A Tale of Two Viruses : Parallels in the Research Trajectories of Tumor and Bacterial Viruses [1 ed.] 9780822987710, 9780822946304

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A TALE OF TWO VIRUSES

A TALE OF TWO VIRUSES PARALLELS IN THE RESEARCH TRAJECTORIES OF TUMOR AND BACTERIAL VIRUSES

NEERAJA SANKARAN UNIVERSITY OF PITTSBURGH PRESS

Published by the University of Pittsburgh Press, Pittsburgh, Pa., 15260 Copyright © 2021, University of Pittsburgh Press All rights reserved Manufactured in the United States of America Printed on acid-free paper 10 9 8 7 6 5 4 3 2 1 Cataloging-in-Publication data is available from the Library of Congress ISBN 13: 978-0-8229-4630-4 ISBN 10: 0-8229-4630-0 Cover art: Clear plaques of bacteriophage formed on a lawn of bacteria growing in a petri dish (top); iconic image of hen with sarcoma in the Journal of Experimental Medicine 12 (1910): Plate LXVI (bottom). Cover design: Joel W. Coggins

To all MY graduate advisors and mentors for giving me what were truly “the best of times” in my life and To my parents, who gave me that life in the first place

CONTENTS preface ix acknowledgments xiii introduction 3



1 Called or Recalled to Life

Discoveries and Conceptions 11



2 Epochs of Incredulity and Belief Reception and Ripples 33

3

What Was a Virus? 57



4

Romancing the Phage 79



5 Reawakenings The Viral Etiology of Tumors 104

6 What Viruses Became New Visions from New Tools 134

7 Knitting Done Lysogeny as Linchpin 160 afterword 195 notes 201 bibliography 257 index 289

PREFACE It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity . . . —Charles Dickens, A Tale of Two Cities

It is inevitable, I suppose, that anybody with the temerity to title a book A Tale of Two anythings would allude to the unforgettable opening lines of Dickens’s novel. Certainly the best-­of-­t imes-­worst-­of-­t imes line has been fluttering at the edges of my consciousness ever since I first began to think of my project in terms of a comparative history of tumor viruses (specifically the Rous sarcoma virus, or RSV) and the bacteriophages, viruses that infect bacteria. At my first ever public presentation of the material—I believe it was at the 2011 History of Science Society meeting in Cleveland—I brought it up sort of hesitantly, almost apologetically, labeling my allusion as a mere literary conceit. A dear friend and fellow historian, Eva Ahrens, told me not to be so diffident, to take ownership of the analogy, advice for which I’ll be eternally grateful. At the time I had quoted only the first two, and most famous, lines of the book. But when I went back to it, both to ensure accuracy and to reacquaint myself with the rest of the opening, I found, much to my surprise and delight, that I had been much closer to the mark than I’d realized. For my tale of two viruses is characterized by the very warring attitudes of belief and incredulity mentioned by Dickens as quoted above and turned my conceit into real history.

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I was first struck by the parallels between the early research trajectories of RSV and the bacteriophages when I began to write my doctoral thesis sometime in early 2005. I can even pinpoint the specific footnote I was writing when the idea first dawned.1 At the time, I couldn’t help but notice a striking similarity between my project and some unrelated research that I had conducted earlier, but it was not until much later in 2011 that I dug it out of the dim recesses of memory and recalled it to life. In addition to the aforementioned HSS conference, that same year I floated the idea as a possibility for a fellowship at the University of Pittsburgh, for the specific purpose of developing a book proposal. As it happened, I didn’t get the fellowship, but as this book bears testimony, some years later I got to write it for their press anyway. Once I took ownership of the analogy to A Tale of Two Cities, it became something of a touchstone that I periodically returned to during the process of writing this book. Aside from the title, there were other Dickensian tropes that suggested themselves to me later during the process of writing, and I’ve tried to incorporate them where I could. Most chapter titles or parts thereof, for example, have been lifted from the classic. The phrase “recalled to life,” which is the title of part I in Dickens’s Tale, is one that applied on more than one occasion to the unfolding events of the story: Peyton Rous, discoverer of the tumor virus that eventually bore his name, recalled to life a theory of cancer causation that many believed had been laid to rest just a year before his announcement. Closer to the end of this book there is another instance of a recall to life, of yet another scientific theory, the details of which I shall defer to the description at the appropriate moment. Another chapter’s title, “The Golden Thread,” is a more than apt expression to describe the nucleic acids—­DNA especially—­t hose chemical threads that form the material basis of information and heredity of different viruses and the understanding of which was crucial to figuring out how bacteriophages, tumor viruses, and indeed viruses in general behave the way they do. Still other titles were borrowed simply because the particular turn of phrase worked. I was particularly delighted to be able to weave—­or I should say knit?—­into my narrative the malevolent Madame Defarge, she who knit the names of guillotine victims into her lethal list and looms high on my list of favorite literary villainesses of all time. Dickens is by no means my only literary inspiration. Nor is he the earliest or most venerated author whom I have invoked here. In fact, the very narrative device that he used to set up his Tale can be traced to

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a biographical technique popularized in antiquity by Plutarch in his famed Lives, which juxtaposed biographies of recent (to him) or near-­ contemporary Romans with ancient and even mythical Greek figures.2 The genius of this parallel approach was that it threw into relief not only the similarities but also the differences in the two biographical subjects. Even today, reading the lives of Plutarch’s Greek and Roman subjects in tandem, the reader is reminded to consider issues about either subject that may have been overlooked altogether in the telling of a single individual’s life.3 Similar benefits are reaped, I believe, in constructing the parallel research lives of the sarcoma agent and the bacteriophages, for when we consider the very different contexts in which these entities were discovered and studied, the similarities in their discoverers’ conceptions seem all the more remarkable. For instance, whereas cancer was a known disease with a centuries-­long presence in human history, and one for which Rous’s putative virus was only one possible cause, both the phenomenon of bacteriophagy and its relevance to human disease were completely unknown at the time of its discovery. Another literary reference to which I must pay tribute in a book featuring both bacteriophages and the Rockefeller University (formerly Rockefeller Institute for Medical Research) is the Pulitzer Prize–winning novel Arrowsmith, by Sinclair Lewis.4 Its influence on me during the writing of this book was perhaps more indirect than the others mentioned here, but no less real for all that. Published in 1925 within a mere decade of the discovery of the bacteriophages, the novel explicitly used these creatures as a major plot device, thus showing off Lewis’s familiarity with some of the cutting-­edge medical research of the day. The book was enormously influential when it was first published, inspiring many a young student to pursue medical research.5 Among these students was Joshua Lederberg, who would many years later go on to become the president of the Rockefeller University, the model for Arrowsmith’s McGurk Institute, where much of the action in the novel takes place.6 Finally, I must also mention my debt to a more contemporary work of fiction, Michael Cunningham’s The Hours, which I encountered while in the throes of writing my dissertation.7 In its own way this book was an homage to the immortal yet all-­too-­mortal Virginia Woolf, featuring as it did both the writer herself as a character and her novel Mrs. Dalloway as a central plot device. In addition to the explicit references to the novel, sprinkled seemingly at random in The Hours were these mo-

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ments—in a sentence here, a turn of phrase there, or a scene somewhere—that exactly paralleled, echoed, or imitated something that appears in Mrs. Dalloway. I am not sure why Cunningham slipped these bits in; for his own amusement, perhaps, or to keep himself immersed in Woolf’s prose and plot?. But I am certain I would have missed this feature altogether had I not had a copy of Mrs. Dalloway at hand while reading The Hours, and frequently interrupted my reading of one to dip into the other. Once I caught the first such parallel, however, I delighted in playing this literary detective game and aspired to imitate the device if and when I got the chance. Thus, readers of A Tale of Two Viruses might, at random places in the book, encounter certain odd sentences with archaic constructions or references. It is simply my attempt, Cunningham-­like, to make the interesting but often heavy business of research lighthearted and fun to write and thereby (I hope) also fun to read. At its core this history is a story about certain curious coincidences, to my mind, in the histories of two viruses, and as a science historian who came into the field from being a writer about science, I have attempted to tell it as such with all the flounces and flourishes that a storyteller might use. But equally it is, I hope, a work of serious scholarship with a definite analytical purpose and theoretical underpinnings, which involved many trips to different libraries and archives in different parts of the world. To these places and the people there, I owe as much, if not more, as I do to the books I have mentioned. They provided me with the raw material, not to mention physical homes at different stages of my research and writing. Before embarking on the tale, therefore, I make a brief detour to thank all these institutions and people without whom this book might yet be a collection of half-­baked ideas and unfinished paragraphs and documents languishing in my head and laptop.

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ACKNOWLEDGMENTS

I begin this vote of thanks with the one person who has been with the project nearly as long as I have—­my editor at the press, Abby Collier. From proposal to print, she has been a magnificent fellow traveler on this journey, reading individual chapters and offering invaluable advice and a keen editorial eye as and when needed. I cannot thank her enough and I won’t even try. Of course, I might never have gotten the chance to work with Abby had it not been for Professor Jim Fleming, who championed my case for fellowship at the University of Pittsburgh, which gave me the gumption to go ahead with the proposal. So he’s the next person to whom I must express my gratitude. Next on this thank-­you list is Ton van Helvoort, whose incredible generosity—­he read every individual chapter in a draft form for no other reason than to help me—­transformed our virtual (email) correspondence and collaborations on papers and books into a real and enduring friendship. Not many people in my line of work can boast bespoke art in their books, but I am privileged enough to have two pieces! First, my old friend Sanja Saftic, microbiological cartoonist par excellence, chan-

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neled her wicked sense of humor to brilliant effect to render Madame Defarge in microbial guise; and second, my beloved niece, Annapoorna Mahesh, patiently dealt with my rambling request to produce a diagram that conveys the message with great clarity. The research and writing of this book has taken me to numerous archives and libraries in different parts of the globe. Heading this list is the American Philosophical Society Library, which was kind enough to award me their Library Research Resident Fellowship twice. It was during my first visit there that I really began the business of research on this book specifically, spending hours sifting through the fascinating papers of various human characters who people its pages. I continue to reach out to the lovely folks at the APS—­especially though not exclusively Patrick Spero, Joe DiLullo, and Adrianna Link—­at the drop of a hat, for help in tracking down a date or a missing resource, and they unfailingly help me. A grant to scholars from the Friends of the Library of the University of Wisconsin–Madison in 2017 gave me much-­ needed funds to visit the Steenbock Library, which houses the papers of Howard Temin, without which I could not have imagined, let alone worked, on the final chapter of the book. The actual completion of the manuscript as well the identification of many of the images I have used herein happened during yet another trip to several archives in both the New York and Philadelphia areas, this one funded by a research fellowship from the Consortium for History of Science, Technology and Medicine based in Philadelphia. There were also visits to the archives of the Pasteur Institute in Paris and to the Wellcome Collection in London, where, as a matter of fact, I am sitting as I type out these lines. Then there are the Burnet papers at the University of Melbourne Archives and the Walter and Eliza Hall Institute of Medical Research (WEHI), which I first visited as a graduate student, and material from which became the meat of what is chapter 4 in this book. Again, Melbourne is a place where I made dear and invaluable friends in our discipline and I should especially like to thank Gavan McCarthy at the University of Melbourne, as well as the staff and faculty at WEHI, for their help and hospitality. I should also mention my graduate university, Yale, which provided me with funds to make that first visit. And of course before all of that there was the Rockefeller University in New York, where—­in what I fondly call my protohistorian days—­I first learned of Peyton Rous and his discovery. They obviously made a lasting impression, because here they are, more than two decades later, as the major protagonists of my first scholarly historical book. The credit for those introductions

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goes to Geoff Montgomery, who I hope will learn of, and enjoy, this book. Early in the game—­even before I had begun to think about a concrete book proposal—­Robin Scheffler and I organized a session on one hundred years of research on viruses and cancer at the 3-­Societies Meeting in Philadelphia during the summer of 2012. The session went on to become a special issue in Studies C with Robin as a very able organizer and editor. I thank him and the other contributors to that issue, for which I wrote the first major stepping-­stone to this this book. More recently at the 2017 HSS meeting, there was another one hundredth anniversary session, this time of the discovery of bacteriophages, led by my doctoral advisor Bill Summers, who ranks first among the unnamed teachers to whom this book is dedicated. That session too, has gone on to become a special issue of the journal Notes and Records of the Royal Society, released in December 2020, and I thank the editors at the journal for the invitation, as well as all the participants for their presentations. A special call-­out is due to Gladys Kostyrka, my coauthor on our joint contribution to that issue, which greatly added to the pleasure of the process and helped me with the conclusion to my own book besides. Dr. Philip Mortimer, a practicing virologist in the UK, whom I first contacted with questions after reading his fantastic review about virus cultivation, offered valuable insights on my writing about that particular topic in chapter 6, just as another virologist, Steve Martin (no, not the actor), and the historian Susie Fisher did for different parts of chapter 7. Professor David Vaux’s enthusiastic response upon reading chapter 4 on Burnet was just the right sort of shot of encouragement for a weary writer nearing the end of the tunnel but just not getting there. Last but not least here, it would be remiss of me to forget the reviewers whose valuable advice and eagle eyes greatly improved the final telling of this tale. Any warts and holes that remain—­and I’m sure there are many—­are my responsibility alone. In no particular order I would also like to thank Pierre-­Olivier Méthot, Michel Morange, Dick Burian, Greg Radick, Greg Morgan, Kersten Hall, Christoph Gradmann, Scott Podolsky, Kersten Hall, Lisa Onaga, Nathaniel Comfort, Vidyanand Nanjundiah, Staffan Müller-­ Wille, Erling Norrby, Warwick Anderson, Rachel Ankeny, Brendan Clarke, Karen-­Beth Scholtof, Jay Malone, and other fellow members of the intellectual community that I call mine for valuable conversations on any number of topics, relating to our field. Many of them, as well as

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Olga Nilova, librarian at the Rockefeller, and Dhananjay Bambah-­ Mukku (neither of whom are historians) also performed the critical service of getting me papers, access to which was severely limited for an itinerant scholar with no institutional affiliation as I was for much of the period that I wrote this book. Being itinerant and unemployed also make the list of friends and family to whom I owe thanks very long indeed. So long in fact, that I won’t even try to name them individually, for then I’d be sure to unintentionally slight someone or the other. Instead, I’ll just offer as much of my heartfelt gratitude and love as it is possible to convey. In different cities in India, the United States, Australia, the United Kingdom, and France, you helped in kind and with kindness, and it is no exaggeration to claim that each one of you has been integral to this book making it to publication. Anyone who happens across this book and these paragraphs, you will know who you are, and I thank you from the bottom of my heart.

