recovery and subsequent immunity remains unknown. Recently, Stapleton et al, (1991 b) has used a sensitive in vitro radio-immunofocus inhibition assay for the detection of neutralizing anti-HAV antibodies in gastrointestinal specimens collected after experimentally induced or naturally acquired hepatitis A. The data obtained suggest that intestinal immunity does not play a significant role in protection against hepatitis A. Although the mechanisms underlying liver injury in hepatitis A are not understood the fact that initial non-cytopathic phase during which the virus replicates and is released as followed by decreased virus multipli-

32

Pathogenesis of hepatitis A virus infection

cation and inflammatory cell infiltration does suggest that immune mechanisms are involved in pathogenesis. In an effort to identify factors contributing to the pathogenesis of autoimmune chronic active hepatitis (CAH) Vento et al. (1991) has recently shown that in two of the subjects of their study specific helper T cells and antibodies to the asialoglycoprotein receptor persisted and increased after acute hepatitis A and autoimmune CAH developed within 5 months. These results suggest hepatitis A virus as a trigger for autoimmune chronic hepatitis type 1 in susceptible individuals.

4 Hepatitis A virus infection: diagnostic tests I. Introduction The transmission of hepatitis A virus (HAV) to certain species of marmosets (Deinhardt et al., 1967; Holmes et al., 1969), visualization of small spherical HAV particles by immune electron microscopy in fecal extracts (Feinstone et al., 1973) and the successful propagation of HAV in 1979 in primary monolayer and in continuous cell cultures of primate origin (Provost and Hilleman, 1979) had major impact on hepatitis A disease research in view of the earlier unsuccessful attempts to identify and diagnose HAV infection. The cultivation in vitro of HAV and the subsequent development of immunoassays for the detection of IgM anti-HAV paved the way for a simple, practical approach to the rapid diagnosis of HAV infection from a single serum sample obtained any time during the acute illness or even perhaps during early convalescence. Molecular cloning of the HAV genome (Ticehurst et al., 1983; Baroudy et al., 1985) has yielded cDNA probes for use in the detection of HAV-RNA by cDNA-RNA hybridization. The currently available diagnostic tests for hepatitis A virus infection are discussed in this chapter.

II. Immune electron microscopy Using the technique of immune electron microscopy (IEM) Feinstone et al. (1973) described the first in vitro test for hepatitis A virus antigen and antibody detection and demonstrated the presence of HAV particles in filtrates of acute phase stool samples incubated with convalescent serum containing antibody to the virus. This provided the basis for testing a serum sample for anti-HAV by incubation with a stool filtrate containing HAV or the testing of a stool samples for the presence of HAV by incubation with standard antiHAV serum. The enhanced sensitivity ( 1000-fold more) ofIEM over conventional electron microscopy for the visualization of virus particles, its high specificity when correctly performed and its usefulness in quantifying both

34

Hepatitis A virus infection: diagnostic tests

HAV and anit-HAV established it as the first in vitro serologic technique for the virologic diagnostic and epidemiologic studies. However, the fact that IEM is rather a cumbersome procedure, requiring not only specialized and expensive equipment but also personnel with expertise and on occasions its interpretation confused by presence in stool samples of contaminating particulate debris, makes it a method unsuitable for large-scale screening or for diagnostic purposes as such.

III. Rapid viral diagnosis In vitro tests in experimental animals to identify HAV antigen and antibody and first-generation and second-generation (complement fixation-CF, immune adherence hemagglutination-IAHA) of in vitro tests contributed to rapid progress in hepatitis A virus infection research during the last decade. However, third-generation radio-immunoassays and enzyme-immunoassay of high sensitivity and specificity have effectively replaced them to our advantage. These techniques can be used to detect HAVin clinical specimens and to monitor the presence of HAV during purification or during in vitro cultivation to detect the presence of total an ti-HAV for seroepidemiologic studies and as an indication of immunity and to detect IgM anti-HAV for rapid diagnosis of acute HAV infection. These tests are simple to perform and assays for anti-HAVare currently commercially available (Hollinger et al., 1991).

A. Serologi,cal identification A diagnosis of acute HAV infection can be made by demonstrating HAV in tissues, body fluids or excretions or by documentation on immune response to HAV. As the viremic phase of HAVis short and the samples are rarely available before the onset of symptoms, serological testing for circulating HAV antigen and/ or particles is of limited value. Attempts of diagnosis based on detection of HAVin the liver, serum, bile or stool have however been successful. Routine rapid diagnosis by tissue culture isolation of HAV from clinical specimens have proved similarly impractical, leaving the diagnosis of HAV by demonstration of a specific immune response as the primary, practical option. Classically, this is achieved by measurement of an increase in an appearance of serum anti-HAVbetween the acute phase ofillness and convalescence with the interval necessary to detect a significant change in antibody titer being 4-6 weeks, if not longer. Due to the unavailability of an appropriately timed convalescent serum sample, a rapid enough diagnosis during the acute illness, without waiting for a convalescent sample, appears to be preferable. Acute HAVis diagnosed by detecting IgM anti-HAVin serum taken during the acute illness (Bradley et al., 1977). It reaches peak values within a few weeks of symptoms onset, declining rapidly thereafter (Flehmig, 1979). Five months from illness onset, 50% of patients have lost IgM anti-RAV (Hatzakis, 1984) and the majority have undetectable IgM anti-HAV within a year.

35

Rapid viral diagnosis ~

ACUTE I INFECTION :

INCUBATION

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Time (Relative) Fig. 5. Serological profile of typical acute hepatitis A virus (HAV) infecting

IgG anti-HAV may be detectable during the acute stage and is always seen during the recovery phase, reaching peak values three to 12 months after onset of illness (Dienstag, 1976). This IgG response persists for life and its presence alone indicates previous HAV infection and immunity to reinfection. Figure 5 illustrates the serological changes found in a typical acute HAV infection. Detection of HAVin stools is oflimited value since viral excretion usually ends at the onset of clinical symptoms.

B. Radio-immunoassay and enzyme-immunoassay techniques Among the most practical methods known to detect IgM anti-HAV are modifications of the classical radio-immunoassay (RIA) and enzyme-immunoassay (EIA) techniques for detection or measurement of anti-HAV. A simplified procedure involves separation of the IgG and IgM fractions of serum by sucrose density gradient ultracentrifugation (Frosner et al., 1979; Mortimer et al., 1981; Swenson et al., 1981) and subsequent ofanti-HAVin the two fractions by EIA or RIA, but the cumbersome steps of ultracentrifugation and fractionation of serum, limits its clinical application. Another approach has been to absorb selectively the IgM or IgG activity from the test serum and subsequent testing of the absorbed serum by RIA for anti-HAV. Another method was used in which IgG from test serum was absorbed by use of staphylococcal protein A with preferential binding for IgG (Bradley et al., 1979). If the level of binding in RIA was not reduced by staphylococcal protein A absorption, the anti-HAV was considered to be primary of the IgM class. Application of this approach has been limited by the suitability of only certain strains of Staphylococcus for IgG absorption, the fact that all IgG subclasses are not absorbed by staphylococcal protein A, poor reproducibility of the test, and high frequency of false positives resulting from high levels of IgA anti-

36

Hepatitis A virus infection: diagnostic tests

HAV in serum (Lofgren et al., 1980). Locarnini et al. (1979) introduced a method whereby the presence of IgM anti-HAV was detected by addition of test serum to a solid-phase microtiter well to which HAV had been bound by linking with anti-HAV. Any presence of IgM anti-HAVin the test serum was detected by incubating with enzyme-conjugated goat anti-human IgM (heavy chain specific) in an otherwise standard EIA procedure. This procedure was found to be more sensitive than the methods utilizing sucrose gradient centrifugation and furthermore limited by detection ofvery low levels oflgM antiHAV and the ratio of optical density in positive specimens to the mean for negative control specimens barely distinguishable from the cutoff value. Currently, an antibody class-capture approach forms the basis for the most popular and widely used assays for IgM anti-HAV. The anti-human IgM is used to coat the solid phase (microtiter well or plastic bead) followed by addition of test serum and subsequently the HAV and enzyme-conjugated or radio-actively labeled 1251-conjugated IgG anti-HAV. Hence, IgM molecules of all antigenic specificities may bind to the solid phase but the presence of only IgM anti-HAV provides the link for the completion of the sandwich immunoassay (Duermeyer et al., 1979). Provided that the anti-human IgM is heavy chain specific, this approach is the most sensitive and specific (Mortimer et al., 1981). Adopted by many investigators in either an RIA or EIA format, this solid phase bound antibody class-capture method is the one that is available commercially (Lemon et al., 1980; Hansson et al., 1981; Mortimer et al., 1981). IgM anti-HAV detectable with this type of assay appears within a few days of the onset of symptoms and reaches at peak within 1 to 3 weeks (Decker etal., 1981). Generally, IgM anti-HAV can be detected for at least 24 months after acute illness and in rare cases as long as 6 and even 12 months to allow optimal discrimination between very recent acute infection and past infection. Decker et al. ( 1981) included a 1 : 4000 dilution of the serum to be tested. Designed as such, the test currently commercially available (HAVABMand HAVEB-M EIA, Abbott Laboratories) is almost universally negative by 3 months after onset of acute illness. Although rheumatoid factor could interfere theoretically, with this type of assay and yield false positive results in rare exceptions (Duermeyer et al., 1979) this has not been encountered and essentially rheumatoid factor levels are rarely high enough to interfere at serum dilution of 1: 4000. Very recently, a fully automated microparticle enzyme immunoassay (MxHAVAB) has been developed for the detection of antibody against hepatitis A (Robbins et al., 1991). MxHAVAB sensitivity reportedly is 18-25 World Health Organization U /I and more sensitive than the commercial RIA or EIA, HAVAB and HAVEB EIA, respectively.

IV. Current trends in molecular biotechniques Molecular cloning of the HAV genome has been performed (Ticehurst et al., 1983; Baroudy et al., 1985) yielding cDNA probes for use in the detection of EAV-RNA by cDNA-RNA hybridization.

Current trends in molecular biotechniques

37

Recently, Karayiannis et al. (1988) has reported a study in which they have used RIA and molecular hybridization to document the presence of HAV and IgA anti-HAV (HAV immune complexes in serial stool specimens from the time of infection to recovery) in experimentally infected tamarins and to correlate them to biochemical and serological findings. HAV-RNA was detected by molecular hybridization on fecal specimens and tissues. HAVRNA was present in feces within 24 hours ofinoculation and then fill until the 9th day when it increased in concentration and continue through the acute disease into the early convalescent phase. Fecal IgA anti-HAV appeared within one week of infection, and immune complexes containing the virus was found to be present until the early convalescent period. In another recent report (Prevot and Kopecka, 1988) two independent methods for HAV detection were used: immunoenzymatic assay (ELISA) using polyclonal and monoclonal antibodies, and molecular hybridization using subgenomic cRNA transcripts (RIBOPROBES) of HAV-cDNA. Both methods reportedly permitted the detection of HAV replication in two different cells (PLC/PRF5 and VC-10). In yet another recently reported study Shieh et al. (1991) have used ssRNA probes to detect hepatitis A virus. Clones of cDNA encoding the 5 'most 1 kb of the HAV and coxackievirus B3 genomes were subcloned into T7 /SP6 RNA transcription vectors.

5 Epidemiology and transmission of hepatitis A virus I. Introduction Extensive epidemiologic observations and experimental studies have closely defined the epidemiological characteristics of viral hepatitis A (MacCallum, 1972) .Judicious use of specific diagnostic laboratory tests have helped differentiate the various forms of human viral hepatitis and provided conclusive answers to questions confronting the epidemiology of hepatitis A virus infection. Despite identification of subtile differences in incubation period, site of replication, route and duration of excretion, a close analogy exists between the epidemilogy of hepatitis A virus and that of poliovirus. Hepatitis A is a disease of global distribution. The prevalence of infection, and hence anti-HAV antibodies has been generally noted to be universally related to standards of sanitation and hygiene. Although HAV may be spred typically through contaminated water supplies and food stuffs, numerous common source epidemics have been described. Most HAV infections are, however, acquired through less-dramatic sporadic or endemic transmission. In countries with poor standards of hygiene and sanitation, infection with HAV has been well known to occur early in life. Oddly enough however, HAV infection now presents a public health concern in many countries, because improvements in living standards have resulted in several countries that would otherwise have been acquired in childhood, being delayed until adult life. The disease has emerged as a health concern in parts of southern Europe (Greece, Portugal), central and southern America (Cuba, Chile) and Asia (China, Korea, Singapore) (Gust, 1988). The epidemiology and transmission of HAV are discussed taking into account several factors.

II. Epidemiologic characteristics of infection Man has been considered the only important host of hepatitis A virus infection. However, the study of existence of animal species is of importance for attempting to explain the epidemiology ofHAV to implement strategies for

Epidemiologic characteristics of infection

39

its control, especially in planning the eradication. Non-human primates in captivity may serve as potential sources of infection of humans. Epidemiological observations have indicated primate-to-primate transmission in captivity and suggest that chimpanzees infected by HAV from humans may spread and become the source of outbreaks among chimpanzee handlers (Hillis, 1961). Nevertheless, non-human primate, although susceptible to HAV infection are indeed epidemiologically unimportant for the spread of the disease and even ecologically insufficient for the survival of the virus in the environment. There is a remote possibility that shellfish sere as an extrahuman reservoir of HAV (Gust et al., 1979; Yang et al., 1988). Extensive epidemiologic observations and experimental studies had defined the major characteristics of the two distinctive types of viral hepatitis (Krugman et al., 1967). Based on these observations, viral hepatitis were classified and reported until recently as infectious (type A) or serum (type B) or hepatitis B following the discovery of hepatitis B surface antigen (HAsAg) by Blumberg et al. (1967). It was shown thereafter that any classification of hepatitis viruses solely based on epidemiologic characteristics could prove to be extremely inaccurate. However, it. was until the availability and use of a laboratory test for the specific diagnosis of HAV (Purcell et al., 1976) that the epidemiologic characteristics of viral hepatitis A could be established and furthermore, led to the designation of some hepatitis cases as non-A, non-B hepatitis. Subsequent epidemiological reports do suggest that non-A, non-B hepatitis may be caused by more than one virus (Gerety, 1982) and that another type of non-A, non-B hepatitis virus similar to but clearly distinct from HAV, may be responsible for large water-borne outbreaks of viral hepatitis in India, Africa and Costa Rica (Khuroo, 1980; Wong et al., 1980; Dienstag etal., 1981; Villarejos et al., 1982). Today these hepatitis are known as viral hepatitis C and viral hepatitis E.

A. Seasonal and geographic variation In the temperate zones, hepatitis A virus infection occurs in seasonal epidemic waves with peaks in late autumn and early winter. The recent decline in incidence has limited HAV infections to specific social groups or to tourists and other travelers and thus seasonal variation has become less obvious. In tropical areas the peak incidence of reported disease tends to occur during the rainly season and a cyclic epidemic pattern with peaks every 5-10 years in similarity to other viral diseases has been observed (McCollum and Zuckerman, 1981). Hepatitis A disease has been reported to occur worldwide and prevalence date indicates the yearly incidence of HAV infection (both clinical and asymptomatic) in Europe between 100 (Sweden) and 7600 (Greece) per 100,000 population (Frosner et al., 1979) and a close association between the prevailing environmental housing and sanitary conditions and the prevalence of anti-HAV antibody has been well documented. Difference indicating an increasing prevalence of anti-HAV antibodies from northern and southern countries have been observed in the Americas (Szmuness et al., 1981). In the Centers for Disease Control approximately 28,000 cases are

40

Epidemiology and transmission of hepatitis A virus

reported as hepatitis A (Francis et al., 1984) and possibly a lot of hepatitis A cases are not reported. Reports suggest that the incidence of hepatitis A virus infection is declining while that of hepatitis B virus is increasing (Hepatitis Surveillance Report, CDC, 1985). Specific risk factors that have been associated with hepatitis A within the United States include contact, with another person infected with hepatitis A virus (26%), homosexuality (15%), illicit drug users (10%), foreign travel (14%), contact with children attending day-care centers (11 %) (Francis et al., 1984; Centers for Disease Control, 1988; Harkess et al., 1989).

B. Age incidence All age groups are susceptible to hepatitis A virus. The highest incidence in the civilian population is observed in children of school age but in many countries in Northern Europe and North America most cases occur in adults. In developing countries where sanitation and hygienic conditions remain very poor, universal exposure to HAV is identified by extremely high prevalence of anti-HAV by the first few years of life and thus age patterns of antiHAV prevalence clearly depend on prevailing socio-economic conditions. The age-related increase in anti-HAV prevalence starts earlier and is more marked in areas with substandard living conditions and thus the epidemiologic pattern of HAV infections is similar to that of poliomyelitis virus infections (Ajdukiewicz and Mosley, 1979). More than 75% of children from parts of Asia, Africa, India, certain Mediterranean countries and South America have anti-HAV antibodies by the age of 5 years (Fagan and William, 1987). Most HAV infections acquired in the first years oflife are asymptomatic or at least anicteric (Prince et al., 1985). In the developed countries, in contrast, the incidence of HAV infection has been declining in recent years and is shifting to older age groups. Direct evidence for declining incidences of hepatitis A virus comes from comparisons of age patterns of anti-HAV prevalences in Australia or Europe (Frosner et al., 1977; 1979; Gust et al., l 978a). In Australia, the morbidity of hepatitis A has declined during the last decades and the proportion of cases among young adults has increased considerably (33% in 1969 to 60% in 1977) (Gust et al., 1978b) and in the United States a steady downward trend in the number of hepatitis A virus infection cases have been occurring since 1972 (Centers for Disease Control, 1988). Despite the diminishing importance of HAV infection in many industrialized countries, it continues to be a problem among certain high risk groups as healthcare workers, food handlers, sanitation workers, drug abusers, travellers to areas of low to high endemicity, nurseries, day-care centers and psychiatric institutions.

C. Inapparent and chronic reinfection There are no universally accepted data regarding the proportion of HAV infections which result in overt hepatitis; the ratio of anicteric to icteric cases reportedly varing from 12:1 to 1:3.5 (Rautenberg et al., 1979). In the

Virus incubation period and transmission

41

Greenland epidemic of 1970-1974, clinical attack rates were found to rise steadily from 10% to 24% for young populations up to 15 years old, without prior immunity (Skinhoj et al., 1977) sugestive of the dramatic rise with age of the rate of clinical to inapparent HAV infection. The existence of human carriers of HAV or of chronic active states of HAV infection would be the most plausible hypothesis to explain the prominent endemic characteristics of hepatitis A. Epidemiologic studies have, however, indicated that chronic hepatitis may develop after HAV infection and furthermore, patients who acquire hepatitis during epidemics of hepatitis A do not generally develop chronic liver disease. Employing specific serologic tests, investigations have indicated that in contrast to hepatitis B and hepatitis C (non-A, non-B hepatitis), persisting abnormalities in liver biochemistry and clinical or pathological signs of chronic active liver disease do not develop in patients with serologically confirmed hepatitis A virus infection (Mathieson, 1981). Chronic liver disease only very rarely, if ever, develops after HAV infection and that a chronic carrier state with continued fecal excretion does not exist. A possibility that may permit the survival of HAVin populations may be the occurrence of brief activation from latency as known to exist for varicella. Reinfection of persons with history of previous hepatitis A infection but who lost detectable anti-HAV antibodies may create new excretors of HAV and thus enhance the potential for amplification in the community. Hereby, reinfection could explain endemicity and especially hyperendemicity. Data from a study of household contacts of acute hepatitis A patients in Costa Rica showed that such reinfection lost detectable anti-HAV antibodies (Villarejos et al., 1982).