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A TALE OF TWO VIRUSES

INTRODUCTION There is some resemblance between the lytic principle of Twort and the Rous principle. —Alexis Carrel, 1925

This tale of two viruses—­bacteriophages, viruses that infect and live at the expense of bacteria, and the Rous sarcoma virus, a causative agent of tumors called sarcomas in chickens—­is long overdue. Although the pairing of these two viruses might seem rather arbitrary, they have shared strangely parallel histories from the time of their respective discoveries in the early decades of the twentieth century until the early 1960s. Nearly a century ago, in 1925, within a few years of their being discovered and decades before they would be acknowledged as viruses, the famed medical researcher Alexis Carrel had already remarked on the similarities in the way that the two “principles” operated. His conjecture was admittedly a stretch, for at the time the two phenomena could not have seemed further removed from one another: one a lysis, or destruction of bacterial cells, and the other the formation of tumors in the connective tissues of certain birds. But, as he noted, both phenomena could be “supposed to develop within a cell under the influence of a metabolic disturbance. . . . Once the process has started, it reproduces itself indefinitely by a mechanism that we do

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not understand, but whose existence is certain.” Of course, Carrel’s observations mark just the beginning of a long and winding tale, and his observations likely would have made little sense to the vast majority of researchers working in one or the other (almost never both) field at the time. It is in the tracking of how the mechanisms were deciphered over the next decades that the parallels in the history of the two viruses becomes evident. And that is the tale laid out in the pages that follow. A reader conversant with viruses might immediately raise the objection that the parallelism constructed here is invalid, for whereas RSV is a specific virus, there are many different types of bacteriophages. It seems a comparison more akin, for instance, to that of apples and all citrus fruit, rather than just apples and oranges. But I defend the ambiguous or fuzzy nature of the comparison on both historiographic and narrative grounds. From a historical point of view, as Carrel’s statement indicates, at the time both viruses were simply “principles,” whose physical, chemical, and biological properties were unknown. The very identity of these principles as viruses—­insofar as viruses had an identity at the time—­was a matter of dispute, the resolution of which forms a significant part of the context for history examined here. It was only over time—­several decades, in fact—­t hat RSV was found to be just one among a very large group of viruses capable of inducing tumors or cancers in the organisms whose cells they parasitize. These viruses in turn, as it turned out, constituted just one type of tumor-­ inducing agent, along with substances such as chemicals and hormones. Although the term tumor virus is a perfectly acceptable label for these viruses, to claim that this book is about the history of this entire category would be misleading. Much like tumors and cancers themselves, tumor viruses constitute too diffuse or heterogenous a group, with many different viruses capable of infecting the cells of humans, other animals—­birds inclusive—­and plants. Studying these different groups requires different techniques and approaches, and input from investigators with different knowledge bases. For the most part, this book follows the specific history of research on RSV, sometimes against the bigger picture of what this research revealed about the viral etiology of tumors. But the bacteriophages, though a diverse population in their own right, constitute a more cohesive group than the tumor viruses, at least in practical terms. They infect bacteria, which, for all their genetic and physiological diversity, are single-­celled organisms that function as a more or less homogenous group of experimental objects that can be studied with essentially the same or very similar laboratory

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techniques. Furthermore, and perhaps of greater relevance to the history followed herein, there is no single bacteriophage that might be considered representative of the category in this history, as RSV stands in for the tumor viruses. Even though their existence as a population of many different types was recognized within a couple of decades of the discovery of the first bacteriophage, the individual identities of different bacteriophages did not appear to impinge significantly on what scientists learned about their behavior or how they were deployed in different studies for the bulk of the period covered in this book. The second virus in the title of this book is therefore best represented by the generic label “bacteriophages.” In a quick aside, Charles Dickens, whose book’s title I lifted and morphed to my own purposes, was also similarly ambiguous in correlating his title to his story. True, the main events of his Tale of Two Cities take place in two cities—­London and Paris—­but it is the countries that he named in those nearly repetitive sentences that follow one another in rhythmic succession at the outset of the book: “There were a king with a large jaw and a queen with a plain face, on the throne of England; there were a king with a large jaw and a queen with a fair face, on the throne of France.”1 In fact, I would argue that my tale is truer to its own title than A Tale of Two Cities is, because people, not cities, were the main characters in Dickens’s novel; my book, in contrast, puts the two viruses of the title and subtitle at front and center of the narrative, albeit through the hands, laboratories, and imaginations of the various scientists who populate the pages. Setting the context for the research trajectories of the two viruses was the evolving understanding of the nature of viruses in general—­t he various cycles of discovery, hypotheses, and experimental testing through which they were defined, refined and redefined, and further refined to their modern guise over the decades between the late nineteenth and mid-­twentieth centuries. Even today, the question, “what is a virus?” is likely to bring forth a flurry of different if not outright contradictory responses. In this day and age of Twitter, Instagram, and other such things, the phrase “going viral” is part of the popular vernacular among English-­speakers the world over as a metaphor for something that spreads very quickly and widely (it has, self-­referentially, gone viral). Most people using the expression would probably not know more about a virus than that it is a disease-­causing agent that spreads very rapidly and easily. Pressed further, some might come up with specific diseases, especially in the present day when COVID-­19 is raging ramIntroduction

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pant. But I think that few of those hailing from outside the medical or medical research position would know or care about facts such as the way in which they are fundamentally different from other disease agents—­bacteria, for instance—­or about the origins of either the word (known) or the entities themselves (many aspects still unknown). What makes the two chosen viruses of this tale particularly apt in this larger context is that they were, in many of the instances as detailed in the chapters that follow, the specific entities that scientists worked with, and over which they discussed and debated the broader issues of viral identity and definition. Having introduced the “what” of this book, I turn to the historiographically more interesting, and dare I say pressing, question of “why.” Actually, I should say questions—­in the plural—­because even I, who am naturally sold on the project, can think of several why’s that various people might legitimately ask right off the bat: Why a history of viruses at all? Why these two viruses in particular? And why now? To address the broader “why” questions first—­why viruses and why now—­I would venture to say that the timing is right because the history of virus research has been given short shrift by historians, philosophers, sociologists, and other humanists and social scientists for far too long. Although the first dedicated histories of virus research appeared as far back as the late 1970s with two complementary treatments, literature on the subject since then has been sparse. The first of those two books, The Virus: A History of the Concept, by Sally Smith Hughes, was an intellectual history focusing on the development of the concept of virus as understood in contemporary times.2 The second, A. P. Waterson and Lise Wilkinson’s An Introduction to the History of Virology, was a somewhat broader look at the development of the field that tied together the ideas that showed “what influenced them and how they interacted with the experiments done and the techniques used.”3 Both were slim volumes, perhaps too slim, as noted by one reviewer (a historian), to cover such a large field, but were lauded as preliminary efforts to “explore a vast and hitherto neglected domain.”4 Certainly they set the stage by identifying questions and gaps for future historical scholarship. A virologist reviewing Waterson and Wilkinson’s Introduction, for example, expressed the hope that having provided a broad introduction to the subject, “the authors will now give us a more detailed history of a single topic or of a few individuals.”5 But although Wilkinson went on to publish a series of papers about the historical importance of several individual viruses, she did not venture into a fuller treatment of

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the sort suggested by the reviewer and such a book did not materialize for many years.6 Indeed the only substantive historical offering in the four decades between the pioneering works of Hughes and Waterson and Wilkinson and the present day has been Angela Creager’s The Life of a Virus, a history of the tobacco mosaic virus (TMV) published in 2002.7 A tour de force by any reckoning, The Life of a Virus was nevertheless just one book, and one, moreover, in which the history of viruses and virology per se was not the primary goal. Rather, it was the story of the “life” of TMV as a “model virus,” which Creager wielded as a historiographic tool to examine larger changes in the conduct of science: “instrumentation, science funding, and postwar biology” from the 1930s through the mid-­ 1960s. It provided a meta-­analysis of “the ways in which key scientific achievements set in motion trends ranging far outside the laboratory walls.”8 In contrast, this tale, despite its focus on the lives of two viruses instead of one, examines the reverberations of scientific achievements within laboratory walls and in the intellectual lives of scientists. It is thus a largely “internalist” history, to use a nearly obsolete term for a way of doing history that trains its gaze on what the philosopher of biology Michael Ruse called “naked science.”9 I do not, of course, ignore the various external factors—­for example, societal and institutional influences, funding issues, and disciplinary ambitions—­but consider them in light of how they shaped scientific content. Thus, I have put my sources in the service of the “inside” of history, which “treats the words and so presumably thoughts, of historical agents.”10 In addition to its historiographic ambit, my Tale also differs from The Life of a Virus in its choice of protagonist, so to speak. As the very first bona fide virus to be discovered in the laboratory and as “almost always the first” in other areas of virus research, TMV definitely deserved to also be the first virus about which a full-­length history was written. But for all its historical significance, it belongs to the group of viruses that infects plants and so falls outside the realm of the virus population that has engaged the largest majority of virus researchers—­ namely, animal viruses involved in human diseases. Beginning sometime in the year 2000 the medical microbiologist Dorothy Crawford may be said to have become something of a one-­stop source of very fine books about medically important viruses.11 But her books are semipopular accounts written from the perspective of a veteran from the trenches, or more accurately in her case, benches. They are completely sound on scientific matters, but they do not purport to be works of Introduction

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deep historical, philosophical, or sociological insights, and hence cannot—­and should not—­be judged on those grounds. Consequently there is a lacuna in the publication of historical accounts of research of animal viruses, a gap that was highlighted more than two decades ago by the historians of science Dan Kevles and Gerald Geison in their astute analysis of the historiography of the life sciences in the twentieth century.12 This Tale of Two Viruses is my first step in the path to addressing that gap. In fact, along with two other recent historical monographs—­A Contagious Cause: The American Hunt for Cancer Viruses and the Rise of Molecular Medicine and Nobel Prizes: Cancer, Vision and the Genetic Code—­and a third historian’s forthcoming book, as well as Discovering Retroviruses: Beacons in the Biosphere, a scientist’s account of the viruses upon which she built her career, this book is perhaps indicative of a trend that medically important viruses, at least the cancer viruses, are finally moving into the spotlight.13 It is probably no coincidence that three out the five of us contributed papers to a 2014 special issue of Studies in History and Philosophy of Biological and Biomedical Sciences that focused on a century of research on cancer and viruses.14 Our books differ quite widely in their approaches, focus, and time periods covered. Therefore, it is to be hoped, they are complementary rather than competing. In this book, for instance, tumor viruses make up only part of this tale. In addition to focusing on viruses of medical significance—­t he particular bacteriophages used in the work described in this book were those that infected pathogenic bacteria—­I use the specifics of research on the bacteriophages and RSV to illuminate details about the broader tale of how viruses, as a class of beings distinct from other infectious agents, came to acquire their modern definition. In this sense, my work, perhaps more explicitly than the other three, aspires to be considered a direct descendant of Hughes’s conceptual history. In order to best achieve my twin goals of cohesive storytelling and historiographic rigor, I have adopted a narrative strategy somewhat akin to a musical composition, in which the linear flow of the central narrative is broken up by interludes or intermezzos. Chapter 1 plunges into the tale, focusing on the discoveries of the two viruses between 1910 and 1917, and their discoverers’ conceptions of their finds. Chapter 2 follows in the same vein, looking at the reception of the ideas about these new discoveries by their respective peer communities within the first couple of decades; namely, into the early 1930s. Chapter 3, the first interlude, takes a step back from the main narrative and goes further

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back in time into the early history of virology to establish the backdrop and contexts for the discoveries and attitudes discussed in the first two chapters. In the next two chapters I resume the main history, but with a slight shift in narrative structure. Instead of following the research trajectories of both viruses in each of these chapters as in the first two chapters, the narrative diverges to consider the unfolding research trajectories of the two viruses separately: chapter 4 focuses on the bacteriophages and chapter 5 on the tumor viruses. I based my decision to separate the narratives in this way primarily on historical circumstance: whereas research on tumor virology was a largely dormant enterprise through the late 1920s and much of the 1930s, research on the bacteriophages continued at a steady pace throughout this period.15 Indeed, it has never really waned since then, experiencing an exponential increase after the end of the Second World War, but the issues at stake in that later work are not really relevant to the history at hand in this book. Tumor virology did not really catch up and experience the same sort of exponential growth until a few decades later, by which time the entire medical research landscape had been drastically transformed. By the mid-­1930s, however, it had begun to experience a level of activity similar to that of the bacteriophages from a decade or earlier, and with similar conceptual advances. This development is the focus of chapter 5. Chapter 6, which is the second intermezzo, returns to the broader backdrop issues, primarily the ongoing negotiation from the 1930s through the early to mid-­1950s of the virus concept, which in fact formed the foundation for virtually the entire parallel history that I have attempted to reconstruct in this book. The main rationale for including this second interlude was based on both chronology and the nature of the advances in the two periods covered in chapters 3 and 6. Whereas the earlier period mainly represents the laying of the theoretical foundations based on rather basic techniques and instrumentation, the latter, post-­1930s, was marked by several technological advances on many fronts, which of course greatly contributed to the understanding of viruses as well as the building of virology as an independent discipline. Finally, chapter 7 concludes with a look at the resolution of a phenomenon that I argue served as the linchpin that brought together the by then desynchronized parallels in the histories of RSV and the bacteriophages. Rather than give away details, which furthermore would only confuse without adequate contextualization, I will now, without further ado, begin the tale of the two viruses. Introduction

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1CALLED OR RECALLED TO LIFE DISCOVERIES AND CONCEPTIONS

The first tendency will be to regard the self-­perpetuating agent active in this sarcoma of the fowl as a minute parasitic organism. —Peyton Rous, 1911 The disappearance of the dysentery bacilli is coincident with the appearance of an invisible microbe endowed with antagonistic properties with respect to the pathogenic bacillus. This microbe, the true microbe of immunity, is an obligatory bacteriophage. —Félix d’Herelle, 1917

On the face of things, the circumstances under which the causative agent of sarcoma tumors in chickens and bacteriophages were discovered could not have been more different. There was a medical pathologist working on problems of cancer causation in a lab in New York City; there was a bacteriological researcher investigating bacterial dysentery in a laboratory outside Paris. For the New Yorker, at some point in the course of his investigations, to claim that he had identified a tumor-­ causing agent, which would later come to be known as the Rous sarcoma virus (RSV), was not in and of itself cause for undue notice, at least not at first. After all, cancer research was what he had been hired to do at the Rockefeller Institute for Medical Research.1 That the researcher in France, as a result of his investigations on bacteria causing human dysentery, announced that he had discovered an entirely new living microbe—­one, moreover, that killed the dysentery bacteria themselves—­was, in contrast, quite startling and so, bound to draw attention. Despite the differences in the specifics of their investigative prob-

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lems and circumstances, the two men, working around the same time on their problems—­during the second decade of the twentieth century—­arrived at rather similar conclusions regarding the objects of their respective investigations. The researcher in New York characterized his find as “a minute parasitic organism,” while the French investigator labeled his discovery “un microbe invisible.” Basically, however, they were both saying the same thing within a few years of each other: that the quite unrelated phenomena discovered by them each were caused by viruses—­namely, pathogenic agents invisible to the naked eye, or indeed, even under a regular microscope. How and why these two men arrived at such similar conclusions in their largely separate worlds is the history set forth in this first chapter.

Tumors Most Fowl “Whatever you do, do not commit yourself to the cancer problem,” was the advice William H. Welch gave to the young Peyton Rous, just as the latter was departing to train as a pathologist at the University of Michigan. To a fresh graduate of the Johns Hopkins University School of Medicine, such advice from one of the founding pathologists of the university appeared very sound, especially since cancer research at the time “seemed indeed the most barren of fields in which to try to make a life by finding out.” But fate, in the guise of Simon Flexner, founding director of the Rockefeller Institute, evidently had other plans for this young man. For when Rous completed his training in Michigan, it was to “attempt research on cancer” that Flexner offered him a position at the Rockefeller. As Rous later recounted: “I wanted some other task. Very quietly [Flexner] asked: ‘What are you working on now?’ and he listened intently as I described a tiny dingle-­dangle. . . . Then he gravely said: ‘Do you consider this the equal of the cancer problem’—­nothing cutting, nothing sardonic, just the question. Thus was the fact borne in upon me that all scientific undertakings are not free and equal, as beginners so readily assume.”2 So, Rous accepted the position and had barely begun the new job when a worried farmer visited the institute with a diseased chicken—­a Plymouth Rock hen—­which had “projecting sharply from the right breast, a large, irregularly globular mass.”3 Others at the Rockefeller seem to have shown little interest in the farmer’s problem, but Rous seized upon it immediately as a new avenue of investigation into cancer, and perhaps even as way to vindicate his choice to go against the advice of his former mentor by breaking new ground in the field.

12

A Tale of Two Viruses

An initial examination of the tumor tissue from the chicken suggested that it was a sarcoma—­a tumor of connective tissue—­of a type hitherto not seen occurring in birds. Further studies revealed that the tumor shared many properties with sarcomas known to occur in other animals. As Rous reported: “It is formed of a single type of cell, only slightly differentiated, resembling young connective tissue cells, and possessed of an enormous proliferative energy which is exercised to the detriment of the surrounding tissues and eventually of the entire host. Growth takes place through infiltration and replacement of normal structures, as well as through expansive enlargement. Metastasis by way of the blood stream is common.”4 When bits of the tumor tissue were transplanted, either to unaffected parts of the same bird or into the breast of a healthy, tumor-­free Plymouth Rock chicken—­it had to be the same species—­a new sarcoma developed in this location. At the end of these first preliminary experiments, Rous concluded: “So far as tested, this avian tumor closely resembles the typical mammalian neoplasms that are transplantable.”5 Upon further investigation, however, Rous found that the new avian sarcoma differed from its other neoplasms in one very significant respect. Where the earlier tumors could be transplanted only when the material used for transplantation had intact tumor cells from the original growth, the chicken sarcoma could be induced to develop in new unaffected birds with cell-­free extracts of the tumor; namely, tissue that had been processed—­ground and passed through filters with pores small enough to retain bacteria—­in such a way as to  ensure that the filtrate was free of intact cells. Such an observation about a tumor was unprecedented; all previous efforts to transmit tumors of mice, rats, or dogs to unaffected animals using cell-­free filtrates of tumor tissue had proven unsuccessful. The implication of such behavior was that whatever the identity of the tumor-­inducing agent, it was extremely tiny. Also, Rous found that with each successive transplant, properties such as the rate of success of transplantation, the growth of the new tumor, and the “frequency, extent and rapidity of metastasis” increased. The results of these early experiments led Rous to the conclusion, cited in the epigraph at the outset of this chapter, that “the first tendency will be to regard the self-­perpetuating agent active in this sarcoma of the fowl as a minute parasitic organism.” But this conclusion was by no means definitive at this early stage, and he also acknowledged that it was “conceivable that a chemical stimulant, elaborated by the neoplastic cells, might cause the tumor in another host and bring about in conCalled or Recalled to Life

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Fig. 1.1 Peyton Rous working at his microscope, 1923. Photograph courtesy of Rockefeller Archive Center.

sequence a further production of the same stimulant. For the moment we have not adopted either hypothesis.”6 By the following year, Rous—­soon joined in his investigations by James B. Murphy, another young pathologist from Johns Hopkins em-

14

A Tale of Two Viruses

Fig 1.2 Iconic image of hen with a sarcoma induced by cell-­free filtrate of tumor tissue, published in the Journal of Experimental Medicine 12 (1910): Plate LXVI. Photograph courtesy of Rockefeller University.

barking on a research career—­had found that cell-­free filtrates of the chicken tumor tissue retained the ability to cause tumors in new birds despite undergoing treatments such as drying, glycerinization, and repeated freezing and thawing, which killed the tumor cells themselves.7 In addition, and perhaps most significantly for Rous, the agent seemed to possess the ability to multiply when transmitted to new tissue: “A very little of it will give rise to a growth from which numerous others may be started, each yielding the agent in abundance.”8 Based on these studies, Rous felt more confident about his “first tendency,” and therefore ended his report with a more definitive statement as to the identity of the causative agent: “Experiments with the chicken sarcoma have not yielded a method whereby a causative agent can be separated from the tumors of rats and mice. But they clearly prove that the characteristics of malignant tumors in general are compatible with the presence of a living causative agent.”9 The finding of two other chicken tumors that were also transmissible to new healthy birds via cell-­free filtrates of the tumors—­a bone tumor and a second sarcoma distinct from the first—­further buttressed Rous’s belief in the living nature of the causative agents, leading him to suggest, “The findings with the chicken Called or Recalled to Life

15

tumors largely demolish the theoretical basis on which objections to an extrinsic cause for cancer have been built up.”10 A couple of years later he followed up with an even stronger declaration, claiming, “It is perhaps not too much to say that their recognition [of the agents of these tumors] points to the existence of a new group of entities which cause in chickens neoplasms of diverse character.”11 Although he seemed quite certain that the agents of the avian tumors were living, ultramicroscopic entities, Rous did not explicitly label the agent a “virus” in his early papers. Between 1911 and 1915, Murphy and Rous coauthored about a dozen publications on the chicken sarcoma. In all of them, Murphy would note later, “we referred to the causative factors by the non-­commital [sic] designation of ‘agents.’”12 In the one instance Rous did use the word virus, the link to it as the identity of the sarcoma agents was an indirect one: “Although the filterable viruses have but recently come to attention, it is known that they are of diverse character and that . . . they can scarcely be discussed together. At present each constitutes a separate problem. This is especially true of the filterable agent which causes a sarcoma of the fowl.”13 Certain historians have suggested that part of the reason for Rous’s reticence in calling the agent a “virus” might have been because Murphy, who had collaborated with Rous on most of the avian sarcoma work until 1915, did not agree with this interpretation.14 But this explanation is not consistent with Rous’s characterization of the sarcoma agent throughout and beyond the period that he worked on the problem. The extent to which Murphy influenced the terminology in their joint papers is unclear, for as Rous observed in a letter to a friend, their disagreements never prevented him from calling “the thing a virus when lecturing.”15 He himself credited a senior colleague at the Rockefeller, T. M. Prudden, with dissuading him from calling the sarcoma agent a virus in his publications; Murphy was not mentioned as an influence.16 Furthermore, although Murphy would distance himself from the virus theory once he and Rous ended their collaboration, there is no evidence of an open disagreement during their collaboration. Indeed, several of the papers they coauthored imply quite the opposite, although it must be admitted that their statements therein make rather softer claims. As they concluded in one paper, for instance, “The relationship existing between the chicken sarcoma and its cause . . . seems to us to furnish some basis for the conception of an extrinsic cause for other sarcomata.” In another report published a week later, they said, “No single attribute among those determined suffices to show the na-

16

A Tale of Two Viruses

ture of the agent; yet taken together, its characters are those we associate with micro-­organisms.”17 Based on the findings of three very different sorts of chicken tumors that seemed to share various properties, Rous and Murphy concluded that the recognition of these tumors pointed toward their causation by “a new group of entities,”18 but, as became evident in the years that followed, the two men’s ideas about the nature of this new group of entities were diametrically opposed. Rous, naturally, believed that the agent was a virus, or a minute living parasite of exogenous origins, as he made clear in his various writings and talks on the topic. Murphy equally firmly disagreed with Rous’s interpretations on the nature of the sarcoma agent, as he made clear in a letter to his colleague Waro Nakahara some years after he and Rous were no longer in collaboration: “I have never believed in the virus theory [of chicken sarcoma], and that was the principle [sic] point of controversy between Rous and myself during the several years we worked together on the subject.”19 But there is little evidence for such a controversy elsewhere, and certainly Murphy does not seem to have openly vocalized his discontent with the virus idea during the period the two men collaborated on sarcoma work. Rous does not seem to have dwelled on the issue much either, although the difference of opinion was certainly acknowledged: “Murphy and I have always been in friendly disagreement as to what the agent is—­a disagreement which may be just as well from the investigative standpoint.”20 For his part, Rous did not pursue research on chicken sarcomas for long, moving at first to work on blood biochemistry, citing among other reasons, both the lack of any positive results or meaningful headway and the “need to broaden scientifically.”21 His bibliography reveals that he completely stopped publishing original papers on the sarcoma agent after 1915.22 But despite later protestations—­“I squirm at having the sarcoma named after me; eponyms are old hat,” he once wrote to the science writer Greer Williams—­he maintained a strong sense of ownership regarding his discovery, as revealed on at least two occasions.23 In a 1929 letter to Simon Flexner in response to the former’s request of Murphy—­by then in charge of cancer research at the Rockefeller—­for a written summary of early work on the chicken tumors, Rous protested rather “vehemently” at the choice of authorship: The more I think the more unendurable does the thought become. That my former assistant should, with the authorization of the Di-

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rector, summarize for our Board and for the world at large, the work for which I have lived, the real sense of the phrase, is beyond the bearing. I find it impossible to enter into an arrangement which would bring, in answer to the inevitable questionings of others, repudiation throughout the rest of my days. . . . Surely you will not ask me to yield to another my scientific identity and integrity, and not for the moment alone but for later time when medical historians will seek the Handbuch to learn at first source of our efforts.24

A couple of months later, following a lecture by Flexner, Rous wrote again objecting to his director’s attribution of the discovery of the chicken tumor to both himself and Murphy. Beginning by thanking Flexner for his “delightful lecture” and generosity, Rous went on to explain his position: One of your statements last night raised an issue which  .  .  . is of great importance to me. I shall discuss it, since doubtless its implications did not occur to you in the press of affairs. You said that Rous and Murphy demonstrated the existence of the filterable agent causing the chicken tumor. Now, the fact is that I carried out this work alone and published alone two papers that embodied its results. . . . Murphy had no hand in the experimental episode which showed an “infinitely little” agent to be the cause of the tumor. . . . When, now, after the lapse of more than nineteen years, you make a statement that Murphy shared in the first demonstration of the agent, you provide ground for an assumption by others that I defrauded a fellow worker in the beginning and have continued to defraud him ever since. Needless to say you would have prevented any such occurrence! But you spoke last night with authority and deliberation, leaving the impression that it had indeed occurred. The point is one so directly affecting my integrity that . . . I am unable to concede it even by keeping silent.

In the rest of the letter Rous went on to lay out a timeline of his investigations on chicken sarcoma from September 14, 1910, to January 26, 1911, where he included the dates of submission of his two single-­ authored papers primarily on filtration work—­January 11, 1911, and February 9, 1911—­as well as the date (October 1, 1910) that Murphy joined the institute and commenced work on the sarcoma project. He also offered to show Flexner the lab protocols corroborating his claims, par-

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A Tale of Two Viruses

ticularly the fact that Murphy “had no share in the filtration work, even in the matter of suggestion.”25 A couple of months later Flexner responded, reassuring Rous that he would be “very circumspect to present the chicken tumor accurately. Your perturbation after my Academy lecture led me to make some discreet inquiries of just what I said about the virus. . . . It seems I made two separate statements. . . . The second one carried your and Murphy’s name. I did not secure a restatement of my exact words [but] you doubtless remember them precisely.”26 The second occasion when Rous felt compelled to defend his priority in the matter of RSV discovery occurred many decades later—­by which time both Flexner and Murphy were deceased—­in response to a book review by the biochemist Joseph Fruton.27 In a manner reminiscent of his letter to Flexner, Rous began by congratulating Fruton on writing the book before going on to spell out his grievances about it. Your account of George Corner’s history of the Rockefeller Institute has delighted him—­and me well who had felt his book to be inexcusably ignored by reviewers.  .  .  . One small inadvertent slip disturbs me: you speak of Rous and Murphy as having together found the chicken tumor virus. This carries by implication an indictment, namely that all along through the years I have ignored the rights of a fellow discoverer; never mentioning him. Actually my demonstration of the existence of a causative virus in the growth had been completed before Murphy entered my laboratory.  .  .  . The rumor that I had been unjust to Murphy was so widely and adroitly spread soon afterwards that on learning about it I felt an imperative need to show my protocols to Simon Flexner. He deemed my evidence sound. Said protocols are still securely on file. This may seem trivial to you, so well have things gone with me since; but I greatly value your regard.28

Fruton was quick to acknowledge his error. “I had not known of the rumor that you had been unjust to Murphy. My esteem and affection for you would make it impossible for me to take such a rumor seriously, but if I had known of it I would have been more careful,” he wrote back contritely in due haste.29 Whereas it is not clear if Murphy knew about the entirety of Rous’s exchange with Flexner on the question of priority, the fact that he harbored some proprietary feelings of his own toward the sarcoma agent, and was furthermore quite prickly on the matter of its viral identity, is evident in comments to various friends and

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colleagues—­both proponents and detractors—­over the years. Rous, in fact, may have been the only person with whom he had no open disagreements on the topic. In the meantime, the main reason why Rous abandoned sarcoma research so relatively early seems to have been the lack of any tangible progress for nearly four years. He made no headway in isolating observable organisms of any sort from the different types of bird tumors, which, furthermore, showed no other signs of infection in the conventional sense. He was also unable to find any examples of mammalian tumors that were transmissible via cell-­free filtrate tissue. “I’d become pinched and parched mentally as a result of continually negative experimentation, and felt that only new outlooks could cure,” he confided to his friend the British virologist Christopher Andrewes many years later. Moreover, he added, Flexner advised him “against publication of the negative findings, saying they would keep others from trying, who might have better luck.”30 Although Rous did eventually return to active cancer research again in the 1930s, the later work was on rabbit papillomas, not chicken sarcomas, and hence only peripherally a part of this history. So for now, I leave the chicken sarcoma, even as Rous did, to discuss the second discovery in this tale of parallels in the vicissitudes of virus research.