III. Virus incubation period and transmission Studies of hepatitis A virus (HAV) epidemics and experiments with human volunteers indicate that the incubation period of acute HAV infection is 14 to 49 days with a mean of 30 days. Hepatitis A virus is spread predominantly by the fecal-oral route, most commonly by person to person contact. Infection is particularly common in conditions of poor sanitation and overcrowding. Common source outbreaks result most frequently from fecal contamination of drinking water and food. The relative importance of water-borne transmission of HAV varies in the different areas of the world and predominates in developing countries and is responsible for infection at an early age and thus occurrence of endemicity rather than clinically recognized outbreaks, rare in these areas. In contrast, water-borne transmission is not a major factor in industrialized communities with adequate water supply and waste disposal with only accidental contamination of water, possibly resulting in extensive outbreaks (Mosley, 1967; Gaon et al., 1982). Although outbreaks of hepatitis A attributable of water contamination have been reported, the impact of water supply characteristics on the incidence of hepatitis remains yet to be adequately documented and this necessitates the application of

42

Epidemiology and transmission of hepatitis A virns

techniques of isolation of HAVin cell cultures currently available to examine the virus content of a water supply prior to and after its treatment and also to measure the effectiveness of chlorination. Recently, in a documented study the water transmission of hepatitis A virus was reported by Mahoney et al. (1992). A multistate outbreak of hepatitis A was traced to a campground in Louisiana where among 822 campers during one weekend, 20 developed hepatitis A. The highest attack rate of 6.4% was for children aged 5-9 years. All infected patients swam in a public swimming pool and have put their heads under water. Due to the design of the pool with a possibility of cross-connection between a sewage line and the pool water intake line, the authors concluded that swimming may serve as a mode of transmission of hepatitis A virus. HAV can be transmitted by food contaminated with feces from an infected person and an increasing number of food-borne outbreaks have been reported in developed countries. This can be attributed to the shedding of large amounts of virus in the feces during the infection period of the illness by infected food handlers (Denes et al., 1977) and the source of the outbreaks can often be traced to uncooked food or food which has been handled before cooking. Uncooked foods have most frequently been associated with food-borne epidemics because normal cooking temperatures inactivate HAV (Peterson et al., 1978). Prepared fruits and vegetables kept frozen for a long period may represent a further widespread source of infection. The most important source of food-borne infection is shellfish. The consumption of raw or inadequately cooked shellfish cultivated in polluted water is associated with a high risk of hepatitis A infection (Yang et al., 1988). An average of only four outbreaks of food-borne hepatitis are reported in the United States. However, for the past few years around 1000 food handlers with non-B hepatitis have been annually reported (Centers for Disease Control, 1982). Most HAV infections are transmitted person-to-person following ingestion of fecally excreted virus. Most often, the source of infection is unrecognizable due to the long incubation period of the disease and the spread of HAV from affected persons prior to appearance of symptoms. Person-toperson spread accounts for the majority of infections in developed countries. Several mechanism in various settings could be responsible for such a spread. i) Intrafamilial spread is common among susceptible individuals with the virus usually introduced by young children and spreading equally among susceptible hosts of all ages, infecting all of them. Overcrowding facilitates person-to-person transmission as in the case with other enteric viruses (Papaevengelou et al., 1980). ii) The role of the school environment in facilitating person-to-person spread of hepatitis A virus is unclear. Schools have been considered the focus of some epidemics in rural areas (Knight et al., 1954). The less importance of the school environment in HAV transmission has been attributed to prevailing immunity from earlier experience or from subclinical infections. HAV spread in school settings could possibly occur when primitive hygienic conditions and crowding prevail or when condi-

Virus incubation period and transmission

43

tions facilitate HAV transmission. Young children attending pre-school day-care centers have frequently been implicated in the transmission of HAV (Benensonetal., 1980; Hadler et al., 1980). Oral behaviour and lack of toilet training promote viral transmission among very young children, who then carry the virus home to older siblings and parents. Although infection is usually not clinically apparent in children under the age of two, the majority of infections in affected parents, older siblings and daycare center staff, are accompanied by jaundice or other evidence of hepatitis A disease (Benenson et al., 1980; Hadler et al., 1980). Hepatitis A virus infection is frequently acquired by travellers from areas of low to areas of high endemicity. A variety of routes could be responsible for travellers hepatitis A with water-borne and food-borne transmission responsible for outbreaks among soldiers involved in almost every military campaign (Lemon, 1982). The incidence ofhepatitisA among travellers depends on the countries of origin and destination of the travellers, whether they travel individually or in groups, the duration of stay in the country of visit and the preventive use of immune globulin (Gerety, 1984). HAV can also be transmitted via the parenteral route but this is rare, usually resulting from transfusion of blood taken from presymptomatic donors. There is no difference in the incubation period. Percutaneous transmission could account for outbreaks of hepatitis A among parenteral drug abusers (Widell et al., 1982). However, the association of HAV infections with intravenous drug abuse might also be due to the poor hygienic conditions in which the drug addicts live. Sexual and especially homosexual transmission plays a very important role in the spread of hepatitis B virus and likewise the incidence of many enteric and other diseases is higher in homosexuals. Therefore, homosexual transmission of HAV would not be unexpected. Sexual transmission of HAV has been shown to be common among homosexual men, especially those who have oral-anal contact (Corey and Holmes, 1980; Szmuness el al., 1981; Christenson et al., 1982). Hepatitis A has occurred in epidemic proportions among some male homosexual populations with a 22% annual seroconversion rate noted during a study conducted in Seattle (Corey and Holmes, 1980). Viral hepatitis occurring in homosexual men who have been immunized with hepatitis B vaccine is most often due to HAV. Thus, HAV should be included among other enteric pathogens that may be sexually transmitted. Most such infections are indeed asymptomatic and of clinical importance (Corey and Holmes, 1980; Szmuness et al., 1981).

6 Hepatitis A: therapeutic approaches, prevention and control I. Introduction Hepatitis A virus disease is a common infection in most parts of the world and even in the highly industrialized countries the disease continues to be a significant problem. The World He>

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162

Diagnosis of hepatitis D virus infection

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later, quite often during the convalescence period. Progressive hepatitis D virus infection in contrast whether acquired by coinfection or by superinfection is accompanied by an increase of IgM and IgG antibodies and the presence of both antibodies in high titres permits the diagnosis of chronicity (Rizzetto et al., 1988). Screening for IgM anti-HD, an excellent marker of chronic HBV/-HDV infections (Dimitrakakis et al., 1986), appears to be critical for differential diagnosis as it is the only test yielding positive results during the clinical period of acute hepatitis D virus disease, and its decline or increase would signal to resolution or progression of the infection. Serological monitoring of IgM anti-HD for periods over several weeks from onset of disease would be a useful prelude towards either diagnosis or exclusion of hepatitis D. Total anti-HD has been shown to be a useful marker of previous HDV infection (Shattock, 1989) and it has also been shown that total anti-HD could indeed be persistent over the follow-up period (around 25 months) in both coinfection and chronic infection. Recently, Jardi et al. (1991) detected two forms of IgM antibody to hepatitis delta virus (HDV) by rate zonal centrifugation. The high molecular weight IgM (19S) form was predominantly detected in acute HDV cases, whereas the low molecular weight IgM (7-8 S) form was found in chronic HDV cases. In acute stage ofHDV infection, the 19 S form was predominant but 6 months later a predominance of 7-8 S IgM was observed. The authors suggest that IgM antibody to HDV antibody forms are different in acute and chronic hepatitis D virus infection and that their detection helps in differentiating an acute infection from a chronic infection, but not a hepatitis D virus - hepatitis B virus - HBV coinfection from hepatitis D virus superinfection in the acute stage of the disease.

Diagnostic techniques

163

III. Diagnostic techniques

A. Radio-immunoassays (RIA) Hepatitis D virus (HDV) infection is recognized by the expression of the HDV antigen-antibody system in the infected host. The antigen is found in the liver in various forms of HDV disease but in the serum, occurs only in the early phase of the primary infection. Thus, serodiagnosis of hepatitis D rests essentially on the tests for anti-HD. Total anti-HD can be measured in competitive immunoassays. A radioimmunoassay (RIA) that has been developed for the measurement of the specific IgM class antibody (IgM anti-HD), is based on the capture of IgM molecules by goat anti-human U-chain antigen linked to a solid phase (Smedile et al., 1982). In a typical RIA procedure, polystyrene beads coated with goat anti-human U-chain specific serum, are incubated at room temperature with serum and after an initial 4 hours incubation, beads are washed with deionized water and reincubated for 18 hours with standard HDAg; washed again and incubated for 2 hours with normal human serum containing 125I-IgG anti-HD. The beads are counted for residual radioactivity and results expressed as ratio of cpm of test sample to average cpm of normal control sera (Aragona et al., 1987). Since sera obtained during the acute hepatitis D virus infection may be reactive in the U-capture assay is believed to measure predominantly the IgG class of antibody. Both the IgM and IgG antibody are consistently detectable in chronic hepatitis D (Farci et al., 1986). In contrast, studies of acute disease have shown a variable response, ranging from the transient production oflgM anti-HD only to the prolonged expression oflgG anti-HD (Smedile et al., 1982). Patients with chronic HDV infection tend to indicate a persistence of HBsAg in serum and high titers of HDV. Titers of anti-HDV greater than 1: 100 by radioimmunoassay often is suggestive of chronic hepatitis D virus disease.

B. Enzyme-immunoassays (EIA) Various enzyme immunoassays (EIA) have been developed for notable detection of antibodies against the hepatitis D virus antigen (anti-HD) and in recent years for the antigen itself (HDAg). Among them is the EIA for HDAg and anti-HD by Organon Teknika, the Delta-assay for HDAg from Noctech and Abbott anti-delta EIA, a test for anti-HD from Abbott Laboratories. The EIA test for HDAg developed by Organon Teknika (Heijtink et al., 1983) is based on polystyrene micro-enzyme-linked immunosorbent assay (micro-ELISA) strips coated with human anti-HD and on a peroxidase-labeled human anti-HD as second antibody. The test procedure recommends treatment of sera with 3 grams ofNonidet P-40 per liter. The test for anti-HD is essentially identical in principle, except for the absorption of sera first with an HDAg preparation purified from chimpanzee liver. An anti-HD positive

164

Diagnosis of hepatitis D virus infection

sample competing with the solid-phase antibody for binding of HDAg results in a reduction in color in comparison with a negative control. The Delta assay for HDAg from Noctech is a direct sandwich EIA based on polystyrene micro-ELISA strips coated with human anti-HD and on a peroxidase-labeled human anti-HD as a second antibody. A delta antigen (HDAg) extraction buffer containing a detergent is used for treatment of the serum. In the Abbott anti-delta EIA procedure recommended, a one-step competitive assay is involved in which serum samples and peroxidase-labeled human anti-HD are incubated with polystyrene beads coated with HDAg (delta antigen) purified from woodchuck liver. In recent years, Matthyssen et al. ( 1988) has reported the development of a simple and easily performed EIA for the detection of anti-HD with the use of the same reagents as for the carrying-out of a direct test for HDAg. The test is an enzyme immunoassay based on a competitive sandwich-inhibition principle with microelisa strips coated with human anti-HD constituting the solidphase antibody and peroxidase-labeled anti-HD as conjugate. The test sample is incubated in the antibody-coated test well together with a fixed amount of a preparation of HDAg from an infected chimpanzee liver, followed by addition of the conjugate and the enzyme reaction. Results could be obtained according to the authors within 3 hours and the final result seen as a distinct color. The present of anti-HD in the test sample gives either no or perhaps a reduced color. The presence of free HDAg would be indicated by measurement of an increase of the color. This assay provides the potential for detection of both HDAg and its antibody. The analytical sensitivity for antiHD, determined with dilution series of human anti-HD positive sera, was found by the authors to be either equal to or better than the Abbott anti-delta EIA, one of the currently available tests. A clinical specificity of99.9% based on assay of 1259 samples from blood donors and non-HBV patients was also reported. This new, easy to perform, EIA for detection of anti-HD with high specificity and acceptable sensitivity, within 3 hours, appears to be promising as well as attractive since the essential reagents are reportedly available currently in a kit combination (Hepanostika anti-Delta, Organon Teknika). An evaluation of the different enzyme immunoassays has been reported by Dubois and Goudeau (1988). The kinetics of delta antigen (HDAg) and anti-HD were analyzed by them in 22 acute hepatitis D virus infections ( 11 coinfections and 11 super-infections) with the EIA developed by Organon Teknika and the two commercially available assays, Delta-assay for HDAg from Noctech and the Abbott anti-delta EIA for anti-HD. The possibility of conflicting results between different assays for HDAg is thus obvious from the study by these authors in which only 17 of 28 samples positive with the Organon HDAg assay were concurrently positive for Delta-assay. The discrepancies appear to be indicative of a greater sensitivity of the Organ on test over Delta-assay rather than bearing any relation to the type ofHDV infection considering the observation that 7 of 22 patients were positive with the Organon test but negative with the Delta-assay. For six of these patients a late sample available showed a seroconversion to anti-HD. There was no evidence of any patients with HDAg positivity by Delta-assay and negativity by Organon

Diagnostic techniques

165

and furthermore, only patients who had been HDAg reactive with the Organon assay during the first few weeks of illness developed a seroconversion to anti-HD in late serum samples. The Organon anti-HD assay based on sandwich inhibition yielded no false-positive reactions but, as illustrated in this evaluation by Dubois and Goudeau ( 1988), appear to be less sensitive in the early weeks of evolution of infection. With the Abbott anti-HD assay, a disappearance of the blind period lasting 2 to 11 weeks between the negativeness ofHDAg and seroconversion to anti-HDigG or co-occurrence ofHDAg and anti-HD was noted. Dubois and Goudeau (1988) also suggest in their paper the utilization of a modified version of the one-step competition assay recommended by Abbott Laboratories, which they classify it as a two-step procedure involving two incubations for 2 hours at 40°C. In the first step, serum samples are incubated with HDAg-coated beads and addition of peroxidase labeled anti-HD following the washing step. This two-step procedure essentially avoided contact between sera and labeled antibody. A combination of Abbott anti-HD assay used with the two-step procedure and the Organon HDAg was found to suppress the HDAg/anti-HD blind period. It, thus, appears that the diagnosis of acute HDV coinfection would be possible at any time provided it is based on the presence of anti-HBclgM and one of the HDV markers. For acute HDV superinfection (anti-HBclgM negative), early diagnosis would be dependent on presence ofHDAg. Tests for HDAg could be reliably utilized for diagnosis during the first 2 weeks of illness given the utilization of a sensitive assay, whereas seroconversion to anti-HDigG could provide the basis for a late diagnosis (2 to 5weeks after onset of illness). Very recently, an interesting study was performed by Shattock and Morris ( 1991), on evaluation of six commercial enzyme immunoassays for detection of hepatitis D antigen and anti-hepatitis D virus (HDV) and immunoglobulin M anti-HDV antibodies. They concluded that for detecting HDAg, the Noctech, Pasteur and Wellcome assays had 100% sensitivity; the Organon reagents gave 59.5% sensitivity without detergent and 64.3% sensitivity with detergent. The Sorin assays gave 23.8% sensitivity. For the detection of antibody to HDV (anti-HDV) all six commercial enzyme immunoassays gave 97.8% to 100% sensitivity reacting with all anti-HDV-positive specimens. For the detection of immunoglobulin M anti-HDV the Noctech, Sorin and Wellcome assays had 100% sensitivity for the all 38 positive specimens confirmed inhouse. The authors found that there has been a substantial improvement over previously evaluated assays. However, still major differences remain with regard to sensitivity among some assays for HDAg detection.

C. Immunofluorescence and immunoperoxidase techniques It remains undetermined yet whether the presence of anti-hepatitis D virus antibodies is always indicative of active viral replication or is merely a marker of previous infection. As a matter of fact, in some anti-delta (HDV) positive cases, HDV antigen may be undetectable in the liver (Craxi et al., 1984).

166

Diagnosis of hepatitis D virus infection

The use of direct immunofluorescence or immunoperoxidase methods has made it possible the demonstration of the presence of HDV Ag in liver tissue biopsy specimens (Smedile et al., 1981; Govindarajan et al., 1984; DiSapio et al., 1988). This provides a reliable confirmation of any chronic hepatitis D virus infection. Liver biopsy specimens fixed and embedded in Araldite are stained for HDAg by indirect immunoperoxidase ( Govindarajan et al., 1984) whereas indirect immunofluorescence, a specific anti-serum is utilized (Craxi et al., 1984).

D. Immunoblot techniques Hepatitis D virus antigen (HDAg) is rarely present in serum when tested by radioimmunoassay, but is frequently detectable by immunoblot techniques. Bergmann and Gerin (1986) developed an immunoblot procedure that is specific for proteins ofHDV and demonstrates the presence of these proteins in the serum and liver of acutely infected chimpanzees and woodchucks, as well as in the serum and liver of patients with chronic hepatitis D. This was the first report detecting HDAg in the serum of patients with chronic hepatitis D. Prior to this, HDAg had never been detected in the serum of chronic carriers of HDV and this was presumably due to the result of complexes formed with antibody (Bonino et al., 1981) because, HDAg could be found in the livers of those patients. The immunoblot procedure in brief, involves reduction with 1 % 2-mercaptoethanol of samples, running them on 12% polyacrylamide gels and transfer to nitrocellulose. Filters are blocked for 3-5 hours with 3% BSA in TN (0.01 M Tris [pH 7.4] and 0.15 M NaCl) and incubated with dilutions of serum (1:500-1:400) orofimmunoglobulinfractions (5-lOmg/ml) in TN containing 1 % BSA and 0.05% Tween 20. Following washing in TN plus Tween 20, filters are incubated for 2 hours at room temperature with 125Ilabeled Staphylococcus aureus protein A diluted in TN with BSA and Tween 20 and then finally autoradiographed following washing and drying (Bergmann and Gerin, 1986). In all serum samples Bergmann and Gerin (1986) tested, the same pattern consisting of two bands, 24 kilodaltons (kDa) and 27kDa of equal intensity was apparent and it is likely that they represent the mature virion proteins ofHDV. It is suggested by the authors that these proteins may be resultant of post-transcriptional processing of a single coding region as found by Wong et al. (1985) with HBsAg. Alternatively, these proteins could very well be the products of different genes. Bonino et al. (1986) have also described an immunoblot assay for HDAg in which they have likewise detected two proteins in the serum from an acutely infected chimpanzee. The decline in HDAg, as measured by RIA, has shown to occur approximately coinciding with the rise in antibody to HDAg in chimpanzees (Rizzetto et al., 1980). While with the RIA, it becomes impossible to determine, whether the loss ofHDAg is indeed real or due to the antigen complexed with antibody following detergent release from virions (Bonino et al., 1981). The immurioblot assay circumvents this potential problem by not being affected by antibody in the sample. The immunoblot assay for HDAg thus being more

Diagnostic techniques

167

sensitive and perhaps even informative than the RIA is for. HDAg can be useful in monitoring the course ofHDV infections and possibly to a greater extent in conjunction with the newly introduced hybridization assay for HDV-RNA. Another attractive feature of the immunoblot assay lies in the possibility of diagnosis of chronic HDV infection in patients with the detection in serum of HDAg without resort to invasive liver biopsy.