“At the expense of bacteria” It was around the time that Rous and the sarcoma virus exited the scene that the first bacteriophage made its first appearance as a relatively unremarkable player in the drama that was human disease. Soon thereafter it would also feature prominently on the broader stage of science. The first person to report the occurrence of the phenomenon we now call bacteriophagy was Frederick Twort, a medical researcher in London who was attempting to cultivate or culture “filter-­passing viruses” from different possible sources in artificial media. Though unsuccessful in this regard, Twort reported “interesting results” when he attempted to culture extracts of calf vaccinia on agar: he found that the material contained a substance that had the apparent ability to dissolve bacteria called micrococci.31 By dissolution, Twort meant that liquid cultures containing micrococci—­normally turbid in appearance due to the growth and multiplication of these bacteria—­turned clear or “glassy,” when incubated with material from the vaccinia cultivations. When this material was inoculated along with bacteria to grow on on a solid medium, the normally dense and continuous “lawns” of bacteria would

20

A Tale of Two Viruses

be riddled with glassy patches. That the dissolution and patchiness resulted from the breakdown or destruction—­called lysis—­of the bacterial cells was evident from the fact that both the cleared suspension and the material from the patches contained very few or no intact bacterial cells, which were very much in evidence in samples from normal turbid cultures or bacterial lawns. In a manner somewhat reminiscent of Rous in his first reports on the transmissible chicken sarcoma, Twort made several suggestions as to the possible cause of the observed bacterial lysis—­which he would later dub “glassy transformation”—­without displaying any obvious preferences.32 “It is clear the transparent material contains an enzyme,” he wrote, on the basis of his observations that the lytic substance could retain its bacteria-­dissolving activity for up to six months and was destroyed by heating. Nevertheless, he conceded, “The possibility of its being an ultra-­microscopic virus has not been definitely disproved, because we do not know for certain the nature of such a virus. . . . On the whole it seems probable, though by no means certain, that the active transparent material is produced by the micrococcus, and since it leads to its own destruction and can be transmitted to fresh healthy cultures, it might almost be considered as an acute infectious disease of micrococci.”33 Twort himself did not pursue this line of research any further, partly due to financial considerations and partly because he was called away soon after to serve in England’s war efforts in Greece.34 One near-­ contemporary, Carroll Bull, would later observe that his article “attracted little attention at the time, possibly because it appeared during the [First] World War or because of the title under which it was published.”35 In Bull’s estimation the title proved detrimental because it afforded no clues that the paper was reporting the discovery of a new hitherto unidentified phenomenon. Consequently, the novelty and implications of the phenomenon of glassy transformation of the micrococci and Twort’s speculations regarding their cause were not recognized for some years. References to his work did not appear until nearly a decade later.36 When these citations did finally appear, they did so in the context of claims made by a virtually unknown scientist named Félix d’Herelle that he had discovered a new entity that he explicitly labeled as a “bacteriophage.”37 Unlike Rous and Twort, both of whom were part of the mainstream medical research establishment, d’Herelle was a relative outsider to the scientific community. Indeed, even the details of his early life are hazy. Called or Recalled to Life

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Based on such documentary history as his passport, wartime identity cards, and his memoirs, earlier biographers identified his birthplace as Montreal, Canada, where he was raised.38 But in 2003 Alain Dublanchet, a French physician, found a birth certificate that places d’Herelle’s birth in Paris, as Hubert Augustin Félix Haerens, the last being his mother’s maiden name.39 It was not until 1901, at the age of twenty-­ eight, that he appears to have settled on the name by which he is known today: Félix d’Herelle.40 There is even doubt as to whether he received any formal education—­t here is some indication that he may have studied medicine, but there are no records of his graduation from any university or medical school.41 He claimed to have taught himself microbiology, obtaining practical experience in a private laboratory that he set up in his home in Montreal in 1897.42 In the early twentieth century, d’Herelle gained recognition within scientific circles by working on a number of diverse problems for various scientific commissions in Latin America and North Africa, notably on pathogenic bacteria and the biological control of insect pests through these pathogens. It was during this time, according to his later autobiographical writings, that he first observed the formation of certain “glassy plaques” (taches vierges) on petri dishes spread with cultures of certain coccobacilli that he found to be infecting locusts and grasshoppers. But he never published anything about these findings in his copious reports of his work that he produced at the time.43 In 1911 he became associated with the Pasteur Institutes and worked at branches in Algiers and Tunisia for some years before moving to the flagship institute in Paris. There he began to work on bacterial dysentery, which led to his discovery of bacteriophagy.44 The result of a bacterial infection that leads to debilitating gastrointestinal symptoms and even death, dysentery had become a matter of great urgency at the Pasteur Institute since the onset of the First World War. In 1915 there was a particularly severe outbreak in the town of Maisons-­Laffitte outside Paris. The specific clinical presentation in this case had led the chief medical investigator, Georges Bertillon, to suspect that the outbreak was not caused by any of the hitherto known strains of the dysentery bacillus, and he assigned d’Herelle to investigate and manage the outbreak. In a relatively short time, d’Herelle completed his assigned task, which he described as a relatively simple undertaking:

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A Tale of Two Viruses

One did not need to be a great hygienist to find the cause: leaves had been thrown into trenches dug less than 20 meters from the kitchen, and flies, present in great numbers due to the proximity of the stables, formed a conduit between the kitchens and leaves, where we saw them placed over bloody stools. One wonders what they taught them about hygiene at the school of military medicine. I advised the filling of these trenches and digging others farther away from the kitchens, taking care to frequently treat them with chloride of lime. Once such action was taken, the epidemic was promptly contained.

Once he had resolved Bertillon’s problem, d’Herelle proceeded to carry forward his own investigations using the pathological specimens from the patients. In his words: Several soldiers were treated at the hospital of Maisons Lafitte; I collected their stool samples for research. . . . I passed an emulsion of the dysentery stools in nutrient broth through a porcelain filter, I mixed the filtrate with a culture of dysentery bacilli and placed the whole mixture in an incubator at 37°[Celsius]; after a few hours of incubation I spread a drop of this mixture on a plate of nutrient agar and incubated it to look for the development of glassy plaques; . . . When spread on agar, on two different occasions, glassy plaques dotted the surface of the dysentery bacilli cultures. Finally I had proof that the phenomenon of glassy plaques was not limited to the coccobacilli of grasshoppers, and that they could occur just as easily in bacteria pathogenic to humans.

But what were the implications of the finding that the lytic principle of bacteria—­what d’Herelle described as “le générateur”—­was transmissible? By his own account, there was a period during the early phase of his experiments when he was unable to achieve any satisfactory consistency or regularity in his results working with specimens from different patients. But evidently this state of affairs soon changed, and dramatically at that, as he eloquently recalled in his memoirs: One day, it was in the middle of September, I was dejectedly reviewing my laboratory notebooks, I wasn’t getting anywhere, when it suddenly occurred to me that it was only after I had examined the stools of the same patient multiple times, that I found the glassy

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Fig 1.3 Félix d’Herelle (standing, center) with three associates working in his laboratory at the Pasteur Institute, Paris, 1919. Copyright Institut Pasteur/Archives Félix d’Hérelle.

plaques, and even then, invariably in the final samples collected around the time just preceding convalescence. The appearance of the glassy plaques seemed to coincide with the end of the disease, and right then an idea came to me: I thought that if the “generator” of the glassy plaques, from the intestines of the grasshoppers or the dysentery patients, was the instrument of sickness, would not it also function as the instrument of healing?45

D’Herelle’s first results were presented by Émile Roux to the French Académie des Sciences in September 1917 and published soon thereafter in Comptes rendu de l’Académie des Sciences.46 There is no reference to Twort or his findings in these reports; to this day the jury is out on whether this oversight was because d’Herelle was unaware of these results or because he, as claimed later, did not think them related to his own discovery.47 Certainly the basic phenomenon described—­namely

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A Tale of Two Viruses

Fig 1.4 Clear plaques of bacteriophage formed on a lawn of bacteria growing in a petri dish. Photograph © 2019 by Steven M Carr, after © 1963 by WH Freeman.

that of a transmissible bacterial lysis—­was the same. But the investigative contexts and interpretations in the two cases were completely different. Twort, as previously discussed, had come across the phenomenon in a search for ways to cultivate viruses. He was primarily concerned with the phenomenon as it appeared in micrococci, although he had extended his studies to other material and had devoted the last couple of paragraphs to describing a “dissolving substance [of] bacilli of the typhoid-­coli group” on which he hoped to continue studies at a later date.48 But where Twort had suggested multiple possible explanations for the phenomenon, d’Herelle, from his very first paper, interpreted his findings only one way and was unequivocal in his statements that they were viruses. True, he used the the phrase “microbe invisible” in his French publications, but he used virus in his earliest English language writings on the bacteriophages. In a discussion of various hypotheses about source of the substance that caused the lysis of bacteCalled or Recalled to Life

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ria, for instance, he suggested that they could be “secreted by an ultramicroscopic virus, which is a parasite of bacteria. This is the hypothesis by which I have held since my first publication.”49 As for the term bacteriophage itself, which d’Herelle introduced in his first paper, it does not seem to have been originally intended as a neologism for a brand-­new discovery. He began his paper with the declaration: “From the feces of several patients convalescing from infection with the dysentery bacillus,  .  .  . I have isolated an invisible microbe endowed with an antagonistic property against the bacillus,” and in his conclusion even gave this putative agent of lysis a name: “This microbe, the true microbe of immunity, is an obligatory bacteriophage.”50 He appears to have used the term in a descriptive sense, as something that lived at the expense of these bacteria in much the same way as the bacteria themselves lived at the expense of their human hosts. As elaborated in his monograph, which d’Herelle published within a few years of his initial discoveries, “The suffix ‘phage’ is not used in its strict sense of ‘to eat’ but in that of ‘developing at the expense of;’ a sense that is frequently used elsewhere in scientific terminology. . . . This is precisely the interpretation to be given the term ‘phage’ in the word ‘bacteriophage.’”51 Although at first it seemed as though d’Herelle and his discovery would fade into obscurity like Twort, the same war that had stalled or stopped Twort proved to be a catalyst for d’Herelle.52 After a short lull of about two years, d’Herelle’s bacteriophage began to garner an ever-­ widening interest from the scientific community, beginning with scientists who “geographically closest to him” and rippling outward to North America and even Australia.53 As bacteriophage researcher Donna Duckworth has pointed out, in those first few years, “Hundreds of people cited d’Herelle’s work, and although he may not have been universally regarded, he was certainly universally acknowledged.” One possible reason why d’Herelle’s work drew more attention than Twort’s discoveries is that in addition to claiming novelty he emphasized the medical implications of his findings, not only for understanding infectious diseases and immunity but also for disease therapy. As Duckworth noted, “although, for a historical record, d’Herelle’s conclusion that he had found a living organism that would grow only in bacteria (a bacterial virus) is the most noteworthy, for d’Herelle and many others it was this latter observation, that this ‘antagonist’ might be the agent of immunity to bacterial disease, that was the most thrilling.”54 Indeed, the potential applications of the antagonist as an immu-

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A Tale of Two Viruses

nizing or therapeutic agent against dysentery was to remain at the forefront of d’Herelle’s interests for many years to come. Twort, on the other hand, had made no connections between transmissible autolysis and immunity or antimicrobial therapy in his paper. Also, after returning from the war, he seems to have returned to his search for ways to culture viruses rather than pursue investigations into the nature of glassy transformation.55 In fact, Twort appears to have remained as unaware of d’Herelle’s initial work as the latter maintained he had been of Twort’s 1915 discoveries, for he made no public comments about it until his own work was brought into the spotlight by the microbiologists Jules Bordet and Mihai Ciuca (as detailed in chapter 2).56 On a few occasions thereafter, however, Twort would vigorously defend the claim that this group had staked on his behalf. “May I point out that that the work of d’Herelle is little more than confirmation of my work,” he wrote to the Lancet in 1921.57 And upon reading a review of Sinclair Lewis’s Pulitzer Prize–winning novel, Arrowsmith, in which the bacteriophage was a major plot device, he sent a similar note again, objecting that “the author gives the credit for the discovery of the phenomenon of bacterial lysis caused by a contagious filter-­passing material to Dr. d’Herelle of the Pasteur Institute.” Furthermore, he added: “At the time of the publication of the paper I was asked to undertake duties in the army in Salonika, and I had no opportunity to work out any additional details connected with the phenomenon, although when in Salonika the whole subject was discussed with the Canadian, French, and British medical officers there. The first work of d’Herelle on the ‘lysin,’ named by him the ‘bacteriophage,’ associated with the dysentery bacilli was not published until nearly two years after the appearance of my paper.”58 The editors of the Lancet seem to have had only limited sympathy for Twort’s claim, responding to it with the terse footnote, “We have referred to the matter in our columns as the Twort-­ d’Herelle phenomenon on more than one occasion.” This note from the editors serves to illustrate the impact that the participation of a famous personage can have on a scientific debate, for before Bordet—­t he sole recipient of the 1919 Nobel Prize—­entered the bacteriophage fray, scientists had used d’Herelle’s label of bacteriophagy to describe transmissible lysis quite unproblematically. After the priority issue was raised, however, the phenomenon came to be called the Twort-­d’Herelle phenomenon for a time. But in the long run it was the shorter label that stuck, and remains in use to this day. Called or Recalled to Life

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The incident also exemplifies, once more, the value of a comparative Plutarchian narrative in showcasing the small details and nuances of an episode that may have been overlooked in individual, perhaps more linear histories. Both in the case of bacteriophagy and in that of the discovery of the chicken sarcoma agent, for instance, the priority issue was spurred by people not directly involved in the actual investigations: Flexner and Fruton, as shown in the instance of Rous, and Bordet’s lab in the case of d’Herelle. But Rous was only charged of wrongdoing by implication—­and inadvertently at that—­by both Flexner and Fruton, both of whom acknowledged their errors. Bordet and his colleagues, on the other hand, claimed that Twort’s work had been overlooked by d’Herelle, albeit unknowingly, and roundly declared that they believed it was “a duty to recognize the incontestable priority of Twort in the study of this question.” In this paper, which was first presented before the Belgian Society for Biology in March 1921, the authors also announced, “The burden of an exact history makes it necessary for us to cite a previous work which d’Herelle has not known and that we ourselves have been ignorant of until now that contains the observations that d’Herelle had made. This remarkable work by F. W. Twort appeared in Lancet in 1915, that is to say, two years before the research of d’Herelle.”59 In contrast to Rous, who had expressed his concerns in private letters to Flexner and Fruton, d’Herelle defended his position publicly many times. The first occasion was at a meeting of the Society for Biology in Paris—­t he French counterpart to the society where Bordet and Ciuca had earlier presented their papers—­and his presentation was later published in the Society’s proceedings.60 In this and other early response to his critics, d’Herelle neither denied nor admitted to having prior knowledge of Twort’s 1915 work; rather, he emphasized the difference in their findings, arguing that Twort’s description of the phenomenon with micrococci was “not a question of a real bacterial dissolution, but a transformation of a normal culture on agar into a glassy and transparent one.”61 For reasons that are not entirely clear, d’Herelle “abruptly left” the Pasteur Institute in Paris in 1921 and over the next two decades or so pursued his studies on bacteriophages, especially their use in therapy against bacterial diseases, in various places, including Leiden, the United States, Egypt, India, and Russia.62 His career trajectory—­t he pursuit of a single topic in many far-­flung places—­presents a striking contrast to that of Rous, who pursued many different topics over the course of his career, but stayed at the Rockefeller throughout. What both men

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A Tale of Two Viruses

shared, however, was a steadfast belief in their interpretation of their findings and an active participation in debates over the viral identity of their respective discoveries for many decades.

Why Viruses? Why did both Rous and d’Herelle think of their discoveries as “viruses”? How exactly did they conceptualize the entities? The answers to these questions are by no means straightforward, for the term virus represents a very good example of a scientific concept that has undergone multiple changes, or “variances,” in meaning over time, to borrow the vocabulary of the historian of science and medicine Ilana Löwy.63 It was only in the 1950s that virus came to acquire the definition we recognize today: an obligate intracellular parasite basically composed of a single type of nucleic acid encased in a proteinaceous coat.64 A survey of earlier literature shows widely varying definitions for the term for at least half a century prior: “Every virus is a microbe,” the famed Louis Pasteur had declared in 1890, whereas just over two decades later, the Harvard bacteriologist S. B. Wolbach was defining filterable viruses far more specifically as “microorganisms which will pass through filters, the pores of which are too small to give passage to ordinary bacteria.”65 As Löwy argued: “Scientists naturally employ the vocabulary of their discipline. But we should not assume that the meaning of the terms remain constant. . . . Within a single scientific community at least, scientists assume that a given term has the same meaning for every potential reader of a scientific work. [They] employ terms to sum up a scientific consensus at a given moment, and, more importantly, they assume that a consensus does indeed exist. Many scientific terms do not, however, possess a single, well-­defined meaning.”66 Rous retrospectively alluded to such a fluidity and change in the meaning of virus when discussing the reasons he had refrained from using the term in his early publications: “I wanted to call the tumor cause a virus, but the crusty, redoubtable, lovable old Secretary of the Board of Scientific Directors, Dr. [T. Mitchell] Prudden, whose wisdom I admired, put his granite foot down against it, suggesting ‘agent’ instead.”67 He also admitted that by not letting him use this term in publications, the “older and wiser” Prudden had done him “a good turn, since the virus proved in some ways so peculiar that not until the time of my Harvey lecture . . . , when the traits of viruses generally were better realized[,] could it safely be called as such.”68 Given the intellectual and institutional contexts of Rous’s discovCalled or Recalled to Life

29

ery, it is actually rather remarkable that he considered viruses or any sort of living parasite as a possible cause of the chicken sarcoma at all. His colleague Murphy’s attitude, discussed in greater detail in later chapters, was rather more typical of what one might have expected from someone trained in medical pathology, especially in the immediate wake of a consensus—­arrived at during an international cancer congress in 1910—­t hat cancer and tumors could not be caused by living organisms or parasites.69 Based on his body of work at the time of the discovery, it is possible to discern that Rous’s definition of a virus at the time of his initial discovery of the sarcoma agent in 1911 was that of an extremely tiny living infectious organism that was invisible under a light microscope and could pass through bacteriological filters impermeable to the smallest known bacteria. Rous’s willingness to accept that a tumor could be caused by such an entity indicates an extraordinarily flexible mind, but one must also consider the influence of his work environment in shaping his ideas. Simon Flexner, who had hired Rous directly and principally to work on the cancer problem, had done so within his own division of pathology and bacteriology at the Rockefeller. In part such a move was due to the fact that Rockefeller had not prioritized cancer research at the time of its founding—­reflecting the aforementioned attitudes of such prominent medical researchers as William Welch, who was also the president of the institute’s board of scientific directors.70 Nevertheless, as Rous and others experienced firsthand, it provided an environment that was particularly conducive to new ideas.71 Flexner, who as founding director of the institute would have has a greater degree of involvement with its day-­to-­day affairs, compared to Welch, was not as pessimistic about the prospects of this field. In fact, he had led the way at the institute with his 1906 discovery of a transplantable tumor of rats.72 Although he never worked on the avian sarcoma problem himself, he remained in Rous’s corner, so to speak, on the matter of the possible viral etiology of the tumor.73 Of the various criteria, it was the living nature of the sarcoma agent that was perhaps the most difficult for Rous to demonstrate. Then, as indeed is the case even today, the “most direct means of proving that the agent is alive is to grow and transfer it in culture,” as Rous and Murphy rightly pointed out in their report.74 But the recognition that viruses are fundamentally different than bacteria and thus would require rather different materials and conditions for growing in culture, was many years away. Consequently when Rous tried different ways to

30

A Tale of Two Viruses

cultivate—­t hat is to say, grow and propagate—­t he sarcoma agent in vitro, he did not succeed. Meanwhile, his reason for thinking that the sarcoma agent might be living was based on numerous pieces of indirect evidence, including its survival—­t he retention of its biological activity under different conditions known to destroy or inactivate other living cells—­and its capacity to multiply in tissues of unaffected birds when injected therein.75 In one of their earliest joint papers, Rous and Murphy had concluded, “No single attribute among those determined suffices to show the nature of the agent; yet taken together, its character are those we associate with micro-­organisms.”76 In a single-­authored publication later that year, Rous cited more specifics regarding the sarcoma agent’s behavior under different physical and chemical treatments, and concluded even more definitively: “The various features seem sufficient to identify it as a living organism in distinction from a ferment.”77 D’Herelle, as seen, had showed not the slightest bit of hesitation in declaring his find “an invisible microbe,” or virus, in both the title and opening sentence of his very first presentation about the transmissible agent of bacterial lysis isolated from the stools of dysentery patients. Relying on much the same line of reasoning as Rous had for the sarcoma agent—­namely, its capacity to multiply in fresh uninfected host cells—he also went on to offer what he believed was “visible evidence” for the lysis being caused by a living agent: “If one adds to a culture of Shiga [the dysentery bacillus] approximately one to a million of an already lysed culture, and if, immediately after, one spreads out on an agar slant a drop of this culture, one obtains, after incubation, a coat of dysentery bacilli showing a certain number of circles about 1 mm in diameter, where the culture is void; these points can only represent the colonies of the antagonistic microbe: a chemical substance would not be able to concentrate at defined points.”78 A few years later, he elaborated his argument with the description of an experiment and the following interpretation of its results: It is the presence of these immutable bare spaces, which are perfectly circular, that characterizes what we have named “bacteriophage.” . . . The number of spaces depends simply on the quantity of filtrate added to the bacterial culture. If into various bacterial emulsions we introduce variable quantities of the filtrate, the number of bare spaces is strictly proportional to the quantity of filtrate added. On the other hand, the number of the bare spaces is inde-

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pendent of the number of bacteria contained in the medium.  .  .  . The phenomenon of these vacant spaces is only comprehensible on the supposition that the bacteriophagic principle . . . the source of the lytic enzymes, is a corpuscle; and that each corpuscle deposited on the agar in the midst of the bacteria gives rise to a colony of these ultramicroscopic corpuscles, such a colony being represented by a bare space.79

In the same paper, d’Herelle added, unconsciously, and almost uncannily echoing Rous in both the nature of evidence and reasoning, “The behaviour of bacteriophage towards physical and chemical reagents is that of a living being, and does not agree with that of an enzyme.” He held tenaciously to this original conception throughout his life, defending his position against challenges and attacks from different fronts and for many years. What is perhaps his most detailed treatment of this issue appeared in his second monograph on bacteriophages, the English translation of which was advertised using d’Herelle’s own words: “Of the present book the author says, ‘I offer physiological proof of the living nature of the bacteriophage, an infravisible parasite of bacteria. Logic demands that the evidence which I have provided be justly evaluated before contrary theories be affirmed.’”80 Despite the differences in the particulars—­of geography, investigative goals, experimental systems, and even of specific terminology that they used—­t hen, Rous and d’Herelle had a fundamental idea in common, one that would stay with them through the decades that followed. Both conceived of their discoveries as “infective agents” of some extrinsic or exogenous origin, which caused their effects by entering, or infecting, the host cells, and somehow disrupting normal functions within—­in other words, as viruses.81

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A Tale of Two Viruses

OF INCREDULITY 2EPOCHS AND BELIEF RECEPTION AND RIPPLES I have never believed in the virus theory. —James B. Murphy, 1928 The invisible virus of d’Hérelle does not exist. —Jules Bordet, 1931

As the focus of the drama surrounding disease causation shifts from discovery and conceptualization to reception, the parallels in the trajectories of the histories of the sarcoma agent and bacteriophage grow all the more remarkable. The reaction and opinions of entire research communities rather than individual discoverers are scrutinized in this chapters. There was little overlap in the disciplinary interests of the two communities—­one interested in cancers and tumors and the other in bacterial infections of the gastrointestinal tract—but their responses were remarkably similar. In both cases there was no disputing the data: as Peyton Rous and his colleagues had shown repeatedly, sarcomas in chickens were clearly transmissible via a filterable “principle” that survived many a harsh treatment guaranteed to destroy whole cells.1 Similarly, the transmissibility of bacterial lysis in the experiments of Félix d’Herelle with dysentery bacilli—­and before him, Frederick Twort with micrococci—­was never questioned, whether it manifested as glassy transformation in liquid cultures or as the glassy plaques (taches vierges) on lawns of bacterial culture on solid agar.2 Both Rous and d’Herelle, however, met with considerable resistance from their

33

respective research communities to their hypotheses on the identity of their finds as living viruses, especially at the outset. In his Nobel lecture more than half a century after his discovery, for instance, Rous would recall that for the most part “the findings with the sarcoma were met with down-­right disbelief.”3 Late in his life, d’Herelle, too, would recall of the years immediately following his discovery, that “at that time there was nobody I think, except Dr. [Émile] Roux, who took the matter seriously.”4 That the immediate reactions to the hypotheses about the causative agents of the chicken sarcomas and transmissible bacterial lysis were largely negative is perhaps not too surprising. After all, both men had made discoveries that ran counter to all that was known about disease and disease agents at the time. Cancer was after all not a contagious disease, so how could it be caused by a virus? As Rous would remark to a friend in response to a congratulatory message when decades later, he was awarded the Nobel Prize for Physiology or Medicine, “I never expected to get the Prize myself because ‘Virus’ is an evil word in every one of its several senses. It took courage for the Nobel Foundation to give it.”5 As for bacteria, microscopic in size, unicellular in form, and asexual in their reproduction, they were not even considered as true living beings by the entire community, and so the idea that they could be subject, in turn, to infections by even smaller versions of themselves was also difficult to accept. Curiously, however, in both cases, what came under fire from their colleagues was the discoverers’ interpretation of their evidence rather than the experimental data. Moreover, the reasons underlying these criticisms were rooted in the similar types or styles of thinking. Once again, the juxtaposition of these histories reveals details that were likely overlooked in the otherwise very fine narratives about the individual discoveries, and provides a better perspective into certain wider paradigms in medical science, which affected the way in which the discoveries were conceptualized by different research communities.