E. Molecular hybridization assays Molecular hybridization for HDV-RNA is the newest assay in obtaining a diagnosis of acute hepatitis D virus infection and substantiating the diagnosis of chronic disease (Rizzetto et al., 1980; Smedile et al., 1987). The hybridization assays currently available are directed towards the measure of the genome of the hepatitis D virus in serum and appear to be the best means of monitoring HDV replication. The demonstration ofHDV-RNA in the serum as a direct marker for the replication of HDV was first extrapolated from the studies of Smedile et al. (1986) in which HDV-RNA was detected in serum samples from a high percentage of patients with chronic HDV infection by use of a nucleic acid hybridization procedure (Northern blot). The hybridization assays available at the present time for directly measuring the HDV genome are based on probing sera with DNA clones complementary to HDVRNA (Smedile et al., 1986; Saldanha et al., 1987) or with a cDNA-derived riboprobe (Smedile et al., 1987). The cloning and sequencing of HDV-RNA had been in earlier studies achieved using chimpanzee serum derived HDV (Denniston et al., 1986; Kos et al., 1986; Wang et al., 1986). Also, the cloning of a cDNA fragment cloned from HDV-RNA obtained directly from human serum has been reported tsaldanha et al., 1988). This cloned fragment, according to the authors, is 380 nucleotides long, has a G+C content of 57% and shares 80% homology with the published sequences. The obtaining of this clones cDNA fragment widens the potential use of cDNA probes in nucleic acid hybridization towards studying the replication of HDV in infected liver tissues and as cDNA primers of the synthetic oligonucleotides. Molecular hybridization-based assays which used a pKD3 HDV cDNA clone and "Northern blotting" of RNA extracted from serum while being successful in detection ofHDV-specific nucleotide sequences in patients with hepatitis D have not, however, appeared as a technique suited to rapid assessment oflarge number of samples or for routine laboratory adoption. In 1988 Rasshafler and collaborators reported the use of a spot hybridization assay with a riboprobe derived from a cDNA fragment of 650 base pairs cloned into plasmid Gemini 2. The authors indicate this spot hybridization assay as a very simple and sensitive method for detection of HDV-RNA. However, in this study despite the use of the Riboprobe system with 32Plabeled RNA, the most sensitive and specific probe for detection of minute amounts ofRNAonly 13 (76%) ofl 7 patients with HDAgin hepatocyteswere indeed found to be positive for HDV-RNA and opens the possibility of

168

Diagnosis of hepatitis D virus infection

questioning whether or not all patients with HDAg in liver could indeed be positive for HDV-RNA in the serum. A spot or dot-blot hybridization procedure has been developed (Gupta et al., 1989) and tested with a new HDV cDNA probe. Their method differed from the earlier reported method of Rasshafler et al. ( 1988) in the use of a larger volume of serum and a superior probe representing a large portion of the HDV-RNA, they found an excellent correlation between the spot and Northern blot hybridization. The method has been reported to be rapid, sensitive and specific, even with use of less ultracentrifugation for recovering RNA. The authors advocate the use of the short ultracentrifugation (two hours instead of five) for initial screening of samples and performance of the elaborate procedure when results for HDVRNA appear to be negative in the initial assay. This would ensure substantial reduction both in time and manpower and allow screening of a large number of samples with rapidity. The usefulness of polymerase chain reaction (PCR) in HDV-RNA detection has been recently studied (Madejon et al., 1991). The results suggest that HDV-RNA detection by gene amplification is 10,000 times more sensitive than slot-blot hybridization and allows the detection of viral replication in patients without other viral replication markers. Recently, Gupta et al. (1991) have evaluated serologic diagnosis of hepatitis D virus by testing HDV RNA in stored sera from 48 patients with acute delta hepatitis who were identified with anti-HD antibodies. Results of HDV RNA and IgM anti-HD tests were found to be concordant in only 40%-50% of instances. The results of this study indicate that serological testing for HDV RNA is direct and will demonstrate HDV replication in a large number of cases with acute delta hepatitis. Testing for IgM anti-HD could provide supplemental evidence of HDV infection. Cariani et al. ( 1992) has very recently reported the development of a nonradioactive assay for detection of hepatitis D virus RNA in serum by combining reverse transcription of RNA, polymerase chain reaction of the resultant complementary DNA and enzyme linked immunoassay detection of the polymerase chain reaction products, using a monoclonal antibody specific for double-stranded DNA. This DNA enzyme immunoassay is reported to have a limit of detection of cloned hepatitis D RNA similar to that of standard PCR followed by Southern blot hybridization (-10 copies sample) and 103 to 144 times more sensitive than direct dot-blot hybridization. The DNA enzyme immunoassay may be a potentially useful method for therapeutic monitoring in chronic hepatitis D virus infection and may even perhaps contribute to a wider application in clinical laboratory of polymerase chain reaction.

References Aceti A, Papro BS, Celestino D, Pennica A, Caferro M, Brilli A, Sebastiani A, Muhamud OM, Abdirahman M, Bite K (1989) Trans R Soc Trop Med Hyg 83: 399-400 Amazigo UO, Chime A (1988) Trans R Soc Trop Med Hyg 82: 907 Andrade ZA, SantosJB, Prata A, Dourado H (1983) Rev Soc Brasil Med Trop 16: 31-40

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Aragona M, Caredda F, Lavarini C, Farci P, Macagno S, Crivelli 0, Maran E, Purcell RH, Rizzetto M (1987) Lancet 1: 478-480 Ashraf SJ, Arya SC, Arendrup M, Krogsgaard K, Parande CM, OrskovB, AgeelAR (1986) Liver 6:73-77 Bergmann KF, GerinJL (1986) J Infect Dis 154: 702-706 Bonino F, Hoyer B, Ford E, Shih HWK, Purcell RH GerinJL (1981) Hepatology 1: 127-131 Bonino F, Hoyer B, ShihJWK, Rizzetto M, Purcell RH, GerinJL (1984) Infect Immunol 43: 1000-1005 Bonino F, Caporaso N, Dentico P, Marinucci G, Valeri L, Craxi A, Ascione A, Raimondo G, Picciaino F, Rocca G, Rizzetto M (1985) J Hepatol 1: 221-226 Bonino F, Herman KH, Rizzetto M, Gerlich WH (1986) J Virol 58: 945-950 Bonino F, Smedile A (1986) Semin Liver Dis 6: 28-33 Butrago B, Popper H, Hadler SC, T Hung SN, Gerber MA, Purcel R, Maynard JE (1986) Hepatology 6: 1285-1291 Caredda F, Rossi E, D' Armino Monforte A, Zampini L, Re T, Meruni B, Moroni M ( 1985) J Infect Dis 151: 925-928 Caredda F, Antinori S, Re T, Pastecchia C, Moroni M ( 1987) Prog Clin Biol Res 234: pp 267-276 Cariani E, Ravaggi A, Puoti M, Mantero G, Albertini A, Primi D ( 1992) Hepatology 15: 685-689 Chao YC, Lee CM, Tang HS, Govindarajan S, Lai MMC (1991) Hepatology 13: 345-352 Cra.xiA, Raimondo G, PasquaP, Giannoli G, DiFranco C, Pagliaro L (1984) Fron GastroentRes 8: 191-196 De Cock KM, Govindarajan S, Redeker AG ( 1987) Prog Clin Biol Res 234: 167-179 Denniston KJ, Hayer BH, Smedile A, Wells FV, NelsonJ, GerinJL (1986) Science 232: 873-875 Dimitrakakis M, Waters MJ, Woolten A, Gust ID (1986) J Med Virol 20: 308-311 Dinter-Gottlieb G (1986) Proc Natl Acad USA 83: 6250-6254 DiSapio M, Caporaso N, Vecchio-Blanco CD, Coltorte M (1988) Liver 8: 236-240 Dourakis S, Karayiannis P, Goldin R, Taylor M, Monjardino S, Thomas HC (1991) Hepatology 14:534-539 Dubois F, Goudeau A (1988) J Clin Microbiol 26: 1338-1342 Farci P, Aragona M, Crivello 0 (1986) JAMA 255: 1443-1446 Fattovich G, Boscaro S, Noventa F, Pornaro E, Stenico D, Alberti A, Ruol A, Realdi G (1987) J Infect Dis 155: 931-935 Gmelin K, RoggendorfM, Schlipkoter U, Theilmann L, Bommer J, Kommerell B, Deinhardt F (1985) J Infect Dis 151: 374 Govindarajan S, Lin B, Peters RL (1984) Histopathology 8: 63-67 Govindarajan S, Valinluck B, Peters RL (1986) Gut 27: 19-22 Govindarajan S, Cassidy WM, ValinluckB, Redeker AG (1991) Prog Clin Biol Res 364: 207-210 Greenfield C, Farci P, Orisna V, McPherson CN, RomigT, Zeyhie E, French M,Johnson B, Tukei P, Winkays BM, Thomas HC (1986) AmJ Epidemiol 123: 416-423 Gupta S, Valinluck B, Govindarajan S (1989) AmJ Clin Pathol 92(2): 218-221 Gupta S, Govindarajan S, Cassidy WM, Valinluck B, Redeker AG ( 1991) Am J Gastroenterol 86: 1227-1231 Hadler SC, deMonzon M, Ponzetto A, Anzolia E, Rivera D, Mondolfi A, Bracho A, Francis DP, Gerber MA, Thung S, GerinJL, MaynardJE, Popper H, Purcell RH (1984) Ann Intern Med 109:339-344 Hedin G, Weiland 0, Ljunggren K, StrombertA, Nordenfelt E, Hannson BG, Oberg B (1987) In: Progress in clinical and biological research, vol 234. Rizzetto M, Gerin JL, Purcell RH (eds) Alan R Liss, New York, pp 309-320 Hedin G, Weiland 0, Ljunggren K, Nordeenfelt E, Hansson BG, LernestedtJO, Oberg B (1988) In: Zuckerman AJ (ed) Viral hepatitis and liver disease. Alan R Liss, New York, pp 94 7-952 Heijtink R, KruiningJ, Krupers L,Jacobs A, Geudens C, Walters G (1983) In: Verme G, Bonino F, Rizzetto M (eds) Viral hepatitis and liver disease. Alan R Liss, New York, pp 263-272 HoofnagleJH, Mullen K, Peters M, Avigan M, Park Y, Waggoner J, GerinJL, Hayer B, Smedile A (1987) In: Rizzetto M, Gerin JL, Purcell RH (eds) Progress in clinical and biological research, vol 234. Alan R Liss, New York, pp 291-298 HoofnagleJH (1989) JAMA 261: 1321-1325

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Jacobson DM, Dienstag JL, Werner BG, Brettler DB, Levine PH, Mushabwar DR (1986) Hepatology 5: 188-191 Jardi R, Buti M, Rodriguez-Frias F, Garcia-Lafuente A, Sjogren MH, Esteban R, Guardia] (1991) Hepatology 14: 25-28 Kos A, Dijkema R, Amberg AC, Van des Meids PH, Schellekens H (1986) Nature 323: 558-560 Liaw YF, Chiu KW, Chu CM, Sheen IS, Huang MJ (1990) J Infect Dis 162: 1170-1172 Lattau IA, McCarthy JG, Smith MH (1987) N EnglJ Med 317: 1256-1262 Madejon A, Castillo I, Bartolome J, Melero M, Campillo· ML, Porres JC, Moreno A, Carreno V (1991)] Hepatol 11: 381-384 Makino S, Chang MF, Shieh CK, Kamahora T, Vannier DM, Govindarajan S, Lai MMC (1987) Nature 329: 343-346 Maestrup T, Hansson BG, Widell A, Nordenfelt F (1983) Br MedJ 286: 87-90 Matthyssen L, Skystra M, Nelissen P, Werff, Walters G (1988) In: Zuckerman AJ (ed) Viral hepatitis and liver disease. Alan R Liss, New York, pp 408-411 Nordenfelt E, Hannsson BG, Al-Nakib B (1983) J Infect Dis 148-168 Pol S, Dubois F, Roingeard P, Zignedo L, Housset C, Brechot C, Goudeau A, Berthelot P ( 1989) Hepatology 10: 342-345 Ponzetto A, Forzani B, Puravicini PP, Hele C, Rizzetto M, Zenetti A (1985) Eur Epidemiol l: 257-263 Price JS, France AJ, Maoven LD, Welsby PD (1986) Lancet 1: 1273 Raimondo G, Smedile A, Gallo L (1982) Lancet 1: 249-251 Rasshafler R, Buti M, Esteban R,Jandi R RoggendorfM (1988) J Infect Dis 157: 191-195 Rizzetto M, Canese MG, Arico S, Crivelli 0, Trepo CG, Bonino F, Verme G (1977) Gut 18: 9971003 Rizzetto M, Canese MG, GerinJL, London WT, SlyDL, Purcell RH (1980)JinfectDis 121: 590602 Rizzetto M, Morello C, Marinucie PM (1982) J Infect Dis 145: 18-22 Rizzetto M, Verme G, Recchia S (1983) Ann Intern Med 98: 437-441 Rizzetto M (1984) Hepatology 3: 729-737 Rizzetto M, Verme G, GerinJL, Purcell RH (1986) In: Schaffner F (ed) Progress liver disease VIII. Grune & Stratton, New York, pp 417-431 Rizzetto M, Macagno S, Chiaberge F, Verme G, Negre P, Marinucci G, DiGiacommo G, Alfani D, Cortesini R, Milazzo F, Dogina M, Fassati R, Galmorini (1987) Lancet 2: 469-471 Rizzetto M, Ponzetto A, Bonino F, Smedile A (1988) In: Zuckerman AJ (ed) Viral hepatitis and liver disease. Alan R Liss, New York, pp 389-394 Salassa B, Daziano E, Bonino F, Lavanni C, Smedile A, Chiaberge E, Rosina F, Brunetto MR, Pessione E, Spezia C (1991) J Hepatol 12: 10-13 SaldanhaJ, Thomas HC, MonjardinoJ (1987) J Med Virol 2104: 35AAbstract 102 SaldanhaJ, Thomas HC, Monjardino J (1988) In: Zuckerman AJ (ed) Viral hepatitis and liver disease. Alan R Liss, New York, pp 198-199 ShattockAG, Irwin FM, Morgan BM, Hillary IB, Kelly MG, FeildingJF, Kelly DA, Weir DG (1985) Br MedJ 290: 1377-1380 Shattock AG (1989) J Virol Methods 23: 233-238 ShattockAG, Morris MC (1991) J Clin Microbiol 29(9): 1873-1876 Smedile A, Dentien P, Zanetti A, Sagnelli E, Nordenfelt E, Acs G, Rizzetto M (1981) Gastroenterology 81: 992-997 Smedile A, Lavarini C, Crivelli 0, Raimondo G, Fassone M, Rizzetto M ( 1982) J Med Virol 9: 131138 Smedile A, Locarnini C, Farci P (1983) AmJ Epidemiol 117: 223-229 Smedile A, Rizzetto M, Denniston K, Bonino F, Wells F, Verme G, Consolo F, Hayer B, Purcell RH, GerinJL (1986) Hepatology 6: 1297-1302 Smedile A, Baraudy BM, Bergman KF, Rizzetto M, Purcell RH, GerinJL (1987) In: Rizzetto M, Gerin JL, Purcell RH (eds) Progress in clinical and biological research, vol 234. Alan R Liss, New York, pp 235-241 Smedile A, Rosina F, Saracco G, Chiaberge E, Lattore V, Fahiano A, Brunetto MR, Verme G, Rizzetto M, Ronino F (1991a) Hepatology 13(3): 413-416

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Part IV Hepatitis C virus, hepatitis E virus and disease

19 Current nomenclature, viral agents, and clinical aspects of hepatitis C and hepatitis E I. Current nomenclature and viral agents

A. Introduction Hepatitis C and hepatitis E (Non-A, Non-B hepatitis-NANBH) are important transmissible diseases whose precise causative agents and diagnosis remain not entirely resolved problems, despite of recent progress. It was first identified as a type of blood-transfusion-associated hepatitis which on serological testing, was unrelated to hepatitis A or hepatitis B infection (Feinstone et al., 1975). In the 1970's during the clinical recognition and description of cases of post-transfusion hepatitis, NANBH, the long incubation hepatitis was noted for its mild, often subclinical presentation but high rates of chronicity and progression to cirrhosis. A similar illness could also be transmitted by blood products such as clotting factors (Dienstag, 1983a). An important series of studies in chimpanzees clearly showed the presence of a transmissible agent in blood products and in serum from carrier blood donors (Dienstag, 1983b). The agent was characterized as being sensitive to organic solvents. Even in the absence of conventional virological studies in vitro or knowledge of the genome, it was possible for Bradley (1985a, b) to make a calculated guess that the NANBH agent could be a small togavirus-like enveloped RNA virus. Presently, the agent of parentally transmitted non-A, non-B hepatitis is known as hepatitis C virus (Choo et al., 1989; Houghton et al., 1991). The recent studies of hepatitis C virus genome revealed three types of HCV distantly related (Takayasu et al., 1992; Chan et al., 1992) and Cha et al. (1992) demonstrated five related but distinct HCV genotypes.

B. Nomenclature: hepatitis C virus and hepatitis E virus Clinical and epidemiologic studies suggest more than one infectious agent is responsible for non-A, non-B hepatitis (NANBH). Three epidemiological types of NANBH has been recognized to date: post-transfusion or parental

176

Current nomenclature, viral agents, and clinical aspects

NANBH (PT-NANBH or hepatitis C), enteric NANBH (ET-NANBH or hepatitis E), and sporadically occurring community-acquired NANBH (SPOR-NANBH).

Hepatitis C. Parenterally transmitted NANBH is known to be the commonest form of post-transfusion hepatitis in most developed countries and may possibly account for more than 90% of cases. Recent work at the Centers for Disease Control and Chirion Corporation (Choo et al., 1989) led to the isolation and characterization of the genome of the major etiologic virus of parenterally transmitted NANBH and has now been designed hepatitis C virus (HCV). HCV represents the first example of successful cloning of a viral genome in the absence of any prior growth in cell culture, nucleic acid or protein sequence information, or identification of virus-specific agents or antibodies during infection in humans. This unprecedented cloning ofHCV was the result of application of improved molecular cloning and selection techniques that enhanced probabilities of cloning and identified a viral nucleicacidpresentatonlyonepartinafewmillionofunrelatednucleicacids. The hepatitis C virus is currently known to be a small (IO kb) enveloped single-stranded RNA virus with a genome of approximately 10,000 nucleotides and a genomic organization that places it in the family ofFlaviviridae. Flaviviruses, formerly called arboviruses, have been reclassified into five different groups. One of these groups, known as "unclassified viruses" are blood-borne causing chronic viremia and include viruses such as the simian hemorrhagic fever virus of primates and now possibly the hepatitis C virus. HCV is believed to be a diameter 50-60nm, sensitive to organic solvents as chloroform and ofrelatively low buoyant density. The particle size ofHCV has however been estimated by filtration through microporous regenerated cellulose fibre to be between 30nm and 38nm in diameter, although the possibility remains that larger HCV particles or HCV aggregates with a diameter more that 39 nm might exist (Yuasa et al., 1991). The genomic organization of the hepatitis C virus (Choo et al., 1989, 1991; Overby, 1990) is shown in Fig. 23. The nucleocapsid (core) protein of HCV is highly antigenic and the antibody may be detectable in either early or late in infection. Likewise, the NS-3 non-structural protein is believed to be highly antigenic (Fig. 24) (Overby, 1990). Recently, Fuchs et al. ( 1991) has characterized the 5 'end and parts of the structural genes of European isolates of hepatitis C virus and compared them with recently published RNA sequences of American and Japanese HCV isolates. The cDNA, obtained by reverse transcription of viral RNA, extracted from different sera, was amplified by nested PCR, cloned and sequenced. The amino acid homology, was between 98%-99% among all published sequences. Gene mapping of the putative structural region of the HCV genome by in vitro processing analysis (Hijikata et al., 1991) was shown recently to generate four major products, gp35 and gp70, p19 and p21. Both gp35 and gp70 could be candidates of initially processed forms of envelope proteins of the hepatitis C virus.

177

Current nomenclature and viral agents

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The existence of multiple agents for parenterally transmitted NANBH is suggested by the occurrence of multiple episodes of biopsy-proven acute hepatitis in some patients by the variation in incubation periods observed in some studies of chimpanzees and by the recent demonstration that 20%-30% of patients with parenterally transmitted NANBH lack serum markers ofHCV infection, (WHO, 1988; Choo et al., 1989; Kuo et al., 1989). However, recent studies demonstrated the importance of the putative polymerase (NS-5) gene product as a new marker for detection ofHCV (Desai et al., 1992; Lesniewski etal., 1992). In addition, the isolation and characterization ofan BCV-specific RNA-dependent RNA polymerase activity (Chung and Kaplan, 1992) will be useful to elucidate the mechanisms of HCV RNA replication.

178

Current nomenclature, viral agents, and clinical aspects

Hepatitis E. A form of the enterically transmitted epidemic non-A, non-B hepatitis virus (or water-borne NANBH) has now been provisionally designated as hepatits E (HEV) (Bradley et al., 1987). The HEV genome is a single positive-stranded RNA of about 7 .6 kb (Reyes et al., 1990). It is spherical, nonenveloped virus with a diameter of 27-34nm. This virus appears to be a member of the Calicivirus family but it shows characteristics similar to the Picornaviridae which includes enterovirus type 72, the hepatitis A virus. A final classification has yet to be made. First reported in India and Pakistan, the hepatitis E virus is now known to be the cause of recent outbreaks of epidemic hepatitis in young adults in South America, Eastern Europe and North Africa. Examination of stool samples from patients affected during this epidemic by means of reverse transcription polymerase chain reaction suggest that HEV indeed caused this large epidemic (Ray et al., 1991). Crossreactivity with acute-phase sera obtained from patients involved in outbreaks of hepatitis in diverse places such as Algeria and Gambia, has been demonstrated with virus-like particles recovered from stools of patients in India and the former USSR. This data strongly suggest that one virus, or a class of viruses is responsible for most cases of enterically transmitted NANBH worldwide (WHO, 1988). However, cross-reactivited studies of sera from patients with enterically transmitted NANBH suggest the existence of a second non-A, non-B hepatitis agent which can be transmitted by the fecal-oral route. It is likely that this agent may be responsible for some cases of enterically transmitted hepatitis infections reported in western countries. An antigen associated with HEV has been identified in the liver of experimentally infected macaques by use of an immunofluorescent probe prepared from human convalescent. serum. Sporadic cases ofNANBH from the United States and Europe were found to be serologically unrelated to those from HEV outbreaks (Krawczynski, 1989).

II. Clinical aspects of hepatitis C and hepatitis E A. Hepatitis C Clinically, non-A, non-B post-transfusion hepatitis (PT-NANBH), the one disease apparently associated with HCV (causing at least 85% of cases of transfusion-associated hepatitis) is similar to hepatitis B individuals. A large percentage of individuals, however, progress to chronic stages in hepatitis C than in hepatitis B. HCV also causes sporadic non-A, non-B hepatitis. The range of symptoms seen in HCV infection is similar to acute hepatitis B. The.early symptoms. (prodrome) include non-specific and gastrointestinal symptoms and are followed by the onset of jaundice and eventual improvement of symptoms in most patients. The incubation period from the time of infection to the development of symptoms or elevation in liver enzymes, ranges from 2-26 weeks with a mean of 8 weeks. In contrast, the mean incubation period for hepatitis B is slightly longer, 12 weeks.

Clinical aspects of hepatitis C and hepatitis E

179

The clinical course ofhepatitis C is less severe than hepatitis B with a higher proportion of asymptomatic cases. Peak levels of ALT (alanine aminotransferase) are lower than for hepatitis A or hepatitis B but rise and fall sporadically throughout the course of the disease -a symptom not observed in other forms of hepatitis (Dienstag, 1983a). More patients with hepatitis B require hospitalization than those with hepatitis C although the relationship of hepatitis C virus infection and the incidence of fulminant hepatitis is not clear (Saracco, 1988).Asmallnumberofpatientswithfulminanthepatitismay represent cases ofHCVhepatitis superinfection ofhepatitis B chronic carriers. Similarly, a small group of hepatitis C patients are associated with the development of aplastic anemia (Tzakis et al., 1988). The mechanism ofinjury to the liver by the virus has been described as antibody-mediated in cases of hepatitis B, whereas studies suggest a more direct role for the virus in hepatitis C. Hepatitis C results in a higher rate of progression to chronicity when compared with HBV infected individuals. It is also interesting that the severity of the initial HCV infection does not appear to predict the development of chronicity, something that may be slightly different in contrast to the case for hepatitis B virus infection. Prospective studies of PT-NANBH show that over 50% of patients with PT-NANBH are HCV carriers and could develop chronic liver disease (Alter and Hoofnagle, 1984; Lettau, 1992). The risks of developing the severe complications associated with chronic hepatitis C have been documented and appear to be similar to those found in chronic hepatitis B. In a study of 189 patients with PT-NANBH, 39% had chronic persistent hepatitis or chronic lobular hepatitis; 40% had chronic active hepatitis and 18% had liver cirrhosis (Dienstag et al., 1983a). Some studies have suggested that the age and route of transmission may influence the severity of the disease. Infection by transfusion may have a higher incidence of chronic active liver disease and cirrhosis than patients with chronic sporadic NANBH (Mattson et al., 1988). Primary hepatocellular carcinoma (PHC) has been casually associated with hepatitis B chronicity and may also complicate cirrhosis following PTNANB hepatitis (Realdi et al., 1982; Lefkowitch, 1987). In a study ofJapanese patients reported in 1988 (Sakamoto, 1988), 13 years elapsed between infection and transfusion and a diagnosis of chronic active hepatitis; 1 7 years to a diagnosis of cirrhosis and 18-24 years to the development of hepatocellular carcinoma. This long suspicion that NANBH infection may occasionally lead to PHC and the development of an assay for circulating antibodies against hepatitis C virus (anti-HCV) has now possibly confirmed that HCV is frequently involved in chronic liver disease. A recent article by Kaklamani et al. (1991) has highlighted the earlier notion that HCV does have an interactive role in the origin of PHC. The results of yet another recent study (Tanaka et al., 1991) further indicated that in Japan, the possible role ofHCV infection in the etiology of PHC is extremely large and may, in fact, be more important than chronic hepatitis B virus infection. The precise incidence of chronic viral hepatitis following sporadic NANB hepatitis still remains unclear, however, it does not appear to be as high as the situation following most-transfusion hepatitis.

180

Current nomenclature, viral agents, and clinical aspects

Recently, the lack of association between circulating HCV RNA and antiHCV positivity in primary biliary cirrhosis (PBC) has been reported (Bertolini et al.,1991). In this study, serum samples from 106 consecutive patients with PBC were tested for anti-HCV by conventional ELISA (Ravitan, Ortho Diagnostics). Positive samples (optical density 2: 0.459) were retested in duplicate and further with the recombinant immunoblot assay (RIBA). To detect the HCV genome, RNA extracted from ELISA-positive samples were reverse-transcribed and amplified by the polymerase-chain reaction (PCR) with primers of the non-structural region. Based on their results where a 3% prevalence of anti-HCV positivity in the group of 88 patients, who could be regularly followed, and none with detectable HCV-RNA among these ELISA positive with serum available for HCV determination, the authors conclude that the frequency of anti-HCV positivity in Italian patients with PBC, appears to be somewhat higher than in the general population. Since none of the patients with PBC tested by PCR unlike those with auto-immune chronic active hepatitis had detectable HCV-RNA in serum, it is unlikely that HCV plays a part in the pathogenesis of PBC. In a recently published clinico-pathological study by Schueuer et al. ( 1992), a histological pattern of mild chronic hepatitis with pontal lymphoid follicles and varying degrees of lobular activity was found in many of the patients. Aggregates oflymphoid follicles in cirrhosis should perhaps alert a pathologist to the possibility of HCV infection. A recent study (Pascual et al., 1990) has suggested an association between HCV and cryoglobulinemia type II. Cryoglobulins have been found in 6%43% of patients with chronic liver diseases. Type II cryoglobulinemia is characterized by the presence in the cryoprecipitate of a monoclonal immunoglobulin that binds to Fe portions of polyclonal IgG; most often this monoclonal component is an IgM.

B. Hepatitis E The clinical features of hepatitis E (ET-NANBH), enterically transmitted hepatitis do not distinguish it from other types of hepatitis (Ramalingaswami and Purcell, 1988). The incubation period is slightly longer than for hepatitis A, ranging from 22 to 60 days. Signs and symptoms are similar to those occurring with other types of hepatitis. Comparison of peak mean bilirubin and ALT levels in cases of hepatitis A, hepatitis Band ET-NANBH hepatitis has not helped to differentiate these diseases. Histopathologic examination of liver biopsy specimens from non-fulminant cases, however, have shown cholestasis not typical of findings in acute viral hepatitis. The disease transmitted by ET-NANBH virus is self-limited and usually moderately severe (Arankalle et al., 1988) with no long-term sequelae or chronic liver disease (Ramalingaswami and Purcell, 1988). Among the key differences between ET-NANBH and HAVis the mortality rate in pregnant women and hospitalized patients. Whereas the mortality from HAVin pregnant women is generally not higher than that in the general population, it has been between 10% and 20% in outbreaks ofET-NANBH (Purcell and Ticehurst, 1988).

20 Hepatitis C and hepatitis E viruses: epidemiology and transmission I. Epidemiology and distribution of infections Non-A, non-B hepatitis (NANBH) was first identified as a type of bloodtransfusion-associated hepatitis (hepatitis C virus infection) unrelated to HAV or HBV infection (Feinstone et al., 1975). Subsequent research and analysis of epidemiological and hepatitis transmission data has helped characterize NANB hepatitis. NANB hepatitis has been reported in practically most parts of the world. Current worldwide epidemiological surveys estimate that approximately 100 million individuals are chronic carriers ofNANB hepatitis. Over 175,000 new cases are reported each year in both the United States and Europe and 350,000 cases per year occur in Japan (Alter et al., 1985). In a recent in tensive surveillance NANBH over a 7-year period conducted by the Centers for Disease Control (Alter et al., 1990), it was found that the incidence of NANBH has remained relatively stable (average 7.1 cases per 100,000) but significant changes in disease transmission patterns were obvious. The proportion of patients with a history of blood transfusion declined from 17% to 6% but the proportion with a history of parenteral drug use significantly increased from 21%to42%. Antibody to hepatitis C virus was found in 45% of patients within 6 weeks of onset of illness and in 68% of patients followed up for at least 6 months. Patients with no history of transfusion were just as likely to be positive for antibody to hepatitis C virus as patients with transfusion-associated hepatitis, indicating that hepatitis C virus is the major causative agent of all non-A, non-B hepatitis in the United States. HCV infection appears to play a relatively minor role in HBsAg-positive liver disease in Taiwan but is strongly associated with HBsAg-negative chronic liver disease and hepatocellular carcinoma. The infection is extremely common in hemophiliacs and parenteral drug users (Chen et al., 1990). The contribution of NANBH to the total number of cases of acute viral hepatitis varies depending on geographical location and source of patients. In Africa and Asia NANBH may possibly account for 50% of cases of viral hepatitis, attributable probably to HEV whereas in contrast in Europe, North

182

Hepatitis C and hepatitis E viruses: epidemiology and transmission

America and Australia, NANBH may account for less than 7% of reported cases of acute viral hepatitis (MMWR, 1988). In Behei province, China, the anti-HCV antibody was found in 90.8% of population of plasma donors during the epidemic of NANBH (Meng et al., 1990). The detection rate of anti-HCV of 26.2% among Egyptians attending blood bank in Riyadh was found in comparison to 2.4% for other expatriate blood donors (Saeed et al., 1992). The ability to identify both hepatitis A and B by the mode of transmission provided the indication that more than one virus may be responsible for NANB hepatitis. Evidence indicates that at least 2 or more NANBH viruses exist with defined and specific serologic markers. A current nomenclature renames these viruses as hepatitis C (HCV) for the blood-borne and hepatitis E (HEV) for the epidemic water-borne agent. Most NANBH cases in the United States were linked to post-transfusion or transplant events. The hepatitis C virus has been recently noted as the major causative agent of all nonA, non-B hepatitis in the United States (Alter et al., 1990). Another set of cases however, was linked to NANBH agents that appeared responsible for viral hepatitis in patients with no history of parenteral exposure from transfusions or percutaneous injections. This set of cases with milder infections had been referred to as "sporadic" NANB hepatitis. Recent data suggest that the causative agent for both types of hepatitis may, in fact, be the same. It has also been proposed that some cases ofNANB hepatitis represent infection by a seronegative variant NANB virus, with different physio-chemical characteristics to HCV, has been reported (Feinstone et al., 1984; Wands et al., 1986). The advent of testing for HAV infection has also made it possible to substantiate that some other form of viral hepatitis could occur in epidemics. In 1980, an epidemic of viral hepatitis in India was evaluated to determine its etiology since some features as the high mortality rate (10%-20%) found among pregnant women and the lack of hepatitis A markers appeared to be not typical of hepatitis A (Khuroo, 1980; Khuroo et al., 1981). An epidemic form of NANBH virus was proposed to exist. Clinically, epidemic NANBH resembles hepatitis A more than hepatitis B with similarities in mode of transmission, length of incubation, clinical features, and liver enzymes test results. Furthermore, like hepatitis A, no evidence of chronic disease is associated with epidemic NANB hepatitis (Khuroo et al., 1981). This human hepatitis virus responsible for epidemic NANB hepatitis appeared to be rather distinct from the post-transfusion NANBH type or types. Epidemics and suspected epidemics of enterically transmitted NANB hepatitis have been recognized in a broad region encompassing much of South-East Asia, Central Africa, Eastern, Northern and Western Africa and recently, North America (Purcell and Ticehurst, 1988), South America and Mexico (Tandon et al., 1982; Belabbes etal., 1984; Kane etal., 1984, Myint et al., 1986; Gust and Purcell, 1987; DeCock et al., 1987; Centers for Disease Control, l 987a, l 987b 1988; Bradley et al., 1988). ET-NANBH has a high incidence in areas where standards of hygiene are poor and where there is shared water supply. Hepatitis C (parenterally transmitted NANB hepatitis) appears to have a global distribution with a prevalence between 0.3% and 1.5%. In developed

Incubation period and mode of transmission

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countries, it is the most common form of post-transfusion hepatitis and may possibly account for more than 90% of cases. The risk factors are probably similar to HBV infection. Some groups like hemophiliacs IV drug abusers and renal dialysis patients are at high risk. Using clinical symptoms and serologic marker exclusion as criteria for a diagnosis of NANBH, a recent CDC study described the prevalence of both NANBH and hepatitis B (HBV) in 2 county health departments representative of the general population in the United States (Alter et al., 1989). Heterosexual transmission may play a previously unidentified role in the spread ofNANB hepatitis. To be noted is the high prevalence of NANBH in the intravenous drug abuser population (34%) and blood tranfusion recipients (13%) and the relatively low prevalence in the hemodialysis patients. Patients with no history of parenteral exposure, classified as "sporadic" or "community acquired" NANBH, constitute 40% of the clinically diagnosed cases. This data reflects earlier CDC surveillance studies ofNANBH in the United States between 1981-1985 and indicates that modes of transmission other than parenteral are indeed possible (Hadler, 1987). Until the case in 1989, community-based outbreaks of hepatitis C (PT-NANBH) had not been reported in the United States. This unusual outbreak, which occurred, provided two very important findings: a high proportion of ill persons were confirmed or suspected - IV drug users and no identifiable common hepatotoxic chemical or drug was found. This finding suggested that the etiologic agent was HCV (parenterally transmitted NANB hepatitis virus). Approximately 90% of post-transfusion hepatitis cases are now known to be caused by HCV (Esteban et al., 1990). Very recently, Poterucha et al. ( 1992) pointed out that hepatitis C virus infection is a major cause of chronic hepatitis occurring after liver transplantation. Thus, not only hepatitis B virus should be taken into consideration in liver transplantation practice. In another study Novati et al. (1992) demonstrated the transmission of HCV from mother to child by women coinfected with hepatitis A virus and human immunodeficiency virus. The HCV viremia in neonates was detected by PCR assay. The weak immunity in hepatitis C virus infection (Prince et al., 1992) plays a role in epidemiology of the diseases. Reexposure to even low doses of HCV results in reinfection as reappearance ofviremia.

II. Incubation period and mode of transmission In hepatitis C (PT-NANBH) the incubation period is intermediate to those of HAVand HBV (19 to 91 days) with a mean of7.8 weeks and 80% to 90% of cases occurring within 5 to 12weeks (peak of 42 days) of transfusion (Alter et al., 1978, 1982). Shorter periods have been reported with hemophiliacs infused with factor VIII concentrate (Bamber et al., 1981), whether this is attributable to an altered host response or a different virus is not clear at the present time. Hepatitis E (enterically transmitted NANB hepatitis) appears to have a mean incubation period between 15 and 40 days (Wong et al., 1980) and is spread similarly to HAV by the fecal-oral route.

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In the United States, Europe and Japan, NANB hepatitis has been associated primarily with parenteral routes such as blood and blood product transfusion, hemodialysis and intravenous drug use. Recent data suggest that heterosexual transmission may also be a route of infection; the efficiency of a sexual mode of transmission remains however, unclear (Vilarejos et al., 1975; Wyke et al., 1979; Alter et al., 1989) but sexual transmission is highly probable (Alter, 1991). It was demonstrated that heterosexuals with multiple partners and homosexual men, who attended clinics for sexually transmitted diseases, had a prevalence of anti-HCV 5-fold to 15-fold higher than control of blood donors (Hess, 1989; Mortimer, 1989; Alter, 1991). Groups at high risk for NANB hepatitis are those where infection via a parenteral route is probably involved and include intravenous drug abusers, and recipients of blood or blood products. Recently, evidence of vertical transmission ofHCV like hepatitis B virus has been provided (Degos et al., 1991; Thaler et al., 1991). The findings suggest that perinatal infection may initiate a silent process or more perhaps a chronic carrier state. Transmission ofHCV within households has been demonstrated, although not consistently (Ideo et al., 1990). The results of a survey have been recently reported (Stevens et al., 1990), carried out on volunteer blood donors in New York Blood Program who were tested for two surrogate markers for NANBH-elevation of alanine aminotransferase level and presence of antibody to hepatitis B core antigen. Serum sample from selected donors were tested for antibody to hepatitis C virus (anti-HCV). Anti-HCV was detected in 0.9% to 1.4% of donors and was higher in black and Hispanic donors in comparison to white donors. AntiHCV prevalence increased with increasing age through the fourth decade of life but decreased thereafter possibly reflecting the disappearance of detectable antibody with time. Anti-HCV correlated with both alanine aminotransferase (ALT) level and the presence or absence of antibody to hepatitis B core antigen. These associations suggest that donor screening for elevation of alanine aminotransferase level and presence of antibody to hepatitis B core antigen can be partially effective in preventing hepatitis C (transfusion associated NANBH). The detection of anti-HCV in donors with neither an elevation of alanine aminotransferase level, non-presence of an antibody to hepatitis B core antigen is strongly suggestive that donor screening for antiHCV will certainly further reduce the risk of transfusion-associated hepatitis. The level of ALT could be used to predict chronicity in post-transfusion hepatitis due to hepatitis C. In hepatitis C cases generally, ALT appears to be higher and the persistence of chronicity no longer than in hepatitis B cases.