“Downright Disbelief” and Other Reactions to Rous In October 1910, just around the time that James B. Murphy joined Rous at the Rockefeller, cancer researchers from different countries in the world were convening in Paris for an international conference, the second of its kind since the turn of the century. The British Medical Journal reported shortly thereafter: “Throughout the whole scientific pro-

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A Tale of Two Viruses

ceedings it was evident that there were two schools of thought, representing respectively those who cautiously, and perhaps more vaguely than in the past, believe in a parasitic etiology, and those who regard such an etiology as quite incompatible with the natural history of cancer.”6 Despite the fact that attendees of the conferences were divided on the specifics of cancer causation, they reached a more or less formal consensus that the cancer was not caused by an infectious agent. The decision was the culmination of a drawn-­out debate over the past decades over what had been known as the “parasitic theory” of cancer; namely, the idea that it was a disease “due to an extrinsic cause.”7 British bacteriologist and cancer researcher H. G. Plimmer, a proponent of such a theory, described it thus: “The idea that cancer is an infectious disease is an old one: so long ago as 1797 it was classed amongst infectious disease by the Prussian sanitary laws. But the question only came actually into practical pathology after the development of bacteriology: then the differences between cancer and the other bacterial infectious diseases gave rise to the view (after a very short-­lived cancer bacillus) that it may be due to an organism of another kind.”8 But others, such as the British surgeon Henry Morris, one of the founders of the International Cancer Research Fund, were skeptical about both the validity and the future of the parasitic theory: “The theory of the microbic origin of cancer still occupies the very forefront of cancer research. There are not wanting, however, indications that it is on the wane and that it may soon come to be described,  .  .  . as ‘belonging to history.’” Following in the wake of the discovery of the tuberculosis bacillus, he explained, “A very large number of scientific workers made it their aim to demonstrate, if possible, the existence of a corresponding micro-­organism as the causative agent of carcinoma and sarcoma.” But despite a number of claims that followed regarding the “alleged discovery” of different bacteria, protozoa, and fungi as possible causes for different tumors and cancers, Morris noted that no one had furnished any repeatable experiments or concrete evidence in support of their claims and theories. As a result, he concluded: “Several eminent observers were arriving at the conclusion that many of the supposed micro-­organisms were the factitious products of faulty technique, and that such micro-­organisms as had been actually discovered played but a very subordinate role, if any, in the evolution of malignant tumours.” 9 In his 1908 Harvey lecture, the American cancer researcher James Ewing reiterated the problems with the parasitic theory identified by Morris, going so far as to provide what he claimed was “a partial list of Epochs of Incredulity and Belief

35

the various structures or micro-­organisms at one time heralded as the cancer parasite, with name of author and date of publication.” In this lecture, Ewing also foreshadowed arguments on the usefulness of tumor transmissibility as evidence for parasitic causation, claiming, for instance, that the mere fact that tumors were inoculable proved nothing about their etiology.10 By the time of the international congress in 1910, then, the failure to find any definitive evidence for the presence of any cancer parasites had led all but a few die-­hard proponents of such theories to arrive at a consensus that tumors and cancers were not the result of extraneous causes. Coming as soon as it did after this conference, Rous’s announcement that the chicken sarcoma agent was a living agent was naturally greeted with skepticism that was likely linked to a reluctance to revive a decades-­long debate that had only just been settled. If such an international consensus of disbelief were not bad enough, Rous faced even stiffer opposition to his idea that right at home, from none other than Murphy, his closest collaborator on the chicken sarcoma work. Exactly why Murphy was so intractable in his opposition to the idea of a sarcoma virus right from the beginning is not clear, in part because there is very little in his personal papers or correspondence with Rous that sheds light on the matter. In fact, he appears to have been so reticent about his disagreement with the virus theory that even colleagues working closely with him were unaware of his stance. For instance, in a 1928 letter to a colleague who had worked closely with him in the cancer laboratory from 1918 to 1925, Murphy said, “I am rather surprised at your expression of surprise that I was opposed to the virus theory of chicken sarcoma.”11 Unless more personal records surface, it is entirely possible that the question about the earliest differences of opinion between Rous and Murphy will remain poorly understood by historians. But the difference in their opinions bears at least a passing mention because the oft-­cited reasons of the formative influences on the research and thought styles of different researchers fall short in credibly explaining the widely divergent views of these two men. Indeed, both come from very similar backgrounds, having studied medicine at Johns Hopkins—­under the mentorship of William Welch, one of the pioneers of medical science in the United States—­and specializing in pathology. Welch was also the chairman of the Board of Scientific Directors of the Rockefeller, where both men then took up positions in quick succession to work on cancer research. If anything, the commonalities in their backgrounds would have led

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A Tale of Two Viruses

one to predict that their views were aligned with rather than opposed to one another. The main discernible difference between the two men seems to have been in their personalities. Recalling his stint as a visiting scientist at the Rockefeller from 1926 to 1928, Hungarian-­American medical researcher Jacob Furth described Rous and James Murphy as “both brilliant contributors to cancer research but mutally [sic] incompatible. Both were great men, but not without human frailty.”12 The impression one gets about Rous from his many letters to various people, as well as reminiscences from friends and colleagues, is that he was gregarious and outgoing with a lively and adventurous sort of mind. At a memorial tribute some months after Rous’s death, James Henderson, a colleague who worked at the Rockefeller from 1957 to 1970, compared the atmosphere in the Rous laboratory to a “playroom [where] his assistants, neighbor children, asked to play for awhile.”13 Rous’s longtime friend and correspondent Christopher Andrewes described him as someone who “was throughout his working life constantly excited by every new fact he brought to light,” with “an insatiable curiosity about the facts of nature,” as well as a keen interest in people, “always wanting to know more about them.”14 In addition, it might be argued, Rous’s role as editor of the Journal of Experimental Medicine gave him more exposure to a wide array of happenings in biology and medicine, which, in turn, would have made him receptive to unusual and heterodox ideas. Murphy, in contrast, seems to have been far more reserved. His obituary describes a “manner of quiet gentleness that won him many devoted friends” with a dominant trait of “great conscientiousness.”15 The meticulousness that seems to have been Murphy’s dominant trait also hints at an imagination that was, perhaps, a bit more conservative and therefore more cautious about embracing the new kids on the block that were the viruses. Additionally there is some some indication that Murphy’s reticence may have stemmed from a lack of confidence in his ability to articulate his ideas as fluently as Rous. Nearly a decade into Murphy’s tenure at the Rockefeller, for instance, Simon Flexner would write to him with the observation, “You still have trouble writing an effective, clear style. I believe the time has come to put yourself in the hands of a tutor. . . . After all, you do wish to write effectively; indeed, it is imperative that you should. And I believe you are quite able to achieve this end under proper guidance. Please think of this suggestion seriously.”16 Unfortunately, the archival materials do not ofEpochs of Incredulity and Belief

37

fer evidence one way or another as to how, or indeed whether, Murphy responded directly to Flexner on this matter. Rous’s recollections that he was met with “downright disbelief” and that his work had not received the attention it deserved from the scientific community may have been somewhat exaggerated.17 Although his ideas certainly went “against the grain” of the thinking of the mainstream of cancer research, he never had to contend with institutional or personal barriers to continuing his research on the sarcoma agent at the Rockefeller.18 For instance, whatever reservations Murphy might have harbored toward the theory, he did not, or could not, prevent Rous from calling the agent a living organism in any of their joint papers. When Flexner advised Rous against publishing negative findings, it was not so much because he had any quarrel with Rous’s theories or experiments—or if he did, I could find no evidence of it in the many letters exchanged between the two men—but rather because he thought “they would keep other workers from trying, who might have better luck.”19 We know that when Rous did quit the field in 1915 it was largely voluntary and that it had no effect on his standing at the Rockefeller. Also worth considering is the fact that despite the consensus at the conference, the various parasitic theories of cancer never entirely lost their presence, even if only within a small minority. We already noted one example in Plimmer; still another researcher who strongly supported such views was the prominent American surgeon Roswell Park, who thought that this theory was “the only one that satisfies the needs of both the pathologist and the clinician.”20 There was even additional evidence for the parasitic theory in the form of an independent discovery by two Japanese scientists of another transplantable sarcoma of chickens, although this discovery seems to have escaped contemporary notice from most of the cancer research community, including Rous.21 For example, although a footnote in a 1912 paper from his laboratory refers to an earlier 1911 report by the same authors of “success in transplanting a myxomas of the fowl,” there is no mention of the transplantable sarcoma as corroboration for his data.22 Exaggerated as his complaints were, Rous was not entirely without justification for his feelings of isolation at the time he put forth the idea of the sarcoma agent. Even though Murphy did not make his disagreements public, his opposition must not have been easy to contend with. By 1911 such leading proponents of the parasitic theories as Plimmer and Park were retired; indeed, neither of them lived for much longer

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and so could not have offered Rous the kind of intellectual support he must have sought. As for the Japanese discovery, perhaps because their work on the filterability of the sarcoma agent seems to have met with disbelief by their local colleagues, the authors, in their English-­ language publications, focused on their more successful work transplanting the tumors across species.23 So we may surmise that Rous found little or no alleviation from his isolation from those quarters either. The majority of the cancer research community went along with the consensus, described by the British delegate to the conference, Ernest Bashford, as follows: “In regard to the causation of cancer, a mass of evidence was presented supporting the view that cancerous tissue is really a biological alteration of the tissue proper to the individual attacked by the disease, and that its peculiar properties may be explained without assuming the intervention of extraneous agencies, such as a hypothetical cancer virus.”24 Bashford’s language in this report is actually somewhat anachronistic, for until Rous reported his finding with chicken sarcoma, the term virus had not been associated with the parasitic theory of cancer. In the extensive “partial list” provided by Ewing, for instance, there were over forty examples of possible parasites reported between 1876 and 1907, but not a single one was identified as a virus.25 Rous had been the first to suggest that a virus—or in his words, a “minute parasitic organism”—might be the cause for certain tumors, but he had done so after the conference.26 It is unlikely, therefore, that anyone at the conference explicitly mentioned viruses as a candidate for cancer causation in any of the formal presentations; certainly the earlier report did not mention anything along these lines. Consequently Bashford’s mention of “a hypothetical cancer virus” in his report, which was published later, might justifiably have seemed to Rous to be have been a pointed reference to his own work suggesting, if not naming, a virus as the possible cause. Further compounding the skepticism or disbelief that greeted Rous’s theory was the fact that many cancer researchers, especially those working with patients, considered his findings to be largely irrelevant to their primary concern, which was the clinical management of patients with cancer. A notable example is Ewing, prominent in the American medical research scene, who by 1910 “had arrived at the conviction that the best hope for advancing knowledge in cancer was to study the disease in man.”27 More than a decade later, the New York City–based German surgeon Willy Meyer would claim that “true canEpochs of Incredulity and Belief

39

cer” occurred only in vertebrate animals.28 Adding more fuel to this fire was the fact that, as discussed in chapter 1, Rous was unable to find examples of any mammalian tumor analogous to the transmissible sarcoma of chickens. Consequently, as Andrewes would later note, Rous was besieged from both ends. “Some argued . . . that the growths were not really cancers; others that the infective agent was not a virus.”29 Perhaps the greatest barrier for the majority of cancer pathologists to accept the possibility that a virus could cause tumors or cancer was the fact that such a theory implied that cancer was somehow an infectious—­t hat is, contagious—­disease. Such an implication flatly contradicted the available evidence on cancer. “The contrast between the transmission of infection and the propagation of cancer is worthy of the serious attention of all those brought into contact with patients suffering from the disease, or engaged in its investigation,” wrote Bashford, comparing, for example, the propagation of tuberculosis—an established infectious disease—to that of cancer from the lesions of an affected patient. In the case of tuberculosis, a new animal implanted with tuberculous tissue clearly acquired the infection in its own tissues while the implant itself died. In contrast, during the experimental transmission of cancer: “The tissues of the new hosts do not acquire any cancerous properties; they merely react to the presence of the cancer cells and supply them with nourishment. The process is fundamentally different and distinguishable from all known processes of infection.”30 Such attitudes persisted for many decades, as evidenced in the writings of the French cancer researcher Charles Oberling: “If cancer were contagious, doctors, nurses, surgeons, pathologists, who come into daily contact with cancerous tissue without taking any special precautions, ought to be particularly liable to it. But they are not.”31 Bashford and Oberling’s remarks underscore their practical concerns about managing the disease in patients and regulating various personnel working with cancerous materials. Even as he acknowledged the potential usefulness of studies on animal models on the transmissibility of cancer, for instance, Bashford expressed concerns about the take-­ home messages such studies offered the general public, explaining: “It would be a grave misfortune if the increasing flood of alleged cures of transplanted cancer in animals led to an augmentation of the number of persons who, disdaining or fearing surgical advice and treatment, prefer ‘treatment’ by some other less efficacious or even useless method, or by some of the new chemical preparations already prematurely placed upon the market.”32

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A Tale of Two Viruses

Although the issue of the potential living nature and of the sarcoma agent and its consequent infectiousness or contagiousness lay at the heart of most of the mainstream cancer researchers’ objection to Rous’s ideas, there were also other hurdles to the acceptance of the virus theory. Chief among such hurdles were those reasons that had also led Rous to quit sarcoma work altogether; chiefly, the consistently negative outcomes of attempts to isolate and propagate an identifiable agent from different transmissible avian tumors. Fortunately, chicken sarcoma research did not die with Rous’s departure from the field. Although he did not do any active research on the subject, Rous did not destroy any of the chicken sarcoma tissue but rather maintained the material in his laboratory and for many years continued to supply dried material to anyone who requested it. Among his papers archived at the American Philosophical Society are several folders of correspondence dealing with requests for the sarcoma material, which date well into the 1930s.33 Although most of these exchanges seem short and impersonal, there are a few revealing snippets, showing that, along with the prominent “epoch of incredulity” that Rous remembered, there also coexisted a small but steady group of people who sustained an “epoch of belief” in his ideas. A good example is to be found in a letter from Stephen Baker, a physician-­researcher at the Middlesex Hospital in London, requesting some experimental material a good decade and a half after Rous had quit the field: “I take it that you (confidentially) believe that the agent in these tumours is a virus though you are guarded on the matter in print?,” Baker wrote. “I don’t think anyone who has worked on the matter could escape from this view, as being the simplest explanation of the facts available at the present time (although Murphy seems inclined the other way).”34 Rous’s response to Baker shows him steadfast in his original views about the living nature of the agent, despite his long hiatus from activity in the field. “My own belief has always been that the agents causing these tumors are viruses, though the statement is confidential to you,” he replied, adding, “To suppose them such is to invoice no new and strange entity but to explain the phenomena in terms of the already known.”35 Another request for material came from British researcher William E. Gye, at the National Institute of Medical Research (NIMR) in London. Trained in chemistry before he went on to study medicine, Gye had become “thoroughly steeped in the cancer problem” at the Imperial Cancer Fund in London, where he was a member of the staff from Epochs of Incredulity and Belief