21 Diagnosis of hepatitis C and hepatitis E virus infections I. Introduction The term non-A, non-B hepatitis was initially applied as a diagnosis to patients whose viral hepatitis, after other causes had been excluded, seemed to have been acquired by the blood-borne route. Currently, two epidemiologically distinct types of non-A, non-B hepatitis (NANBH) have been identified worldwide and are believed to be probably caused by different viruses. One type is transmitted parenterally and is associated with acute hepatitis that develops after transfusion and sporadic cases occurring primarily in developed countries. The other type is transmitted by the fecal-oral route and is primarily associated with epidemic cases of acute hepatitis in developing countries. The characteristics of both types ofNANB hepatitis are essentially indistinguishable but the chronic consequences however, appear to be markedly different. A case definition ofNANBH in use over the years have entailed serologic exclusion of hepatitis A, hepatitis B, hepatitis D viruses, cytomegalovirus, Epstein-Barr virus as well as the exclusion of other possible non-viral causes ofliver inflammation including drugs, Wilson's disease, and alpha-1-antitrypsin deficiency. The acute hepatitis panel is often used in the initial diagnosis of patients who present with signs and symptoms of viral hepatitis of unknown origin. This panel consists of three tests for the markers HBsAg, anti-HBclgM, and anti-HAV IgM. When HBsAg and anti-HBclgM are both negative, hepatitis A virus is the cause and when anti-HAV IgM is not detected, hepatitis A virus is not the cause of hepatitis. By exclusion of these markers from the clinical diagnosis and with ALT levels at least 2.5 times normal, a diagnosis of NANB hepatitis is made. Parenterally transmitted hepatitis C (PT-NANBH) irrespective of this sourcer is clinically indistinguishable from other types of viral hepatitis. The incubation period spans those of hepatitis A and hepatitis B (Dienstag, 1983). Several reports have indicated that the mean ALT and bilirubin levels of patients with hepatitis Care significantly lower than those of hepatitis B patients but the extensive overlap of the ranges precludes identification of this type of viral hepatitis by these labora-

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tory tests alone (Dienstag, 1983b; Sampliner et al., 1984; Hoofnagle et al., 1985). The patterns of ALT elevations - monophasic, fluctuating, plateaulike have been found to differ among persons with hepatitis C (Seeff et al., 1975; Aach et al., 1978; Alter et al., 1978; Clemens et al., 1992). The most significant feature of hepatitis C has been noted to be the frequency with which patients develop chronic liver disease and about 50% of patients seem to have persistently elevated ALT level (Dienstag and Alter, 1986). Enteric non-A, non-B hepatitis (ET-NANBH) or hepatitis E virus (HEV) infection acquired by the fecal-oral route first identified during the course of investigations in developing countries has occurred in epidemic or sporadic fashion in parts of Asia, Africa and Mexico and its agent is serologically distinct from other known hepatitis viruses (Wong et al., 1980; Kane et al., 1984; Centers for Disease Control, 1987 a, b; Alter, 1990). HEV particles 27 to 32nm have been identified from the feces of patients with ET-NANBH using immune electron microscopy (Bradley et al., 1988; Arankalle et al., 1988). Currently, the primary method of identification of a case of HEV infection rests on exclusion by serologic testing other causes of viral hepatitis with confirmation obtained by examination of stool by immune electron microscopy in the presence ofa reliable reference antiserum. Solid-phase immune electron microscopy has recently been used also to identify the hepatitis E virus in fecal extracts. It is a simple, highly sensitive assay and rapidly performed. Recombinant DNA technology has enabled the cloning and sequencing of hepatitis C virus (Choo et al., 1989). The development of a serologic assay to detect antibody to a part (epitope) of this virus (anti-HCV) (Kuo et al., 1989) have been major breakthroughs in the long search for a causative agent of NANB hepatitis. The implications of these results for the diagnosis of hepatitis C is discussed in subsequent sections of this chapter.

II. Antibody assays and seroprevalence Characterization of hepatitis C virus (HCV), the new agent of PT-NANBH, was initiated in 1984 at the Centers for Disease Control (CDC) using material obtained from a primate infected by plasma from a case of transfusion associated hepatitis. Human Factor VIII concentrate had been implicated in this case of NANBH. Plasma taken from the infected animal in the chronic phase of NANB hepatitis was injected into another primate, and the plasma drawn from the second animal to obtain a pool infectious material (Bradley, 1985a, b). A concentrated infectious plasma pool subsequently enabled the extraction of nucleic acids, transcription onto complimentary DNAs (cDNAs) which were synthesized from both the RNAand the DNA via the reverse transcriptase. The resulting cDNAs were cloned and a cDNA library generated. The library was screened for expression of an antigen detectable by serum from a chronic NANB hepatitis patient but who was also negative for HBV and HAV. It was only after screening around one million clones that it was possible to find one to produce a protein that reacted with the patient's

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serum. The 155 base-pair insert in this clone was cut out and used as a probe to extract a larger overlapping clone. This was the stepping stone for the ultimate isolation and cloning in 1987 of a fragment of the viral genome and reported by Chiron Corporation scientists in 1988. This reactive clone produced a recombinant viral antigen ( C 100-3) that was capable ofreacting with immunogenic material (antibody) in clinical specimens from NANB hepatitis patients and allowed the development of an immunoassay system (AntiHCV) to detect HCV antibody (Choo et al., 1989; Kuo et al., 1989). The use of molecular biology techniques for the development of the HCV recombinant based antibody assay was a definite deviation from the conventional technique of developing immunoassays by isolation and purifying proteins from the virus itself. The Cl00-3 was used as the antigen source in a radioimmunoassay and enzyme immunoassay to detect HCV antibody. Using a known, pedigreed panel of specimens, the validity of this immunoassay was tested successfully; 6 of 7 infectious primate samples showing reactivity. The only specimen that was negative was obtained from the acute phase of the disease. Normal control and disease control samples were negative (Kuo et al., 1989). Clinical evaluation of the recombinant based assay for HCV antibodies was initiated in August 1988 and preliminary seroprevalence studies of several European, US and Japanese populations have been reported (Kuo et al., 1989; Alter et al., 1989; Esteban et al., 1989; Van der Poel et al., 1989; Kuhnl et al., 1989; Roggendorf et al., 1989). These studies indicate that the test detects as reactive 10%-29% of the clinically diagnosed acute cases of HCV hepatitis, less than 90 days after infection, between 67%-85% of the clinically diagnosed cases of chronic HCV hepatitis and cases of convalescent HCV hepatitis. Between 0.2% and 1.2% of random blood donors were found reactive for HCV antibody. In the prevalence study for the presence of HCV antibody in various groups of specimens from 676 patients in Spain (Esteban et al., 1989), 62% of transfusion related NANBH, 70% of hemophiliac patients, 20% ofhemodialysis patients, 70% of intravenous drug users and drug addicts, and 8% of homosexual males showed evidence of prior HCV infection of individuals with liver disease, but at low risk for NANBH, 38% were found to reactive for HCV antibody whereas, antibody was detected in only 1.2% of low-risk healthy subjects. In a prospective study carried out over a two year period in the Netherlands of multiple transfused heart transplant patients, the HCV antibody assay identified those blood products associated with symptoms of NANB hepatitis in patients receiving transfusions. The assay sensitivity was 67% and HCV seroconversions occurred in 44% of recipients within 6 months of transfusion. The anti-HCV EIA ( Ortho Diagnostic Systems with recombinant antigens from Chiron Corp.) was used to investigate 3123 randomly selected German blood donors. Although no urban/rural differences were detectable in this multicenter German Blood Bank study (Kuhnl et al., 1989) a North/South trend in increased prevalence ofHCV antibody (0.13%-0.83%) was noted in donors. A study of blood donors demonstrated a 0.5%-1.0% prevalence of

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HCV antibody and while the authors (Contreras and Barbara, 1989) indicate the Ortho-ELISA for anti-HCV as being specific for the major agent causing post-transfusion hepatitis C, they point out that the time of the performance of the test (3 hours) would make its introduction in routine donor screening as logically difficult. The second generation HCV-ELISA assay from Abbott Diagnostics, in which recombinant H CV antigens (core, 33c, Cl 00) are used, (Figs. 25 and 26) reduced the test performance time. All these seroprevalence studies thus point towards the existence of a strong correlation between the presence ofHCV antibody in specific groups of individuals as measured by the Cl00-3 and "pre-HCV antibody assay" prevalence studies ofNANB hepatitis based on a clinical diagnosis alone. The failure of the currently available HCV antibody assay to detect 100% of NANBH however, entails the need for a confirmatory or even perhaps a supplementary test for HCV antibody (Contreras and Barbara, 1989) . The ability to detect anti-HCV antibodies, generally only several months after acute infection, is an important advance that apart from providing a clinical diagnostic test could also in effect entail a screening procedure for blood donations thereby improving the safety of transfusion of blood and blood products. Preliminary serological surveys of healthy blood donors

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Antibody assays and seroprevalence

189

indicate average rates of anti-HCV antibodies in 0.5% to 1.5% and more in industrial countries using entirely voluntary blood donations (Zuckerman, 1990). Since HCV antibody is not detected until an average of 15 weeks (range 4-32 weeks) after the onset of hepatitis, the results of this assay should essentially be interpreted with caution and reference to the patients' clinical and social history. In particular, HCV infection cannot be excluded in those whose serum is antibody-negative up to 6 months after the onset of symptoms (Editorial, 1990a). The anti-HCV test which is currently beign routinely utilized measures an IgG antibody. This antibody is obviously not neutralizing because serum containing anti-HCV is infectious. The antibody may disappear with time in cases of resolving hepatitis and may be present in those with no biochemical evidence of ongoing hepatic inflammation. This obviates the need for the test to be interpreted in conjunction with the patients' history, blood test results or liver biopsy, to determine the significance of a positive result. The difficulty in interpreting a positive anti-HCV result is analogous to hepatitis B virus, in which the presence of an isolated IgG antibody to hepatitis B core antigen does not enable one to determine whether the patient has active or resolved infection or even a false positive result (Editorial, 1990b). There is evidence, in fact, that some factor in the globulin fraction of serum may interfere with the anti-HCV assay, leading to false-positive reactions (Colombo et al., 1990). Gray et al. ( 1990) improved the specificity by modifying the Ortho ELISA to include a wash step incorporating 8 mol/l urea, which disassociates lowavidity non-specific antibody from the antigen. Ikeda et al. (1990) suggest that the non-specific reactions might possibly be related to the superoxidedismutase component in the Ortho ELISA antigen. A newer ELISA test, more sensitive and specific, was described as HCV C200 C22 EIA, which uses three HCV antigens instead of one as in the original CIOO ELISA (Bassetti et al., 1991). The goal is to develop a broad diagnostic and blood screening assay for hepatitis C virus infection with viral proteins C, NS-3, NS-4, which are self conserved between different HCV groups. Such assay may permit a broadly reactive and sensitive diagnosis. Enhanced detection of HCV infection using antigens from the putative polymerase NS-5 gene product have shown that antibodies to this region can be detected at high frequency in patients chronically infected by HCV (Lesniewski et al., 1992). The study point to the utility ofNS-5 as a new marker for detection of previously unrecognized HCV infection. Supplementary investigations could include a neutralization test (Abbott) on a recombinant immunoblot assay (RIBA, Chiron Corp) that tests for two epitopes (Editorial, 1990c). The RIBA test detects antibody directed against three recombinant antigens: the BCV-derived 5-1-1 polypeptide in a fusion protein with superoxide dismutase; Cl 00-3, the antigen present in the enzymelinked immunosorbent assay; and the superoxide dismutase protein itself. Preliminary experience with this test indicates that it is generally possible in patients with an unequivocally positive anti-HCV ELISA. The recombinant

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Diagnosis of hepatitis C and hepatitis E virus infections

immunoblot assay seems to be a strong predictor of infectivity. Skidmore ( 1990) in a limited evaluation ofRIBA test suggests that it may be more specific than Ortho ELISA and suitable as a confirmatory test for blood donor testing. A new second generation RIBA (4-RIBA) has recently been developed by Chiron Corporation in which two additional HCV recombinant antigens have been added to the CIOO RIBA. One antigen is from the non-structural NS-3 region (C33c) and the other is an HCV-core-associated antigen (C22). Both antigens have been expressed in yeast. Van der Poel et al. (1991) has very recently evaluated this new four-antigen recombinant immunoblot assay and compared the results with PCR analysis. The results obtained validate the 4-RIBA as a candidate confirmation test to discriminate between infective and non-infective HCV-CIOO ELISA positive blood donors. In a recent study the IgM antibody response in acute hepatitis C virus infection was evaluated (Clemens et al., 1992) by IgM dot blot immunoassay (Abbott MATRIX), which uses HCV antigens spotted on nitrocellulose. Cl 00 antigen expressed in yeast as a fusion protein with human superoxide dismutase (SOD) polypeptides corresponding to putative HCV capsid (core) NS-3 and NS-4 sequences consisted the test panel. IgM anti-HCV core was detected in 13 of 15 post-transfution patients. In 9 of these patients acutephase IgM anti-HCV core was found coincidentally or earlier than active IgM anti-HCV core response. The IgM anti-HCV core reactivity duration was 8.1±3.7 weeks. Late IgM anti-HCV was also detected in some post-transfusion patients. The authors concluded that IgM anti-HCV core is a useful marker for acute HCV infection. As the IgM response does not generally precede the IgG response the IgM detection is unlikely to narrow the window of seronegative infectivity existing between the time of exposure to HCV and the first appearance of antibody. Anti-HCV IgM, particularly anti-core IgM, can be used as an acute marker ofHCV infection (Meisel et al., 1992). In another study Okamoto et al. (1992) developed an immunoassay to detect antibodies against oligopeptides deduced from the putative core gene of HCV. Its performance was compared with that of the commercial immunoassay for antibodies against the product of non-structural regions of HCV (anti-CI00-3). The authors concluded that antibodies against antigenic determinants of the hepatitis C virus core would complement anti-CI00-3 for the diagnosis of HCV infection.

III. Detection of hepatitis C virus Although it would be prudent to regard all HCV antibody-positive blood as infectious, this may not always be the case, since HCV antibody assays cannot distinguish reliably between infectious and non-infectious blood. Sensitive assays for detection of HCV RNA has been described (Kato et al., 1990; Weiner et al., 1990; Kaneko etal., 1990; Hagiwaraetal., 1992). HCVRNAwas detected in serum samples from experimentally infected chimpanzees in the absence of HCV antibody. This type of assay may prove to be invaluable for the identification of infectious patients. The experiments of Weiner et al.,

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( 1990) focused on the development of a sensitive, semi-quantitative cDNA/ polymerase chain reaction to detect HCV RNA in liver and plasma or serum based on the nucleotide sequence ofHCV cDNA clones. A strong correlation was found between the presence of circulating antibodies to Cl00-3 and the presence of HCV sequences in the livers of patients with chronic NANBH. Their study provided molecular basis for the chronic carrier condition caused by chronic NANBH infections and a further evidence that anti Cl 003 being a marker of infectivity. A significant correlation between C33c antigen reactivity and HCV RNA positivity was found by Martinot-Peignoux et al. (1992) in anti-HCV positive individuals. To detect HCV RNA in clinical samples, a very promising signal amplification method based on branched oligodeoxyribonucleotides (bDNA) has been developed recently (SanchezPescador et al., 1992). Garson et al. (1990) examined anti-CI00-3 positive samples in a prospective study by means of a new assay for the detection of hepatitis C virus RNA sequences. The assay was based on a modification of the polymerase chain reaction (Sniki et al., 1985). The "nested PCR" assay used refers to the use of two successive rounds of amplification, with the second round using primers internal to, or nested within, the original two primers. Only 6of1,000 donor blood units were found to be repeatedly positive for antibodies to the Cl003 antigen. Only one the these six donor units was found to contain HCV RNA by the "nested PCR" assay. The authors conclude that anti-CI00-3 may not in fact be a good predictor of infectivity in blood donors and perhaps direct identification of viral RNA ofHCV may be a requisite. In the absence of a test for antigenemia, the "nested PCR" assay, a PCR based technique, nevertheless appears to be a substantial step forward, since it enables the accurate identification of the small proportion of anti-ClOO-positive, carriers of the virus. Modification of the PCR technology may enable adaptation in future to mass-serening laboratory needs. According to Garson et al. ( 1990) PCR may be valuable in defining the time course of the viremia on infected subjects and may allow rapid diagnosis of acute hepatitis C, weeks or months before diagnosis by CI00-3 serology is possible. A 44% positivity in patients who are Ortho HCV antibody positive was detectable by PCR (Editorial, l 990c). The detection of HCV RNA in serum of patients by PCR, before the appearance of anti-HGV, especially in blood donors and drug abusers was performed by Cha et al. (1991) and Waxman et al. (1991). In a recent study with 156 patients with chronic NANB hepatitis Hagiwara et al. (1992) detected by PCR the hepatitis C virus RNA in 121of129 (93.8%) patients positive for anti-Cl00-3. However, they found also HCV RNA in 15 of 27 (55.6%) patients negative for anti-Cl 00-3. It seems that replication of HCV continues to occur in advances liver disease patients positive but also negative for antiCl 00-3. The PCR technique also was applied to detect HCV RNA in patients after liver transplantation (Poterucha et al., 1992). HCV could be considered as a major cause of chronic hepatitis occurring after liver transplantation. In another study Chou et al. (1991) using reverse transcriptase PCR (RTPCR) method found cDNA of HCV genome in patients with primary hepatocellular carcinoma. Of 16 samples tested from each of 8 patients with PHC - the

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Diagnosis of hepatitis C and hepatitis E virus infections

HCV cDNA was detected in 6 patients by RTPCR. These data are additional evidence of a casual link between HCV infection and the development of hepatocellular carcinoma as previously pointed out (Kiyosawa et al., 1991). Interpretation of PCR data in clinical studies requires the consideration of issues related to methodology PCR entails the use of oligonucleotide primers, which are homologous to known nucleic acid sequences to enzymatically amplify the DNA between the two primers. The method is sensitive enough to amplify a single DNA molecule to easily detectable levels. The method could possibly be modified to amplify RNA sequences by first synthesizing complementary DNA (cDNA) with the enzyme reverse transcriptase. Because of the equisite sensitivity of PCR, the possibility of false-negative results may often be overlooked. It has been suggested that in an evaluation of the sensitivity of PCR assays, the reverse transcription reaction be included perferably in the assessment (Lucey and Traber, 1991). For universal adaptation of the PCR technology, entire procedures could be automated, and the possibility of contamination of samples might be eliminated in the future. Until then, steps towards achieving an effective quality control by taking positive and negative controls would appear to be a requisite for every laboratory conducting PCR.

IV. Diagnosis of hepatitis E virus Hepatitis E virus (HEV) is a cause of enterically transmitted endemic hepatitis mainly recorded in developing countries and affecting young-to-middleaged adults (Zuckerman, 1990; Goldsmith et al., 1992). The diagnosis of hepatitis E virus infection has been based until now, on the detection by immune electron microscopy of virus-like particle of 2734nm in diameter, in fecal specimens from infected patients and serologically negative to other viral hepatitis (Bradley et al., 1987). Some progress was registered toward a diagnosis test with the identification of HEV type-common epitopes (Yarbough et al., 1991) who developed an enzyme-linked immunosorbent assay based on clonal recombinant HEV antigen to detect HEV-IgM and HEV-IgG. Recently, Goldsmith et al. (1992) diagnosed the HEV infection using ELISA assay with four HEV antigens for anti-HEV IgG and anti-HEV IgM. The serum was considered reactive for anti-HEV if the ELISA signal to HEV-glutathione-S-transferase fusion proteins were three times higher than that with non-recombinant glutathione-S-transferase proteins. The authors used HEV clones producing Mexico (M) antigens M406.3-2, M406.4-2 and isolated from a lambda gtl 1 cDNA library constructed from a human stool sample collected during NANBH in Mexico. Two other similar epitopes, HEV 3-2(B) and 4-2(B), were obtained by polymerase chain reaction with HEV Burma(B)-derived primers followed by cloning into the expression vector lambda gtl 1. These four antigens of HEV were expressed in the pGEX vector system as fusions with the C terminus of Sj26, and 26-kDa glutathione-S-transferase protein. The four antigens are known to react

Diagnosis of hepatitis E virus

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specifically with acute-phase and convalescent phase sera from hepatitis E virus infected patients and with sera from experimentally infected cynomolgus macaques. The described ELISA reported by these authors detects both anti-HEV IgM and anti-HEV IgG and is recommended as convenient method for the diagnosis of acute or past hepatitis E virus infection.

22

Hepatitis C and hepatitis E: prevention, prophylaxis and treatment I. Prevention and prophylaxis Control measures for enterically transmitted hepatitis E virus infection (ETNANBH) would essentially be similar to those of hepatitis A virus (HAV) and presumably include the requisite of clean water, sanitary disposal of human excreta and an improvement in personal as well as food hygiene. It has been reported that passive immunization with immunoglobulin prepared from healthy donors originating in countries infected by the disease may protect vulnerable groups - particularly pregnant women. Parenterally transmitted/transfusion-associated hepatitis C virus infection (PT-NANBH) is known to result in chronic liver disease and possibly even hepatocellular carcinoma. In the absence of serological markers, preventive measures have centered on the use of "surrogate" markers in screening blood and blood products and viral inactivation. In recent years, surrogate testing for NANBH in the United States has been shown to be successful in achieving a reduction in the total number of transfusion associated hepatitis cases. It is anticipated that an implementation of screening blood donors for hepatitis C virus (transfusion-associated NANBH) antibody would definitely result in a further reduction. Various methods of inactivating NANBH viruses, in particular the parenterally transmitted HCV, such as heat treatment, lipid solvents and photochemical decontamination have been investigated. A report (Study Group, UK Hemophilia Centre, 1988) indicates severe dry heating at 80°C as being highly effective in activating NANBH virus in coagulation factor concentrates. Serial measurement of serum aminotransferase levels for 4 months revealed no patterns of rises attributable to NANBH in the thirty-two patients (coagulation factor deficient) investigated. According to the authors, this severe dry heating methods appear to have reduced the risk ofNANBH transmission from about 90%. No evidence of infection with hepatitis B virus or human immunodeficiencyvirus (HIV) was obvious in these recipients. A combination of specific assays for HBV, HIV, HCV and viral inactivation would thus increase and further ensure the safety of the blood supply (Alter, 1988).

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The effectiveness of immune globulin (IG), also known as immunoglobulin or immune serum globulin (ISG) in the prophylaxis of transfusionassociated hepatitis C virus infection remains as yet to be firmly established. A recent study found ISG effective in preventing hepatitis C (TA-NANBH), however, more investigations are required (Sanchez- Quijano et al., 1988). In this five-year study, Spanish heart surgery patients were assigned to either a control group of to a group that received IG. The IG was commercially available in 16% solution obtained from donors and given in a dose of 10 ml a day prior to and a week after surgery. Transfusion-associated hepatitis C virus infection was significantly more common in the control than in the IG group. Although more cases of hepatitis B virus infection occurred in the control than in the IG group, the difference was not statistically significant. In contrast, transfusion-associated hepatitis C appeared to be statistically less significant in the IG group and the incubation periods were noted to be shorter and the peak aminotransferase levels lower in the few recipients ofIG with hepatitis than in the controls with hepatitis. The authors do suggest that IG could serve as an inexpensive form of immunoprophylaxis against TANANBH. It appears, however, that the efficacy of such passive prophylaxis cannot be properly assessed until serological markers are available for diagnosing infection and the antibody in the immunoglobulin measured. Prospects of a vaccine remain dependent on the isolation and characterization of the virus( es) causing transfusion-associated NANB hepatitis. Recently, the isolation of hepatitis C virus, one of the agents of post-transfusion associated NANBH has been reported (Choo et al., 1989), and this may very well be the stepping stone towards a possible vaccine. In this study by Choo and colleagues, a radom-primed complimentary DNA library was constructed from plasma containing the uncharacterized NANBH agent and screened with serum from a patient diagnosed with NANBH. A complimentary DNA clone was isolated that was shown to encode an antigen associated specifically with NANB hepatitis infections. The clone was not derived from host DNA but from an RNA molecule present in NANB hepatitis infections consisting of at least 10,000 nucleotides and positive-stranded with respect to the encoded NANB hepatitis antigen. Their data appeared to be consistent with the clone being derived from the genome ofNANB hepatitis agent, the hepatitis C virus.

II. Treatment of hepatitis C Hepatitis C virus infection has a marked propensity to progress to chronic liver disease and in about 60%-70% of cases of post-transfusion NANB hepatitis and the patients have been noted to have elevations in serum aminotransferase levels for more periods beyond a year (Aach et al., 1981; Alter et al., 1981). Despite the fact, many cases of chronic hepatitis C turn out to be asymptomatic and mild about 15%-25% does however, lead to pronounced liver injury with an insidious progression to cirrhosis, portal hypertension and hepatic failure (Berman et al., 1979, Koretz et al., 1980). There

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Hepatitis C and hepatitis E: prevention, prophylaxis and treatment

is currently no established therapy for chronic hepatitis C. Corticosteroids have yielded disappointing results and do not appear to be beneficial (Alter and Hoofnagle, 1984). Acyclovir has likewise produced no apparent shortterm or long-term benefits (Pappas et al., 1985). The availability of highly purified and potent preparations of recombinant human interferon-alpha has however, allowed trials of this antiviral and immunomodulatory agent in the treatment of chronic hepatitis C. Both recombinant interferon alpha and human lymphoblastoid interferon have indeed been shown to be effective in chronic hepatitis B infection (Thomas and Scully, 1985) and delta hepatitis (Hoofnagle et al., 1985). In one of the pilot studies for the evaluation ofrecombinant human IFNalpha as a potential antiviral agent for hepatitis C, Hoofnagle et al. (1986) reported eight patients responding with a fall of serum aminotransferase (ALT) activities to normal or near normal; 2 patients relapsed after stopping therapy but responded after its resumption. Liver biopsy specimens obtained after one year of therapy demonstrated marked improvement in hepatic histology, even with the use of low doses of IFN-alpha. Although not adequately controlled, the overall response was regarded as very encouraging. In the second study reported by Thomson et al. ( 1987) all three treated patients had a similar complete biochemical remission during treatment. This study just like the other one also happended to be not adequately controlled. Since wide fluctuations in serum ALT activities occur in chronic hepatitis C, controlled studies would be worthwhile improvement. A prospective randomized controlled parallel group study of active treatment with low dose inferferon alpha (human lymphoblastoid interferon) to determine its effect on ALT activities in chronic hepatitis C virus infection versus no treatment has been reported Qacyna et al., 1989). Though all fourteen treated patients showed a reduction in aspartate aminotransferase activities, in two patients values did not return to normal. Whether the failure of treatment in these two cases was related to the type of NANB hepatitis or misdiagnosis of chronic hepatitis C or even a reflection of individual difference in response to interferon was not obvious. This nevertheless happens to be the first controlled study that shows that IFN-alpha could rapidly return aspartate aminotransferase activity to normal in most patients with chronic hepatitis C. Besides confirming the earlier pilot studies by Hoofnagle et al. (1986) and Thomas et al.(1987) it being a controlled study further solidifies the potentiality of IFN-alpha in treating diagnosed chronic hepatitis C virus infection. In another study the efficacy of interferons alpha and gamma was compared by Sayez-Royuela et al. (1991) and authors concluded that IFN-alpha therapy is effective in controlling chronic hepatitis C contrary to IFN-gamma. The long term response rate after IFN-alpha therapy was 30% at 18 months. High doses of IFN-alpha do not add further benefit in the response rate or relapse rate. However, Marcellin et al. (1991) found that higher dose of recombinant IFN-alpha (3MU) was superior to lMU dose given three times weekly for 24 weeks in inducing improvements in serum ALT levels and liver histological examinations in group of 60 patients with chronic hepatitis C.

References

197

A quite similar data were obtained by Causse et al. (1991) who found that 3MU ofIFN-alpha-2b is more effective dose than lMU for controlling disease activity in NANB chronic hepatitis patients. However, it should be underligned that interpheron alpha is not effective in all patients. Several previous studies demonstrated that only 20% to 25% of patients have had a sustained response to INF-alpha therapy (Di Bisceglie, 1991). If most of the patients tolerate well the IFN alpha treatment, several patients are subject to serious side effects like some degree of malaise, feverishness, muscle aches, poor appetite and concentration problems. Several patients can develop psychological side effects like anxiety, panic attacks, depression, suicidal thoughts and in some case a frank psychosis (Renault and Hoofnagle, 1989; Kurstak, 1991). Recently, Omata et al. (1991) have indicated that a eight-week course of interferon alpha induces biochemical and histological improvement in more than half the patients with chronic hepatitis C. Chayama et al., (1991) also found a therapeutic effect of IFN-alpha in patients with chronic hepatitis C. In 13 of16 patients receiving treatment, HCVRNA became undetectable and improvement in ALT levels in several patients was observed. In another study Boyer et al. ( 1992) assessed the response and tolerance of recombinant IFNalpha in patients with chronic hepatitis C and human immunodeficiency virus infection. They concluded that the response and tolerance were not different from those usually found in patients with chronic HCV infection without HIV infection and recommend the recombinant IFN-alpha for treatment of patients with such infections. Recently, also oral ribavirin therapy for chronic HCV infection has been evaluated in a pilot study with 10001200mg/dayin two devideddosesfor 12weeks (Reichard et al., 1991). These results offer a potentially effective treatment for chronic hepatitis C.

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Alter MJ, Hadler SC, Judson FN, Mares A, Alexander J, Hu PY, Miller JK, Moyer IA, Fields HA, Bradley DW, Margolis HS (1990) JAMA 264: 2231-2235 Alter MJ (1991) Hepatology 14: 389-391 Arankalle VA, TicehurstJ, Srenivasan MA, Kapikian AZ, Popper H, Pavri K, Purcell RH (1988) Lancetl:550-554 Bamber M, Murray A, Arborough BAM (1981) Gut 22: 854-857 Bassetti D, Cutrupi V, Dallago B, Alfonsi P (1991) Lancet 337: 912 Belabbes H, BenatallhaA, Bourgemouth A ( 1984) In: Vyas GN, DienstagJL, Hoofnagle JH (eds) Viral hepatitis and liver disease. Grune & Stratton, Florida, pp 637-639 Berman M, Alter HJ, Ishak HG, Purcell RA,Jones EA (1979) Ann Intern Med 91: 1-6 Bertolini E, Zermiani P, Battezzati PM, Bruno S, Villa E, Manneriti F, Marelli F, Moroni GA, Zuin M, Podda M (1991) Lancet 337: 675-676 Boyer N, Marcellin P, Degott C, Degos F, Saimot AG, Erlinger S, BenhamouJP (1992) J Infect Dis 165: 723--726 Bradley DW (1985a) J Virol MethodslO: 307-319 Bradley DW (1985b) Gastroenterology 88: 773--789 Bradley DW, Krawczynski K, Cook EH (1987) Proc Natl Acad Sci USA 84: 6277-6281 Bradley DW, Andjaparidze A, Cook EH (1988) J Gen Virol 69: 731-738 Brotman B (1989) J Med Virol 28: 13--15 Causse X, Godinot H, Chevallier M, Chossegros P, Zoulim F, Ouzan D, HeyraudJP, Fontages T, Albrecht], Meschievitz C, Trepo C (1991) Gastroenterology 101: 497-502 Centers for Disease Control (1987a) MMWR 36: 241-244 Centers for Disease Control (1987b) MMWR 36: 597-602 Centers for Disease Control (1988) MMWR 37: 110-114 Cha TA, Kolberg], Irvine B, Stempien M, Beall E, Yano M, Choo QL, Houghton M, Kuo G, Han JH, Urdea MS (1991) J Clin Microbiol 29: 2528-2534 Cha TA, Beall E, Irvine B, KolbergJA, Urdea MS (1992) Proc European HCV Council Group Meeting, Venice,July 1992 Chan SW, Holmes EC, McOmish F, Follett E, Yap PL, Simmonds P ( 1992) Proc European HCV Council Group Meeting, Venice, July 1992 Chayama K, Saitoh S, Arase Y, Ikeda K, Matsumoto T, Sakai Y, Kobayashi M, Unakami M, Morinaga T, Kumada H (1991) Hepatology 13(6): 1040-1043 Chen DS, Kuo GC, SiungJL, Lai MY, Sheu JC, Chen PJ, Yang PM, Hsu HM, Chang MH, Chen CJ, Hahn LC, Choo QL, WangJH, Houghton M (1990) J Infect Dis 162: 817-822 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M ( 1989) Science 244: 359-362 Choo QL, Richman KH, Han JM, Berger K, Lee C, Dong C, Gallegos C, Coit D, Medina-Selby A, Barr PJ, Weiner AJ, Bradley DW, Kuo G, Houghton M (1991) Proc Natl Acad Sci USA 88: 2451-2455 Chou WH, Yoneyama T, Takeuchi K, Harada H, Saito I, Miyamura T (1991) J Clin Microbial 29(12): 2860-2864 Chung RT, Kaplan LM (1992) Proc European HCV Council Group Meeting, Venice, July 1992 Clemens JM, Taskar S, Chau K, Vallari D, Shih JWK, Alter HJ, Schleicher JB, Mimms LT ( 1992) Blood 79(2): 169-172 Colombo M, Riumi MG, Romeo R, Sangiovanni A (1990) J Natl Cancer Inst 335: 609-610 Contreras M, BarbaraJAJ (1989) Lancet 11: 505 DeCock KM, Bradley DW, Sandford NL, Govindarajan S, MaynardJE, Redeker AG (1987) Ann Intern Med 106: 227-230 Degos F, Maisonneuve P, Thiers V, Noel L, Erlinger S, Brechot C, BenhamouJP (1991) Lancet 338: 758 Desai SM, Dailey SH, CaseyJM, Rupprecht KR, Lesniewski RR, Devare SG ( 1992) Proc European HCV Council Group Meeting, Venice, July 1992 DienstagJL (1983a) Gastroenterology 85: 439-462 DienstagJL (1983b) Gastroenterology 85: 743-768 DienstagJL, Alter HJ (1986) Semin Liver Dis 6: 67-81 Editorial (1990a) Lancet 335: 1431-1432 Editorial (1990b) Mayo Clin Proc 65: 1373--1376

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Editorial (1990c) Lancet 136: 1158-1160 EstebanJL, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, Roget M, Vargas V, Genesca I, Buti M (1989) Lancet 2: 294-297 Esteban JI, Gonzalez A, Hernandez JM, Viladomiu L, Sanchez C, Lopez-TalaveraJCL, Lucea D, Martin-Vega C, Vidal X, Esteban R, Guardia] (1990) N EnglJ Med 323: 1107-1110 Feinstone SIM, Kapikian AZ, Purcell RH, Alter HJ, Holland PV (1975) N EnglJ Med 292: 767-770 Feinstone SM, DienstagJL, HoofnagleJH (1984) N EnglJ Med 311: 185-189 Fuchs K, Motz M, Schrerer E, Zach oval R, Deinhardt F, Roggendorf M ( 1991) Gene 103: 163-169 Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara JAJ, Contreras M, Aloysius S (1990) Lancet 335: 1419-1422 Goldsmith R, Yarbough PO, Reyes GR, Fry KE, Gabor KA, Kamel M, Zakaria S, Amer S, Gaffar Y (1992) Lancet 339: 328-331 GraylJ, WrightTG, FriendPJ, WightDGD, Sundaresan V, Caline RY(l990) Lancet335: 609-610 Gust ID, Purcell RH (1987) J Infect Dis 156: 630-635 Hadler SC (1987) In: Evans AE (ed) Viral infections of humans. Williams and Wilkins, Baltimore, pp 110-116 Hagiwara H, Hayashi N, Mita E, Hiramatsu N, Ueda K, Takehara T, Yuki N, Kasahara A, Fusamoto H, Kamada T (1992) Gastroenterology 102: 692-694 Hess G (1989) Lancet 2: 987-988 Hijikata M, Kato N, Otsuyama Y, Nakagawa M, Shimotohno K (1991) Proc Natl Acad Sci USA 88: 5547-5551 Hoofnagle JH, Ponzetto A, Mathiessen LR, Waggoner JG, Bales ZB, Seeff LB ( 1985) Dig Dis Sci 30: 1022-1027 HoofnagleJH, Smedile A, Mullen KD (1985) Gastroenterology 88: 1665-1671 HoofnagleJH, Mullen DK, Jones BD, Rustgi V, Bisceglie AD, Peters M, Waggoner JG, Yoon K, Park RN, Jones EA (1986) N EnglJ Med 315: 1575-1578 Houghton M, Weiner A, HanJ, Kuo G, Choo QL (1991) Hepatology 14: 381-388 Ideo G, Bellati G, Pedraglio E, Buttelli R, Dunzelli T, Putignano S (1990) Lancet 335: 353 Ikeda Y, Toda G, Hashimoto N, Kunokawa K (1990) Lancet 335: 1345-1346 Jacyna MR, Brooks MG, Loke RHT, MainJ, Murray-Lyon IM, Thomas HC (1989) Br MedJ 298: 80-82 ' Kaklamani E, Trichopoulos D, Tzonou D, Tzonou A, Zavitsanos X, Koumantaki Y, Hatzakis A, Hsieh CC, Hadziyannis S (1991) JAMA 265: 1974-1976 Kane MA, Bradley DW, Shrestha SM, MaynardJE, Cook EH, Mishra RP,Joshi DD (1984) JAMA 252: 3140-3145 Kaneko S, Unoura M, Kobayashi K, Kunok, Murakami S, Hattori N (1990) Lancet 335: 976 Kato N, Yokosuka 0, Omata M, Hosoda K, Ohto M (1990) J Clin Invest 86: 1764-1767 Khuroo MS (1980) AmJ Med 68: 818-823 Khuroo MS, Teli MR, Skidmone S, Sofi MA, Khuroo ML (1981) J Med 70: 252-255 Kiyosawa K, Sodeyoma T, Tanaka E (1991) Gastroenterology 100 (4): 1145--1146 Koretz RL, Stone 0, Gitnick GL (1980) Gastroenterology 79: 893-898 Krawczynski K (1989) 23rd Meeting Association for Study of Liver, Leuven Kuhn! P, Seidl S, Stangel W, Beyer J, Sibrowski W, FlikJ (1989) Lancet ii: 324 Kuo G, Choo QL, AlterJH, Gitnick GL, Redeker AH, Purcell RH, Miyamura T, DienstagJL, Alter MJ, Stevens CE, Tegtmera GE, Bonino M, Colombo M, Lee WS,Juo S, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M (1989) Science 224: 362-364 Kurstak E (1991) Psychiatry and Biological Factors. Plenum Medical, New York, 1-311 LefkowichJH (1987) Ann Path Lab Med 111: 170-173 Lesniewski RR, Dessai SM, Johnson RG, Nelson LR, Schlauder GG, Sant CL, Mushahwar IK (1992) Proc European HCV Council Group Meeting, Venice, July 1992 Lettau L (1992) Inf Control Hosp Epidemiol 13 (2): 77-81 Lucey M, Traber PG (1991) Hepatology 13: 193-195 Marcellin P, Boyer N, Giostra E, Degott C, Courouce AM, Degas F, Coppere H, Cales P, Couzigou P, BenhamouJP (1991) Hepatology 3: 393-397 Martinot-Peignoux M, Marcellin P, Xu LZ, BernuauJ, Edinger S, BenhamouJP, Larzul D ( 1992) J Infect Dis 165: 595-596