41

1913 to 1914. He joined the NIMR in 1919 and had started to work on the Rous sarcoma agent sometime in 1923. Not long after his first set of experiments, he wrote to Rous again requesting for and offering information about the “virus of the ‘Rous sarcoma,’” having heard rumors about its successful cultivation at the Rockefeller. Two years later, Gye published his first paper on the chicken sarcoma agent in the prestigious British medical journal the Lancet. Beginning with a description of Rous’s 1911 discovery, Gye went on to describe his experiments, largely corroborating Rous’s findings that the agent was a “living but extremely small microorganism,” and concluded with the statement, “These researches have led me to look upon cancer—using the term in its widest sense—as a specific disease caused by a virus (or group of viruses).”36 Although profuse in offering thanks for Gye’s “generosity for some of the thrilling moments of a lifetime,” Rous, doubtless based on his own experience, predicted that Gye was likely “in for a fight that may last for years. The methods by which you prove your points are so exacting, so bound to fail in the hands of the inexpert, that you will have to be prepared for a shower of critical and contradictory papers. I wish you more than well in the fight.”37 Trouble of the sort Rous predicted began even as he was writing his letter, and likely before Gye had even seen it. The editors of the Lancet had published Gye’s paper in their July 18, 1925, issue, along with a related but independent paper on the visualization of the agent by ultraviolet microscopy by an NIMR colleague, Joseph E. Barnard, heralding the pair as marking “an event in the history of medicine [that] may present a solution of the central problem of cancer.”38 This was the kind of language bound to incense the cancer research traditionalists, as indeed it did; in America, the July 29, 1925, issue of the New Republic ran an article titled “Cancer Yields a Secret,” which declared that with their publications and the claims and conclusions therein, Gye and Barnard had “produced a furor that would do credit to a minor war. Half a dozen small nations would be required for a brawl of equal news value.” Just a few weeks later the same magazine published a piece by Francis Carter Wood, an eminent cancer researcher at Columbia University, deeming—or damning—both papers as “somewhat disappointing.” In Wood’s opinion, both papers were “obscure, apparently hastily written, and there is not as much in them that is new as might have been expected from the cabled reports. . . . His statement that the cause of human cancer is ‘almost certainly a virus’ is not only not proved by doctor Gye but the evidence in his paper is not even sufficient to make it probable that

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it is true.” Poking several holes in Gye’s experimental conclusions and theories, Wood concluded in a manner consistent with the attitude of clinical pathologists working on cancer at the time: “The most that can be said is that Doctor Gye’s paper contains some new observations obtained by the most painstaking work, but upon which has been pyramided a threefold hypothesis: that he has discovered the organism related to the Rous chicken sarcoma; that this organism has been photographed; and that on the basis of his work a germ cause may be assumed for cancer in general. [It] might also be well to wait until a firmer foundation is placed under the germ theory of cancer before changing our present view of the disease.”39 Despite, or perhaps because of, this sort of negative reaction, Gye’s work continued to capture attention and became one of the hottest topics of discussion at the annual meeting of the American Zoological Society, held in New Haven in December 1925. It was here that Murphy seems to have first gone public with his opposition to the virus theory of cancer, raising some of the very objections that Rous had warned Gye against. “Am I wrong in supposing that in your cultivation work you employ a fresh piece of embryonic tissue at every transfer?” Rous had asked Gye. “The question will be raised whether in doing so you have not merely caused this tissue to become sarcomatous.”40 Sure enough Murphy homed in on this very point: It must be said that as the work stands at the present time [Gye’s work] is open to entirely different interpretations than the one he places on it. . . . In subcultures of chicken tumors he always adds a generous supply of fresh embryonic tissue. It has long been known that embryonic tissue may assume a malignant character under the influence of the filtrate from the chicken tumour and in time cells are capable of producing more of the active agent. . . . This point has not been considered by Gye. Nor has he considered the possibility of the actual survival of the tumor cell in the culture.41

From these passages one can see that that Murphy objected more to Gye’s interpretation of the results than to his experimental approach or data. In Murphy’s view a living virus was certainly not the only possible explanation for the ability of the agent to maintain transmissibility over several successive generations (Gye had demonstrated the ability of subcultures of a tumor to induce sarcomas through at least five successive passages). The wider medical research establishment also reacted uniformly negatively to Gye’s work: at an international conference in Epochs of Incredulity and Belief

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1927, cancer researchers even passed a formal resolution declaring that “it may be accepted for all practical purposes that cancer is not to be looked upon as contagious or infectious.”42 In the face of such formidable opposition, it is a wonder that theories of viral etiology survived at all, but as we know now, the virus theory not only survived but eventually emerged triumphant, resulting in a Nobel Prize for Rous—but only in 1966, more than half a century after his discovery. For much of the intervening period Rous was in a lonely minority of researchers—albeit never entirely alone—who believed that viruses did have an important role to play in originating tumors. Most of his support came, as I explain further, from virologists who, because they worked with different viruses, were perhaps more willing than cancer researchers to accept the idea that such beings could induce disease in their hosts without said disease being contagious. The staunchest among these supporters were doubtless Andrewes and Gye in England, both of whom maintained lifelong correspondences with Rous wherein the viral theory was heavily discussed. But Rous also had other supporters for the virus theory of tumors closer to home. Perhaps the most notable was Thomas Milton Rivers, the “apostolic father” of the virology of his time.43 In 1928 Rivers published a seminal review on the pathological features of various viral diseases, in which he strongly supported the notion of the viral etiology of tumors. In fact, he cited it as an example of one of two types of pathological changes that resulted from viral infections. While the first category took the form of degenerative or destructive changes, similar to those induced by other pathogens such as bacteria or protozoan parasites, the second class of changes manifested in proliferation or growth, resulting in “pathological conditions such as warts, molluscum contagiosum, and tumors.”44

Debating and Defending the “So-­Called” Bacteriophage In the very next sentence in the same review article, Rivers went on to say that if, instead of proliferation, “destructive or retrograde changes prevail, diseases such as varicella, vesicular stomatitis, and lysis of bacteria are the consequence.” While this latter comment may give the impression that by the late 1920s the bacteriophage issue was resolved, such an impression would be no more correct than to imagine that the inclusion of tumors in the earlier set of examples meant that the idea of viruses as agents of carcinogenesis was acceptable to cancer researchers. Indeed, an address by the Belgian microbiologist Jules Bordet in 1931 shows him staunch in his opposition to the idea that the phenom-

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enon of bacteriophagy was caused by a virus of any sort. Although he acknowledged that the “virus-­t heory of d’Herelle appears truly seductive, by reason of its great simplicity,” he nevertheless reiterated his belief that it was not a correct interpretation of the facts: “The invisible virus of d’Hérelle does not exist. It is the bacteria themselves, subjected to the lysis, which reproduce the lytic principle. Bacteriophagy is accordingly a case of autolysis.”45 Compared to the well-­populated and widespread cancer research community, the community of researchers who were interested in bacteriophagy—a brand-­new discovery—was understandably quite small during the early decades of twentieth century. While most of these early groups were concentrated in Europe, there was a smattering of interest from the United States as well. As detailed by various historians, including Alan Varley and William Summers, and as corroborated in the exhaustive bacteriophage bibliography compiled by Hansjürgen Raettig, the initial round of reactions to d’Herelle, consisting of twenty-­ one papers published sometime in the third year after his initial 1917 announcement, came from geographically close quarters—at least half of them by colleagues within the Pasteur Institute itself.46 The first person to express disbelief in the idea that bacteriophages were viruses—“invisible microbes,” in d’Herelle’s words—seems to have been a visiting collaborator, Tameza Kabeshima. Sometime in 1919, Kabeshima, a microbiologist interested in vaccinations and the prevention of epidemic diseases, such as dysentery and cholera, who was visiting the Pasteur from Japan, had begun to collaborate with d’Herelle on his bacteriophage investigations. But despite some early success in using the lysis-­inducing substance for precisely the purpose that d’Herelle had touted—namely, its ability to protect infected animals from the lethal effects of their bacterial pathogens—Kabeshima argued against the microbial identity of the agent of lysis. He even went so far as to label the agent as the “so-­called filterable bacteriophage of d’Herelle” (du dit ‘microbe filtrant bactériophage’ de d’Herelle) in the title of his paper. Although he began by presenting d’Herelle’s opinion that the clear plaques or points of lysis that appeared on a bacterial lawn were “colonies of the bacteriophage microbe [because] he thinks that a chemical substance cannot concentrate itself into such definite points,” Kabeshima went on to counter that viewpoint, arguing: “In my opinion, however, their action [that of the lytic agent] greatly resembles that of a ferment [enzyme]: An extremely small quantity suffices, in a very short period of time, to dissolve a relatively large quantity of Epochs of Incredulity and Belief

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bacteria; this fact can be considered as a phenomenon of digestion.” He even added a footnote at the conclusion of this paper advising readers that “the expression ‘the microbe or the principle of d’Herelle’ used in my previous reports should read as ‘solution of bacteriolytic immune enzyme.’”47 Kabeshima’s rather pointed attempt to distance himself from d’Herelle was but the first volley in a protracted battle over the nature of bacteriophage, which would last for several years. But save for one follow-­up publication later that year, Kabeshima himself does not appear to have pursued the problem of bacteriophage identity after leaving the Pasteur Institute to return to Japan.48 D’Herelle’s most formidable and influential opponent on the matter of bacteriophage was Bordet, who fired his opening shots at a meeting of the Belgian Society of Biology on October 21, 1920, and whose opposition, as indicated earlier, would remain just as fierce more than a decade later.49 Several historians, including Varley, Summers, and Ton van Helvoort, have persuasively demonstrated that Bordet’s attention to d’Herelle’s work on bacteriophage was very likely drawn by the bold claims the latter made about the role of bacteriophage in immunity.50 Recall from chapter 1 that d’Herelle had labeled the bacteriophage as the “true microbe of immunity,” based on his belief that that it could confer immunity against dysentery bacteria to an infected organism (e.g., humans) by causing these bacteria to lyse.51 To Bordet, an established immunologist who won the 1919 Nobel Prize for his work on the specific substances that animals (mammals) produced in response to microbial infections in order to protect themselves, d’Herelle’s announcement would have seemed to blatantly challenge his authority.52 This challenge was exacerbated in 1921 with the publication of d’Herelle’s first book, Le bactériophage; son rôle dans l’immunité, in which he explicitly took on the very theories of immunity to infection for which Bordet had received his Nobel.53 Primarily d’Herelle challenged Bordet’s idea that bacterial lysis in infected animals was the result of the combined action of a specific antibody and a nonspecific element called complement. According to d’Herelle, there was little evidence for such a mechanism either from infected animals or from experiment. Not only had no one been able to directly observe bacteriolysis in the bloodstream of animals with infections such as typhoid fever but in laboratory experiments bacteria always remained “alive in the antibody-­ complement mixture for much longer than in pure physiological sa-

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line.” Consequently d’Herelle argued against “the reality of the phenomenon,” as described by Bordet, suggesting instead: “Could it not in reality be played by the bacteriophage, that principle endowed with a powerful bacteriolytic action, operating upon the most varied bacteria?”54 Perhaps in reaction to such challenges—many admittedly far-­ fetched—Bordet’s back was up, so to speak, against d’Herelle right from the outset, and he would remain flatly opposed to all of the latter’s ideas, even those with evident merit. To the last, he only ever referred to the bacteriophages in his publications with the descriptor “so-­called” before the label. More immediately, he responded to d’Herelle’s first claims about the possible nature of bacteriophages as viruses by conducting his own investigations, along with his Romanian colleague Mihai Ciuca, on the nature of the immune response to the dysentery bacilli, which Ciuca obtained from d’Herelle’s lab.55 They found that when an animal was injected with the bacteria, the fluids secreted by the animal’s leukocytes (white blood cells) in response to the infection could trigger the lysis of new cultures of the same bacterial species, and furthermore, that the lytic activity was serially transmissible in animals, exactly as d’Herelle had claimed. But while they conceded that d’Herelle’s results were accurate, they claimed that “his hypothesis of a live bacteriophage virus is not.” Embedded in the context of Bordet’s immunological theories, they interpreted their results thus: “Considering that dysenteric stools are rich in leukocytes, and that the lytic power of the feces is observed only around the time of convalescence, we asked ourselves whether the phenomenon did not result from an immune reaction of the organism, specifically, of a particular activity of the leukocytic exudate, which has the effect of determining in the microbe, a hereditary nutritive vitiation consisting of the production, by the latter, of a sort of lytic ferment [enzyme] capable, moreover, of diffusing into the surrounding medium and consequently, to impress in the same way some normal microbes of same species.”56 What exactly Bordet meant by “nutritive vitiation” is not clear. Perhaps the closest translation is that of “malnutrition” provided by the French microbiologist André Lwoff, but that definition would not arrive for many years.57 As Varley contended, the meaning of the phrase was opaque to Bordet’s contemporaries, even to those who supported his opposition to d’Herelle’s views.58 Bordet’s own explanation in English does not give too much insight either, although it does fill out the overall picture of his conception somewhat: “According to our conception Epochs of Incredulity and Belief

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the microbic lysis may be regarded as the consequence of a nutritive vitiation of the bacterial metabolism, primarily induced by some external disturbing influence. This vitiation would have as an effect the production of a lytic chemical principle . . . capable of communicating the same vitiation to new microbes belonging to the same species brought into contact with them.”59 The main thrust of the argument, as can be seen, bears no small resemblance to objections of the opponents of the virus theories of cancer, who disputed the notion that cancer could be caused by an external living agent rather than something that originated from within the organism’s own cells: “According to d’Herelle, the lysis is due to a living being, to a filtering virus. We, on the contrary, believe that the lytic principle originates from the bacteria themselves.”60 The Belgian interpretation of the bacteriophagy phenomenon proved a double-­pronged thrust at d’Herelle, for not only did it incorporate the finding into an existing framework of immunity, it also denied his claim to a novel discovery.61 Busy with work-­related travel in the Indochine as well as with completing his monograph, and with the growing uncertainty of his position at the Pasteur in Paris, d’Herelle did not respond directly until the April 1921 meeting of the society and then via a short communication in what Summers describes as a tone “of respectful and scholarly disagreement.”62 Even before d’Herelle had time to frame his response, however, Bordet and Ciuca, joined by André Gratia, a former student at Bordet’s institute who was at the time at the Rockefeller Institute in New York, mounted what can only be viewed as a full-­fronted offensive against d’Herelle and his theory of the bacteriophage. At the March 26, 1921, meeting of the Belgian affiliate of the Societé Biologie de Paris, they presented a total of seven papers—Gratia’s four papers were read by Bordet—discussing different aspects of the phenomenon, and all opposing d’Herelle’s conception.63 D’Herelle reacted to this new challenge at the May 21, 1921, session of the Societé de Biologie in Paris, in a markedly more reactionary manner than before, raising the question about whether the phenomenon described by Twort in 1915 in micrococci was the same as his own 1917 discovery in dysentery bacilli: “Is the bacteriophage involved in the phenomenon, also very interesting, described by Twort?” he asked rhetorically, only to deem it “peu vraisemblable,” implausible.64 Save for a minor difference in the heat resistance of his and Twort’s discoveries, d’Herelle’s reasons for his verdict seems to have been “largely intuitive” and unsurprisingly found no purchase with the Belgian team.65 As mentioned in the previous chapter, d’Herelle had drawn an