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Mattson L, Weiland 0, Glaumann H (1988) Liver 8: 184-188 Meisel H, Reimer K, Reip A, Pelzer C, Klarmann R, Troonen H, Lennartz L (1992) Proc European HCV Council Group Meeting, Venice,July 1992 Meng ZD, Sun YD, Chen XR, Wang SY, Sun DG, Chen Z, Liu CB, Zhuang H, Xu ZY (1990) In: Proc 2nd Intern Symp Viral Dis. Beijing, p 34 Mortimer PP (1989) Lancet 2: 798-799 Myint H, Soe MM, Khin T, Myint TM, Tin KM (1986) AmJ Trop Med Hyg 34: 1183-1189 Novati R Thiers V, d'Arminio Monforte A, Maisonneuve P, Principi N, Conti M, Lazzarin A, Brechot C (1992) J Infect Dis 165: 720-723 Okamoto H, Tsuda F, Machida A, Minekata E, Akahane Y, Sugai Y, Mashiko K, Mitsui T, Tanaka T, Miyakawa Y, Mayumi M (1992) Hepatology 15: 180-186 Omata M, Ito Y, Yokosuka 0, Imazeki F, Tagawa M, Takano S, Hosoda K, Tada M, Ohto M, Ito K, Okuda K (1991) Dig Dis Sci 36: 1217-1222 Overby LR (1990) In: Hepatitis CVirus. Proc Symposium, Can Dis Wkly Rep., Vol 17 S5, pp 3539 Pappas SG, HoofnagleJH, Young N, Straus SE, Jones EA (1985) J Med Virol 15: 1-9 Pascual M, Perrin L, Glostra E, SchifferliJA (1990) J Infect Dis 162: 569-570 Poterucha IJ, RakelaJ, Lumeng L, Lee CH, Taswell HF, Wiesner RH (1992) Hepatology 15(1): 42-45 Prince AM, Brotman B, Huima T, Pascual D,Jaffery M, Inchauspe G (1992) J Infect Dis 165: 438443 Purcell RH, TicehurstJR (1988) In: Zuckerman AJ (ed) Viral hepatitis and liver disease. Alan R Liss, New York, pp 131-137 Ramalingaswami V, Purcell RH (1988) Lancet i: 871-873 RayR,AggarwalR, SalunkeDN, MehrotraNN, TalwarGP, Naik SR (1991) Lancet338: 783-784 Realdi G, Alberti A, Rugge M ( 1982) Gut 23: 270-275 Reichard 0, Andersson], Schvarcz R, Wieland 0 (1991) Lancet 337: 1058-1061 Renault PF, HoofnagleJH (1989) Semin Liv Dis 9: 273-277 Reyes GR, Purdy MA, KimJP (1990) Science 247: 1335-1339 Roggendorf M, Deinhardt F, Rasshoper R, Eberle J, Joph U, Moller B, Zachoval R, Rape G, Schramm W, Rommer F (1989) Lancet 2: 324-325 Saeed AA, Al Rasheed AM, Rankin D, Kaiser P, Sickinger E, Troonen H (1992) Proc European HCV Council Group Meeting, Venice, July 1992 Saez-Royuela F, Porres JC, Moreno A, Castillo I, Martiner G, Galiana F, Carreno V (1991) Hepatology 13: 327-331 Sakamoto M (1988) Cancer Res 48: 7294-7297 Sampliner RF, Woronow DF, Alter MJ (1984) J Med Virol 13: 125-130 Sanchez-Pescador R, Sheridan PJ, DetmerlJ, Hunt WP, Chan CS, WilberJC, Neuwald PD, Urdea MS (1992) Proc European HCV Council Group Meeting, Venice,July 1992 Sanchez-Quijano A, PinedaJA, Lissen E (1988) Lancet 1: 1245-1249 Saracco G (1988) Ann Intern Med 108: 380-383 Schueuer PJ, Ashrafzadeh P, Sherlock S, Brown D, Dusheiki GM (1992) Hepatology 15: 567-571 SeeffLB, Wright EC, Zimmerman HJ (1975) AmJ Med Sci 270: 355-362 Skidmore S (1990) Lancet 335: 13-16 Sniki RK, Scarf S, Faloona F ( 1985) Science 230: 1350-1354 Stevens CE, Taylor PE, PindyckJ, Choo QL, Bradley DW, Kuo G, Houghton M (1990) JAMA263: 49-53 Study Group UK Hemophilia Centre (1988) Lancet 1: 814-816 Takayasu D, Nobuo T, Shujiro T, Akira T (1992) Proc European HCV Council Group Meeting, Venice,July 1992 Tanaka K, Hirohata T, Koga S, Sugimachi K, Kanematsu T, Ohryohji F, Nawata H, Ishibachi H, Maeda Y, Kiyokawa H (1991) Cancer Res 51: 2842-2847 Tandon BN,Joshi YK,Jain SK, Gandhi BM, Mathiesen LR, Tandon HD (1982) IndianJ Med Res 75:739-741 Thaler MM, Park CK, Landers DV, Wara DW, Houghton M, Wauters GV, Sweet RL, Han JH (1991) Lancet338: 17

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PartV Different forms of viral hepatitis

23 Differentiation between viral hepatitis I. Introduction Hepatitis is a general term for inflammation of the liver due to a variety of causes, for example: several different viruses, bacteria, toxins, drugs or excessive intake of alcohol. Only in recent years the viral etiology of hepatitis has been established. Viral hepatitis still remains to be a global problem. There are at least four major types of hepatitis caused by different hepatitis viruses. These are hepatitis A (infectious hepatitis), hepatitis B (serum hepatitis), hepatitis D (delta hepatitis) and hepatitis C (non-A, non-B hepatitis).

II. Hepatitis viruses Hepatitis A (HAV) is single stranded linear RNA enterovirus, 27-32 nm in diameter, a member of the Picornaviridae family while hepatitis B (HBV) is a double stranded circular DNA virus, 42nm in diameter, and a member of the Hepadnaviridae (Blumberg et al., 1970; Melnick, 1988; Kurstak, 1992). Hepatitis Bis responsible for the most serious form of hepatitis and can cause a spectrum of inflammatory liver diseases ranging from acute to chronic hepatitis, cirrhosis and primary liver cancer. It is estimated that currently there are 350 million carriers ofHBV worldwide (WHO Press Release, 1992). Apart from hepatitis A and hepatitis B viruses, several other virologically and immunologically identified agents exist including the delta agent or hepatitis D virus (HDV), a defective, single-stranded circular RNA virus, 35nm-37nm in diameter, requiring the presence ofHBV fodts replication and expression (Rizzetto et al., 1977; Smedile et al., 1991). Clinical and epidemiologic studies suggest that more than one infectious agent is responsible for non-A, non-B hepatitis (NANBH), so named because of the absence of serologic markers to hepatitis A or hepatitis B in clinically diagnosed cases of hepatitis A virus. Associated with the parenteral transmission ofNANBH it has been recently classified as hepatitis C virus (HCV), most probably, 30nm-

15-45 days (mean 30 days)

Acute (usually)

10%

Incubation period

Onset

Jaundice

Food/water borne

Other routes

Mortality

0.1%-0.2%

No

Not yet known

Carrier

Hepatitis

Sequelae

Usually

Rare

Oral (fecal)

Parenteral

Transmission

Single-stranded RNA enterovirus, 27-32nm in diameter (Picomaviridae)

Virus

Hepatitis A

0.5%-2.0% in uncomplicated cases

Chronic viral he pa ti tis (important cause)

5%-10%

Sexual contact, transfer through fluids to mucosa! surfaces or under skin, perinatal

Usually

No

20%

Insidious (usually)

30-120 days (60-110 days usually)

Double-stranded DNA virus, 42nm in diameter (Hepadnaviridae)

Hepatitis B

30% in chronic cases

Chronic viral hepatitis (can cause)

Yes

Sexual contact less efficient than for HBV

Usually

No

Variable

Acute (usually)

21-90 days (overlaps that of HB usually)

Single-stranted circular RNA virug, 35-37nm in diameter, defective and HBV dependent forreplication and expression

Hepatitis D

1 %-2% in uncomplicated cases

Chronic viral hepatitis (important cause)

Around 50%

Perinatal

Usually (transmission associated 90% of cases)

No

25%

Non-specific symptoms followed by jaundice

1-5 months (intermediate to those of HA and HB)

Single-stranded RNA virus, 30-38nm in diameter, small lOkb, (Flavivirus)

Hepatitis C

Non-A, non-B hepatitis

Table 9. Some aspects ofvirological, clinical and epidemiological differentiation between viral hepatitis types

1%-2% in general, higher in pregnant cases

Not yet known

No observation

Food or water borne-transmission in developing countries

No observation

Usually

No observation

Acute (usually)

22-60 days (mean 15-40 days)

Non-enveloped spherical, single-stranded RNA virus, (Calicivirus)

Hepatitis E

Clinical features of viral hepatitis

207

38 nm in diameter, and yet a form of hepatitis spread enterically and previously known as epidemic or water-borne NANBH is presently termed hepatitis E (HEV) virus infection (Bradley et al., 1987; Choo et al., 1989; Ray et al., 1991). The characteristic of the different hepatitis viruses are summarized in Table 9 and also shown are some aspects of the clinical and epidemiologic differentiation.

ID. Clinical features of viral hepatitis Irrespective of the cause, the clinical symptoms of acute viral hepatitis are similar. Classic, symptomatic hepatitis includes jaundice, fever and abdominal pain. Some patients tend to have only mild flu-like symptoms. Generally, only minor differences are noticeable between hepatitis infections due to the different hepatitis viruses.

Hepatitis A About a third of patients infected with hepatitis A virus (HAV) have mild symptoms and in the presence of symptoms the onset is abrupt. Around 10% of symptomatic patients develop jaundice. Hepatitis B About one-half of patients infected with hepatitis B virus (HBV) show symptoms. The onset of symptoms tends to be slow unlike the sudden onset of hepatitis A. Symptoms last 1-4 weeks and normally after an HBV infection usually after 6 months. About 20% of patients with symptomatic HBV develop jaundice. HepatitisD Symptoms of hepatitis D virus infection are in fact similar to that of hepatitis B and thus a differentiation is difficult. In the case of a chronic HBV carrier co-infected with HDV, the symptoms appear to be rather severe compared to those with hepatitis B virus infection alone. HDV is highly pathogenic but requires HBV for its own replication. Hepatitis C Symptoms caused by parenteral hepatitis C virus (HCV) infection tend to be similar to those associated with hepatitis B. The clinical cause oftransfusionassociated hepatitis C is less severe than that of hepatitis B. HepatitisE Symptoms caused by hepatitis E virus (HEV) infection appear to be similar to those due to hepatitis A. Characteristic feature of hepatitis E has been noted in its high mortality rate in infected pregnant women and as one of the leading causes of acute viral hepatitis in young to middle-aged adults in developing countries (Alter, 1989). No reports of chronic liver disease with hepatitis E yet exist in literature.

208

Differentiation between viral hepatitis

IV. Epidemiology of viral hepatitis Hepatitis caused by viruses in all forms pose a serious public health problem worldwide. Hepatitis B virus infection is endemic with a high prevalence in Africa, Eastern Europe, the Mediterranean basin, Middle East, Asia and in certain regions of South America. Hepatitis D virus infection is detected only in areas with high hepatitis B virus prevalence. The incidence of hepatitis A virus has been noted worldwide with prevalence varying as a function of increasing age and decreasing socio-economic status. Hepatitis E virus infection (NANBH) likewise appears as epidemic in areas of overcrowded living conditions,unsafe and shared water. Recent epidemiologic date indicates evidence of prior infection with hepatitis C virus in 0.2%-1.5% of blood donors in United States, Europe and Japan in high prevalence rates in high risk groups intravenous drug users (Kuo et al., 1989; Esteban et al., 1990). In developing countries it is the most common form of post-transfusion hepatitis and may account for more than 90% of cases. There is a strong association between the presence of antibody to HCV (anti-HCV) and primary hepatocellular carcinoma (PHC). In fact, recent evidence does highlight that HCV infection does have an interactive role in the origin of PHC (Kaklamani et al., 1991). Hepatitis A and NANB hepatitis E viruses are known to be transmitted by the oral-fecal route. The viruses in such cases are excreted in the feces and possibly transmitted to others through sub-standard personal hygiene, oralanal sexual contact or sharing of cooking utensils. Hepatitis B, hepatitis D and NANB hepatitis C viruses are transmitted via parenteral routes through exposure to contaminated blood, blood products, particularly through transfusion of infected blood or sharing of needles by drug abusers. Sexual transmission is also a major mode of spread of hepatitis B virus. Perinatal transmission ofHBV is of importance in hepatitis B. Recent evidence suggests a similar situation with regard to hepatitis C virus (Degos et al., 1991; Thaler et al., 1991).

V. Diagnosis of viral hepatitis The clinical features of acute hepatitis are determined from the patient's history. Physical examination and elevated liver enzymes do not appear to be adequate since different types of hepatitis share too many similar characteristics and symptoms. Specific serologic tests with a variety of markers are used to confirm the type of viral hepatitis. A carefully detailed patient history may give clues to the possible differential diagnosis: occupation (workers with organic solvents liable to toxic hepatitis sewage, workers liable to leptospirosis), sexual bias (homosexuals liable to hepatitis A and hepatitis B), medications (methylidopa and isoniazid causing hepatitis), blood transfusions, intravenous drug abuse (hepatitis B, NANBH) and alcohol consumption. Recent foreign travel to areas of endemic hepatitis should make one suspicious of HAV, HBV, HCV, HEV or yellow fever virus infection. Clinical

References

209

examinations rarely are helpful in distinguishing between the causes of acute hepatitis. Laboratory tests appear to be the most useful diagnostic tools. An inclusion of total blood count would be helpful since white blood cell count is raised in toxic and leptospiral hepatitis and also urea being raised in leptospiral as well as in toxic hepatitis. Hepatic biochemistry would show greatly raised aminotransferases in acute hepatitis with a raised bilirubin but would be not helpful in distinguishing the causes. Acute hepatitis A virus infection is diagnosed mainly by the presence of IgM to HAV and hepatitis B virus by detection of IgM to hepatitis B core antigen. Acute NANBH previously diagnosed only by exclusion of hepatitis A or B are now detectable using recombinant HCV antigens in enzyme immunoassay. IgM anti-HCV can now be detected by a modified immunoassay for anti-HCV (Quiroga et al., 1991). Hepatitis E virus is detected by means of reverse transcription polymerase chain reaction (Ray et al., 1991). Test for IgM anti-cytomegalovirus (CMV) and antibodies to the Epstein-Barr virus (EBV) should preferably be performed so to exclude CMV and EBV hepatitis. An auto-antibody profile would exclude auto-immune lupoid hepatitis in which the anti-nuclear and anti-smooth muscle antibodies are usually raised.

References Alexander GJM, Williams R (1988) AmJ Med 85 (Suppl 2A): 143-146 Blumberg BS, SutnickAI, London WT (1970) AmJ Med 48: 1-8 Bradley DW, Krawczynski K, Cook EH (1987) Proc Natl Acad Sci USA 84: 6277-6281 Carman WF, Zanetti AR, Karayiannis P, Waters], Manzillo G, Tanzi E, Zuckerman AJ, Thomas HC (1990) Lancet 336: 325-329 Caselman \], EisenburgJ, Hofschneider PM, Kishy RI (1989) Gastroenterology 96: 449-455 Choo QL, Kuo G, Winger AJ, Overby LR, Bradley DW, Houghton M (1989) Science 244: 359362 Degos F, Maisonneuve P, Thiers V, Noel L, Erlinger S, Brechot C, BenhamouJP (1991) Lancet 338:758 Dindzans \],Cai MY, Levy GA (1985) Med North Am 21: 2770-2779 Esteban JI, Gonzalez A, Hernandez JM, Viladomiu L, Sanchez C, Lopez-TalaveraJCL, Lucea D, Martin-Vega C, Vidal X, Esteban R, Guardia] (1990) N EnglJ Med 323: 1107-1110 HenriettaJH, Lelie PN, Wong VCW, Kuhns MC, Reesink HK ( 1989) Lancet i(8635): 406-409 Hollinger FB, Lemon SM, Margolis H (1991) Viral hepatitis and liver disease. Williams and Wilkins, Baltimore Hoofnagle JH, Alter HI ( 1984) In: Vyas GN, DienstagJL, Hoofnagle JH (eds) Viral hepatitis and liver disease. Grune & Stratton, Orlando, pp 97-113 HoofnagleJH (1990) N EnglJ Med 323: 337-339 HsuHC, Wu TT,SheuJC, Wu Cy, Chiou TJ,LeeCS,ChenDS (1989) Hepatology9 (5): 747-750 Kaklamani E, Trichopoulos D, Tzonou A, Zavitsanos X, Koumantaki Y, Hatzakis A, Hsieh CC, Hadziyannis S (1991) JAMA 265: 1974-1976 Krugman S (1985) In: Gerety RJ (ed) Hepatitis B. Academic Press, Orlando, pp 1-4 Kuo G, Choo QL, AlterJH, Getnick GL, Redeker AH, Purcell RH, Nikamura T, DienstagJL, Alter MJ, Stevens CE, Tegtrnera GE, Bonino M, Colombo M, Lee WS,Juo S, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M (1989) Science 249: 362-364 Kurstak E (1992) Control of virus diseases, 2nd edn. Marcel Deckker, New York, 1-448

210

Differentiation between viral hepatitis

Mazella G, Rizzetto M, Amed MA, Quiritela G, Rosina F, Barbara L, Roda E ( 1988) Am J Med 85 (Suppl 2A): 141-142 McLachlan A (1991) Molecular biology of the hepatitis B virus. CRC Press, Boca Raton, 1-312 MelnickJL ( 1988) In: Kurstak E (ed in chief) Applied virology research, vol 1: New vaccines and chemotherapy. Plenum Medical, New York, pp 1-14 QuirogaJA, Campillo ML, Catillo I, Bartolome], PorressJC, Carreno V (1991) Hepatology 14: 38-43 Ray R, Aggarwal R, Salunke DN, Mehrota NN, Talwar GP, Naik SR (1991) Lancet 338: 783-784 Rizzetto M, Canese MG, Arico S, Crivelli 0, Trepo CG, Bonino F, Verme G (1977) Gut 18: 9971003

Smedile A, Rosina F, Chiaberge E, Lattore V, Saracco G, Brunetto MR, Bonino F, Vermo G, Rizzetto M (1991) Prog Clin Biol Res 364: 185--195 Thaler MM., Park CK, Landers DV, Wara DW, Houghton M, Wauters GV, Sweet RL, Han JH (1991) Lancet 338: 17 World Health Organization (1992) Press release WH0/12, February 21 Zuckerman AJ (ed) (1988) Viral hepatitis and liver disease. Alan R Liss, New York, 1-1136

Subject index

Active immunization in control and prevention of HAV infection 48-52 inactivated vaccines 48-50 live attenuated vaccines 50-51 other strategies for vaccine development 51-52 in control and prevention of HBV infection 130-132 administration route 131 adverse reactions 131 antibody persistence and booster doses/ reactivation 131-132 dosages and schedules 131 indications 130 safety of the vaccines 130-131 Acute HAV infection 207 Acute HBV infection 69-74 clinical pathology 71-72 clinical presentation 69-71 icteric phase of infection 71 prodromal phase of infection 70 different clinical forms 72-74 cholestatic-type form 73 fulminant infection 73-74 inapparent and anicteric forms 72-73 serology 96--97 treatment 119-120 of diet 120 of drugs 120 of restricted activity 119-120 Acyclovir 121-122 Adenine arabinoside (vidarabine) 121-123 combined with corticosteroids 126

Age, incidence ofHAVand 25, 40 Anicteric form of acute HBV 72-73 Anorexia 70 Antibody test for hepatitis C virus (antiHCV) 1-2 Antigens of HBV 62-63 Anti-HBc (serological marker of HBV) 94 detection of 101-102 possible interpretation of 99 Anti-HBe (serological marker of HBV) 94 detection of 102-103 possible interpretation of 99 significance of 98 Anti-HBs (serological marker of HBV) 94 detection of 100-101 possible interpretation of 99 significance of 98 Anti-HBx (serological marker of HBV) 98 Anti-idiotype antibody as prospective HBVvaccine 110, 135-136 Anti-Pre Sl (serological marker of HBV) 94 Anti-Pre S2 (serological marker ofHBV) 94 Arthralgia 25, 70 Asymptomatic cases of HAV infection 26--27 Blood transfusion of blood products, transmission of HBV via 86--87 Bouyant density ofHAV 16-17

212 Carrier state of HBV 90-92 definition, duration and prognosis 90 epidemiological aspects or carrier state and management 91-92 markers of virus infectivity and replication 90-91 Cellular immunity, HBVand 108-109 Chemically synthesized HBV vaccines 135 Children horizontal transmission of HBV and 89-90 polyphasic course of HAV infection in 27 transmission of HBV from mothers to infant 87-89 Chills 25 Cholestatic-type form of acute HBV 73 Chronic active hepatitis (CAH) 32 Chronic HBV infection 75-82 chronic active hepatitis B 77-80 causes 77 clinical picture of infection and diagnosis 78-79 treatment and prognosis 79-80 chronic persistent hepatitis B 76-77 clinical course of infection and diagnosis 76 prognosis and management of treatment 76-77 in special high risk patients 80-82 hemodialysis and renal transplant patients 80-81 hemophiliacs 81 homosexuals and drug addicts 81-82 interactions between HIV-I, HDV and HBV infections in chronic carriers of HBV 82 serology of 97-98 therapeutic approaches to 120-123 acyclovir 122 adenine arabinoside 122-123 suramin 121-122 three levels of response 121 thymosins/prostaglandins 123 three clinical syndromes of 75 Chronic reinfection ofHAV 40-41 Combined hepatitis A and B virus vaccine 136

Subject index Constipation 70 Corticosteroids 126 Cytomegalovirus ( CMV) 207 Day-care centers, HAV transmission in 40,43 Delta hepatitis s. Hepatitis D virus (HDV) Diagnostic techniques for HAV 33-37 current trends in molecular biotechniques 36-37 immune electron microscopy 3334 rapid viral diagnosis 34-36 for HDV 163-168 enzyme-immunoassays 163-165 immunoblot technique 166-167 immunofluorescence and immuno-peroxidase techniques 165-166 molecular hybridization assays 167-168 radio-immunoassay 163 forHEV 192 Diagnosis of viral hepatitis 206-207 Diarrhea 25, 70 Diet in treatment of acute HBV 120 DNA HBV DNA (serological marker) 4, 105-106 detection of 103-104 recombinant DNA vaccines for HBV 133 Drugs s. also Intravenous drug abuse in acute HBV therapy 120 in chronic HBV therapy 120-123 acyclovir 122 adenine arabinoside 122-123 suramin 121-122 thymosins/prostaglandins 123 drugs combination therapy in HBV 125-126 Enteric NANBH (ET-NANBH) s. Hepatitis E virus (HEV) Enzyme-immunoassay (EIA) for HAV detection 35-36 for HDV detection 163-165

Subject index Enzyme-linked immunosorbent assay (ELISA) 6, 192 for HAV detection 37 Epidemic hepatitis 1 Epidemic jaundic 28 Epidemiology 206--208 ofHAV 38-41, 206 age incidence 25, 40 inapparent and chronic reinfection 40-41 seasonal and geographic variation 39-40 of HBV 83-86, 206 risk factors 84 worldwide prevalence 84-86 ofHCV 181-183, 206 ofHDV 156-158, 206 ofHEV 181-183, 206 Epstein-Barr virus (EBV) 209 Familial incidence of primary hepatocellular carcinoma and HBV 114-115 Fecal contamination of drinking water and food 41-42 Fever 25 Flue-like symptoms 70 Food contamination with feces 41-42 Foreign travelers, HAV transmission among 40, 43, 206 Foscarnet (trisodium phosphonoformate) 120 Fulminant hepatitis 27, 73-74 Genomes ofHAV 18--20 ofHBV 5, 64-67 ofHDV 6 Geographic distribution association between primary hepatocellular carcinoma and HBV 114-115 HAV infection 39-40 HBsAg 85 Glutamate dehydrogenase (GLDH) 72 HBcAg (serological marker of HBV) 101 HBeAg (serologicalmarkerofHBV) 94 detection of 102-103

213 possible interpretation of 99 significance of 98 HBsAg (serological marker ofHBV) 94 detection of 99-100 monoclonal antibody to 126-127 possible interpretation of 99 significance of 98 HBV DNA (serological marker ofHBV) 94 detection of 103-104 HBxAg (serological marker ofHBV) 98 Headache 25 Hemodialysis patients, chronic HBV in 80-81 Hemophiliacs, chronic HBV in 81 Hepatitis, definition of 1 Hepatitis A virus (HAV) 1, 204 active immunization 48--52 inactivated vaccines 48-50 live attenuated vaccine 50-51 other strategies for vaccine development 51-52 asymptomatic and fulminant infections 26--27 clinical features 24-25, 206--207 combined hepatitis A and B virus vaccine 136 diagnosis 208-209 diagnostic tests for 33-37 current trends in molecular biotechniques 36--37 immune electron microscopy 33-34 rapid viral diagnosis 34-36 epidemiologic characteristics of infection 38-41, 206--208 age incidence 25, 40 inapparent and chronic reinfection 40-41 seasonal and geographic variation 39-40 immunopathogenesis 30-32 incubation period and transmission 26,41-43 infection incubation period 26 measures for prevention of virus transmission in hospitals 45-46 nature of the virus particle 15-18 morphology and size 15-16

214 reactivity of physical and chemical agents 17-18 sedimentation coefficient and buoyant density 16-17 passive immunoprophylaxis 46-48 pathologic features of the disease 28-29 polyphasic course ofinfection in children 27 RNA molecule of 18-20 specific risk factors associated with 40,43 virological, clinical and epidemiological characteristic of 206 virologic events of infection 29-30 virus genome and structural proteins 18-21 virus replication cycle 22-23 Hepatitis B immunoglobulin (HBIG) 129-130 Hepatitis B virus (HBV) 1, 205 s. also Acute HBV infection; Chronic HBV infection clinical features 205-207 control and prevention of infection 128-147 active immunization 130-132 HBVvaccines 132-136 passive immunization 129-130 universal HBV vaccination 136137 diagnosis of 206-207 drugs combination therapy for 125126 epidemiology and characteristics of infection 83-86, 208 risk factors 84 worldwide prevalence 84-86 genome of 5, 64-67 immunology 105-110 anti-idiotypic response 109-110 cellular immunity 108-109 humoral immunity 107-108 live antigens 106-107 interferons in treatment of 7-8, 123-125 interleukin-2 therapy 127 monoclonal antibody to HBsAg 126-127

Subject index preventive immunization for 7-8 primary hepatocellular carcinoma (PHC) and 111-118 clinical aspects 112 geographical localization and familial incidence 114-115 molecular biology perspectives 116-118 pathological aspects 112-114 serological association of HBV markers and PHC 115-116 serological markers in course of infection 93-95 subtypes of infectious virus 68 techniques for virus marker detection 99-104 detection of anti-HBc 101-102 detection of anti-HBs 100-101 detection ofHBcAg 101 detection ofHBeAg and anti-HBe 102-104 detection of HBsAg 99-100 detection ofHBVDNA 103-104 transmission of 86-90 horizontal transmission 89-90 incubation period and mode of transmission 86-87 sexual route and transmission through fluids 87 vertical transmission, 87-89 vaccines for 9-10, 132-136 anti-idiotype antibody as a prospective vaccine 135-136 chemically synthesized vaccines 135 combined hepatitis A and B virus vaccine 136 hybrid vaccinia virus vaccines 133-134 polypeptide vaccines 133 recombinant DNA vaccines 133 safety of the vaccines 130-132 universal vaccination program 136-137 vaccines using hybrid particles 134-135 variants of 2 virological, clinical, and epidemiological characteristics of 206 virus carrier state 90-92

Subject index definition, duration and prognosis 90 epidemiological aspects of carrier state and management 91-92 markers of virus infectivity and replication 90-91 virus particles and genomes 61-67 virus genomic organization 64-67 virus structure and antigens 62-63 Hepatitis C virus (HCV) 1-3, 6, 8-9, 175,205-209 clinical features 178-180, 207 diagnosis 185-193, 208-209 antibody assays and seroprevalence 186--190 detection ofHCV 190-193 epidemiology and distribution of infection 181-183, 206--208 genome of 6 incubation period and mode of transmission 183-184 nomenclature 176-177 prevention and prophylaxis 194-195 RNA of 6-7, 8-9 treatmentof 195-197 virological, clinical, and epidemiological characteristics of 206 Hepatitis D virus (HDV) 1-3, 5-6, 205 characteristics and replication 151153 clinical causes ofinfection 153-155, 207 diagnosis of 160-1 71 diagnostic techniques 163-168 serological identification 161-162 epidemiology and virus distribution 156-158,206--208 infection incubation period 156 interactions between HN-1, HBV and, in chronic carriers of HBV 82 mode of transmission 156 prevention and treatment 158-159 virological, clinical and epidemiological characteristics of 206 Hepatitis E virus (HEV) 1, 175, 178, 205 clinical features 180, 207

215 diagnosis of 185-193, 208-209 antibody assays and seroprevalence 186--190 detection ofHEV 192-193 epidemiology and distribution of infection 181-183, 208 incubation period and mode of transmission 183-184 nomenclature 177-178 prevention and prophylaxis 193-194 virological, clinical and epidemiological characteristics of 205-206 Hepatocellular carcinoma s. Primary hepatocellular carcinoma (PHC), HBVand Heterosexual transmission of HDV 155-156 High risk patients, chronic HBV in 80-82 hemodialysis and renal transplant patients 80-81 hemophiliacs 81 homosexuals and drug addicts 81-82 interactions between HN-1, HDV and HBV infections in chronic carriers of HBV 82 Homosexual transmission ofHAV 40, 43 ofHBV 43, 81-82, 87 Horizontal transmission ofHBV 89-90 Hospitals, preventive measures for HAV transmission in 45-46 Human immunodeficiencyvirus-1 (HN-1) interactions between HDV, HBV and, in chronic carriers of HBV 82 Humoral immunity, HBV infection and 107-108 Hybrid vaccinia virus vaccines for HBV 133-134 Icteric phase of acute HBV infection 71 IgG antibody to HCV (IgG anti-HCV) 7 IgM anti-HBc (serological marker of HBV) 94 possible interpretation of 99 Immune electron microscopy (IEM) 33-34

216 lmmunoblot techniques 166-167 Immunofluorescence 165-166 Immunoglobulin (IG) for HAV 46-48 for HBV 129-130 Immunoperoxidase 165-166 Inactivated HAV vaccines 48-50 Inapparent form of acute HBV 72-73 Inapparent HAV reinfection 40-41 Incubation period for HAV 26, 41-43 for HBV 86-87 for HCV and HEV 183-184 forHDV 156 Infants, transmission of HBV from mothers to 87-89 Infection with chronic active HBV clinical picture 78-79 treatment and prognosis 79-80 with chronic persistent HBV clinical course 76 prognosis and management 76-77 Infectious hepatitis s. Hepatitis A virus (HAV) Interferon-alpha 7-8 Interferon (IFN) therapy 121 for HBV 7-8, 123-125 for HCV 194-197 for HDV 158-159 Interleukin 121 interleukin-2 in HBV therapy 127 Intrafamilial spread ofHAV 42 Intravenous drug abuse HAV transmission due to 40, 43 HBV transmission due to 81-82 Lethargy 70 Live attenuated HAV vaccines 50-51 Liver, effect of acute HBV infection on 71, 106-107 Liver cancer 2 s. also Primary hepatocellular carcinoma (PHC), HBV and Malaise 70 Metoclopramide 120 Molecular biology studies of relationship between PHC and HBV 116118

Subject index Molecular hybridization assays 167-168 Monoclonal antibody to HBsAg 126127 Mothers, transmission ofHBV to infants from 9, 87-89 Myalgia 25, 70 Non-A, non-B hepatitis (NANBH) s. Hepatitis C virus (HCV); Hepatitis E virus (HEV) Nosocomial transmission of HAV 45-46 Parenteral transmission ofHAV 43 ofHCV 185-186 Passive immunization in control and prevention ofHBV 129-130 Passive immunoprophylaxis of HAV 46-48 Pathogenesis of HAV 28-32 immunopathogenesis 30-32 pathologic features of the disease 28-29 virologic events of infection 29-30 Pharyngitis 25 Polypeptide vaccines for HBV 133 Polyphasic course of HAV infection in children 27 Post-transfusion NANBH (PT-NANBH) s. Hepatitis C virus (HCV) Prednisone 121 Prenatal transmission of HBV 9 Primary hepatocellular carcinoma (PHC), HBVand 111-118 clinical aspects 112 geographical localization and familial incidence 114-115 molecular biology perspectives 116-118 pathological aspects 112-114 serological association ofHBV markers and PHC 115-116 Prodromal phase of acute HBV infection 70-71 Prodrome rash 25 Prostaglandins 123 Radio-immunoassay (RIA) 35-36, 163 Recombinant DNA vaccines for HBV 133

Subject index Renal transplant patients, chronic HBV in 80-81 Replication cycle of HAV 22-23 Restricted activity in the treatment of active HBV 119-120 Risk factors associated with RAV 40 associated with HBV 84 RNA molecule ofHAV 18-20 ofHCV 6-9 Safety ofHBVvaccines 130-132 School environment, HAV transmission in 42-43 Seasonal variation in HAV infection 39-40 Sedimentation coefficient of HAV 16-17 Serological identification of acute HBV 96-97 of chronic HBV 97-98 ofHAV 34-35 ofHBV association of HBV markers and PRC 115-116 serologic markers in course of infection 93-95 ofHDV 161-162 Serum hepatitis s. Hepatitis B virus (HBV) Sexual transmission ofHAV 40, 43 ofHBV 43, 81-82, 87 ofHDV 155-156 Sporadically occurring communityacquired NANBH (SPOR-NANBH) 175 Structural proteins of HAV 20-21 Suramin 121-122 Symptoms of viral hepatitis 25, 70-71, 205 Thymosins 121, 123 Transmission ofHAV 41-43 ofHBV 86-90

217 horizontal transmission 89-90 incubation period and mode of transmission 86-87 sexual route and transmission through fluids 81-82, 87 vertical transmission 87-89 ofHCVandHEV 183-184 of HDV 155, 156 Universal HBVvaccination program 136-137 Vaccine development for HAV 45, 4852 combined hepatitis A and B virus vaccine 136 inactivated vaccines 48-50 live attenuated vaccines 53-51 other strategies for development 51-52 Vaccines for HBV 9-10, 132-136 anti-idiotype antibody as a prospective vaccine 135-136 chemically synthesized vaccines 135 combined hepatitis A and B virus vaccine 136 hybrid vaccinia virus vaccines 133-134 polypeptide vaccines 133 recombinant DNA vaccines 133 safety of the vaccines 130-132 universal vaccination program 136-137 vaccines using hybrid particles 134-135 Variant of hepatitis B virus (HBV-2) 1 Vertical transmission ofHBV 87-89 Vidarabine 121-123 Vomiting 70 Water-borne transmission of HAV 41-42 World Health Organization (WHO) 44 Worldwide prevalence ofHAV 44 ofHBV 83-86

David H. Walker (ed.)

Global Infectious Diseases

Prevention, Control, and Eradication With a Foreword by Thomas N. James

1992. 33 figs. XIV, 234 pages. Soft cover DM 148,-, oS 1.036,-. ISBN 3-211-82329-8 The subject of the book is global infectious diseases and includes 12 entities that were carefully selected for diversity of epidemiology, transmission, pathogenesis, and immune mechanisms. Each topic (schistosomiasis, malaria, filariasis, arboviruses, diarrheal diseases, AIDS, hepatitis, fungal diseases, Chagas' disease, rickettsial diseases, Lyme disease, and cysticercosis) is particularly interesting in its own right. Each entity will be reviewed by an expert who perceives the big picture and will cover the epidemiology, ecology, pathogenesis, immunity, and relevant molecular data pertinent to the etiologic agents and their hosts. Each expert will then express his opinion as to the best approach to eradicate, control, or contain the disease in the near future by attacking the most vulnerable point in the interaction of the etiologic agent and the host or the environment. Molecular approaches will be described which point towards novel methods to stimulate effective immunity, eradicate a vector, render it genetically incompetent, or develop a new therapeutic intervention. The ultimate standard and the reasons for its success and the failure of other eradication campaigns will be posed by Dr. D.A. Henderson, leader of the successful campaign to eradicate smallpox and currently a high level advisor in the White House. In preparation:

Edouard Kurstak (ed.)

Measles and Poliomyelitis

Vaccines, Immunization, and Control

1993. 49 figs. Approx. 400 pages. ISBN 3-211-82436-7

Springer-Verlag Wien New York