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analogy between the bacteriophage plaques and bacterial colonies in culture. He regarded the linear relationship between the dilution of a solution of lytic principle and the number of plaques it produced when plated after being mixed with bacterial cultures as strong evidence for the particulate nature of the agent. By his reasoning—based on the premise that a single bacterial cell was infected by a single bacteriophage—the more a solution was diluted, the fewer particles there would be to infect bacteria, resulting in fewer plaques. But the Belgian team was unconvinced by his reasoning. Gratia, for instance, believed that the results could just as soon be explained on the basis of the sensitivity of the bacteria to lysis and that dilution was accessory: “We are inclined . . . to search for the immediate source of the phenomenon in the relative resistance of the colon bacilli. If this is so, we should expect the similar production of small areas of clarification even with undiluted lyric agent on submitting to its action cultures of greater resistance. As will be shown later, this is precisely what we have often observed.”66 In a lecture given before the University of Edinburgh, Bordet offered yet another interpretation, based on the principles of natural variation in populations, which assumed that “all of the bacteria are not equally endowed with the capacity to anchor very rapidly the lytic principle. Then clear spots appear only at the spots where such bacteria have been deposited, and which have absorbed a sufficient quantity. . . . The very slight amount of lytic agent will indeed be distributed between so many microbic individuals that each of them, being not sufficiently touched, will not reproduce the principle.”67 This behavior clearly excluded the possibility that the lytic principle was a living virus, which would have presumably multiplied with greater facility (and hence produced more plaques) in the presence of more food—the host bacteria. Perhaps the heaviest piece of ammunition that Bordet’s group obtained against d’Herelle’s conception of the bacteriophage as virus was the phenomenon that they called lysogeny. Bordet and Ciuca first introduced this term—­meaning the genesis of lysis—­to describe the seemingly spontaneous ability of certain normally growing bacteria to undergo lysis without any exposure to bacteriophage material such as stools from convalescents.68 The material from these lysed bacteria was capable of transmitting the lytic ability to new unexposed bacteria in the same manner as d’Herelle’s bacteriophagy. Although the appearance of lysogeny was unpredictable, Bordet argued that its very occurrence was evidence against a possible viral identity of the bacteriophage, for it was impossible to imagine that the lysogenic bacteria had Epochs of Incredulity and Belief

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harbored viruses for generations without manifesting any signs of infection, and that it suddenly underwent lysis due to the action of those selfsame viruses.69 His own explanation for the phenomenon fit well into his picture of bacteriophagy or transmissible lysis as originating within the bacterial cell; he believed that that the ability to produce bacteriophage was “inscribed into the heredity of the bacterium.”70 D’Herelle, in turn, had his own theories regarding lysogeny: at first inclined to dismiss it as an artifact of contamination of the bacterial cultures with bacteriophage, he later explained it as the manifestation of a symbiosis in a mixed culture of bacteriophages and bacteria and thus as a variation of the basic phenomenon of bacteriophagy: “I have applied the term ‘mixed cultures’ to cultures derived from secondary cultures when resistant bacteria and virulent bacteriophage corpuscles co-­exist. . . . A mixed culture results from the establishment of equilibrium between the virulence of the bacteriophage corpuscles and the resistance of the bacterium. In such cultures a true symbiosis obtains in the true sense of the word; parasitism balanced by the resistance to infection. . . . Such mixed cultures, symbiotic in nature, can be subcultured indefinitely.”71 Although his basic tenet—that the origins of lysogeny were distinct from classical bacteriophagy and in no way refuted his theory of bacteriophage as virus—remained intact, lysogeny would remain a major thorn in d’Herelle’s side for many years, primarily because so many microbiologists of the era were persuaded by Bordet’s reasoning about it. Meanwhile, d’Herelle’s virus theory was not entirely without its supporters. A team of scientists, coincidentally also from Belgium, headed by Richard Bruynoghe, at the Catholic University in Louvain, was perhaps the earliest group to have looked into the problem of bacteriophagy and publish their support for the virus theory. In a recent historical investigation of Bruynoghe’s work, the Belgian microbiologist Alfons Billiau identified several lines of evidence, which appear to have persuaded these scientists of the viral identity of bacteriophage.72 One piece of evidence was the adaptation of the power of the lytic agent upon serial passages in different bacterial species. For instance, when passaged in cultures of the bacterial genus Shigella, the lytic principle derived from E. coli grew more prolific, whereas it became less so through successive cycles of growth in E. coli itself.73 Bruynoghe concluded that such an adaptation could “more easily be explained when one considers the bacteriophage as an autonomous being that, like microbes, is able to modify its properties (virulence) by successive passag-

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es.74 Even more persuasive was the conservation of antigen specificity of the bacteriophages despite the adaptation of their lytic capabilities.75 As Bruynoghe and his colleagues argued, “that the bacteriophage, as a lytic ferment secreted by microbes, does not display any specificity finds its explanation when one considers the ferment not as a product of secretion of microbes, but as a substance formed by the same single virus able to parasitize diverse species of microbes.”76 Bruynoghe also observed the development of bacteriophage resistance by certain bacteria, which he, like d’Herelle, interpreted as evidence of an acquired immunity by these bacteria to the infective parasite.77 In addition, he agreed with d’Herelle’s interpretation of experiments on the behavior of bacteriophage upon serial dilution, even devising an experiment to circumvent objections by the likes of Gratia and Bordet, explaining, “The bacteriophage is a figured element that, once the limit dilution is reached, does not dissociate itself further and is thereby spread irregularly in the liquid of subsequent dilutions. This result is not obtained with enzymes. When one dilutes them beyond their activity, one can take as many samples of this dilution as one would like without ever finding the active enzyme in any of them.”78 Although Bordet did, in fact, respond to Bruynoghe at a 1923 meeting of the Royal Academy of Medicine, his talk either “lacked specificity with respect to Bruynoghe’s arguments” or “disregarded [his] other points of evidence.”79 Instead, Bordet chose to focus on his argument that there was still insufficient evidence for bacteriophages to be living. Younger than Bordet by almost a decade and less internationally experienced, Bruynoghe was also naturally, considerably less influential.80 Furthermore, as Billiau has pointed out, although it was original enough in different aspects, Bruynoghe’s work did not gain notice from other scientists compared to the work that emerged from Bordet’s laboratory, and soon became an overlooked chapter in the history of the bacteriophage. The response of Bordet’s younger colleague André Gratia to the bacteriophagy phenomenon presents an interesting case in the history of ideas about the bacteriophage, particularly with regard to the opposition to d’Herelle. According to his son, Gratia first became interested in bacteriophages in 1920 while working at the Rockefeller. Leafing through a volume of the Lancet in search of an article about meningococcus one day, he chanced upon Twort’s 1915 article: “Reading it, he became convinced of a link between Twort’s ‘glassy’ transformation of Epochs of Incredulity and Belief

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Micrococcus colonies by a filtrable agent contained in a ‘vaccinia lymph’ (his words) and the bacteriophage of d’Hérelle.”81 Like Bordet and Ciuca, Gratia soon became convinced of the sameness of the two phenomena, declaring, “The Twort phenomenon and the d’Herelle phenomenon are identical. They are two different aspects of one and the same phenomenon: the transmissible lysis of bacteria.”82 Whereas at first Gratia was inclined to agree with Bordet and Ciuca that their observations “make very doubtful the parasitic nature of the bacteriophage,” he gradually became persuaded otherwise, and by 1931 confessed, “Je crois devoir remettre au point quelques notions relatives à la bactériophagie” (I believe I have to revise some notions about bacteriophagy).83 Despite this flip, however, he remained ever adversarial against d’Herelle, with whom he also sustained a long disagreement about the plurality and widespread nature of bacteriophages.84 He continued to champion Twort and gave him credit for all ideas about the bacteriophage, even that of its identity as a virus, even though Twort had only mentioned it as one possibility in his first paper and never expanded on the idea in his future writings. Perhaps the most measured response to both d’Herelle and Bordet came from Eugène Wollman, a Russian-­born biologist at the Pasteur Institute in Paris since 1909, who made many important contributions to understanding bacteriophages and lysogeny. Indeed, Wollman, together with his wife and collaborator Élisabeth Wollman, might well have occupied a place of even greater prominence in the history of bacteriophages than they already do, had it not been for personal tragedy. In December 1943, first Élisabeth and then a week later Eugène were arrested by the Nazis, and after being interred at Drancy were deported to Auschwitz and never heard from again.85 Wollman most likely learned about bacteriophagy from d’Herelle himself relatively soon after the latter had taken up his research work in the Pasteur Institute laboratories in Paris. In a 1925 paper Wollman mentioned being struck, in 1919, by the analogies between different transmissible phenomena that had been attributed to filterable agents—­ specifically the tobacco mosaic disease, chicken sarcomas, and bacteriophagy—­which had led him to accept the idea that they were all viruses.86 But unlike d’Herelle, he neither dismissed lysogeny as an artifact of contamination nor regarded it as symbiosis between the bacteriophages and resistant bacteria, considering such a generalization “extremely artificial.”87 Between 1925 and 1940 he produced a total of six memoirs on bacteriophagy and lysogeny, often working in collabora-

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tion with his wife, attempting to clarify the various problems posed by the bacteriophages. Interest in the bacteriophage phenomenon gradually rippled outward from France and Belgium to other centers of Western medical research including the United States, the United Kingdom, and Germany, though it seems to have remained the province of a few select groups of researchers in the first decades after its discovery.88 In the introductory section of a 1929 paper offering a refinement on d’Herelle’s method for studying bacteriophage multiplication, Frank Macfarlane Burnet identified six different extant theories on the nature and identity of bacteriophages.89 Although not in agreement with one another as to what the bacteriophage actually was, the scientists advocating those various theories were in perfect accord on one point: that it was not a virus.

Commononalities in the Resistance to Viral Causes of Sarcomas and Bacteriophagy As remarked upon by scientists and historians alike, a flavor of negativity—concerning what things were not viruses and what viruses themselves were not—was infused into much of the discussion around viruses at the time that Rous and d’Herelle offered up their ideas as to the identities of their discoveries.90 Thomas Rivers, for instance, opened his classic review on the nature of viruses with the observation that viruses then were typically characterized by “three negative properties, namely, invisibility by ordinary microscopic methods, failure to be retained by filters impervious to well-­known bacteria, and inability to propagate themselves in the absence of susceptible cells.”91 His observation bears out the fact that for most of the medical research community in the 1920s and 1930s the virus was still an idea in flux. Consequently it is not too surprising that so many researchers, especially those who were not primarily concerned with viruses anyway, preferred the various, more concrete possibilities for the identities of the agents of sarcoma and bacteriophagy/transmissible bacterial lysis, such as enzymes or growth factors. Interestingly, even though the objections of researchers within both the bacteriophage and cancer research communities were quite varied, one can find clusters of objections in each of the two groups that bear a close resemblance to each other.92 The basis for these similarities is the fact that the ideas were rooted in very similar lines of thinking about broader biological problems. Especially striking are the Epochs of Incredulity and Belief

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Fig. 2.1: Jules Bordet circa 1920. Copyright Institut Pasteur/Archives/Archive Jules Bordet.

similarities in the arguments offered by Bordet vis-­à-­vis bacteriophagy and by Murphy on the matter of sarcoma agents. Bordet, for example, thought that bacteriophagy—transmissible autolysis—represented “the pathological exaggeration of a function belonging to the physiology of

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the bacteria,” and that it had “some relation to the phenomenon of mutation.”93 It should be noted that although the use of the term mutation in the context of microbiology was contested at the time, Bordet was clearly using it to denote a “hereditary variation,” in bacteria, in the same way it was used to describe inheritable variations in plants.94 A very similar explanation was being put forward for the chicken sarcoma agent by Murphy, when he suggested that it had “the ability to induce permanent inherited changes” in their host cells.95 Both men even had similar thoughts on the evolutionary implications of such mutations: Bordet speculated that the lytic power of bacteria was a “weapon” that endowed an evolutionary advantage against their competitors, and Murphy carried this line of argument even further, reasoning that cellular transformation was a type of “physiological evolution” and therefore all the more difficult to explain in terms of the activity of “ordinary parasites, even endocellular agents.”96 Another interesting parallel may be seen in the way in which Bordet and Ewing approached the problems of bacteriophagy and tumor causation, respectively. In an international conference on cancer in 1927, Ewing had articulated the problem of the origin of tumors as “wholly different from the problem of their continued growth. Two distinct questions must, therefore, be considered in discussing general etiology. One concerns the existing factors, the presence of which initiates the tumour process, while the other relates to the nature of the tumour process itself. The former may be called the causal genesis, the latter the formal genesis.” Ewing even acknowledged that viruses might be able to instigate tumors—­t hat is, play the role of one of many possible agents of causal genesis. But he opposed the ideas of such proponents of the viral theory as Rous, Andrewes, and Gye, strenuously denying the possibility that viruses could be involved in the formal genesis or maintenance of tumors. Of the results of experiments on the basis of which Gye had defended the virus theory, Ewing simply argued that they could just as easily “be explained on the basis of a growth stimulating enzyme.”97 In his discussion of bacteriophagy, Bordet had similarly focused on the importance of separating the question “How is the nutritive vitiation primarily induced?” from the issue of how it—­t he vitiation—­was propagated in successive generations of bacteria: “Under some disturbing influence . . . a nutritive vitiation of the bacterium is primarily induced, testified by the appearance of the lytic agent. After this the interference of the external influence is no longer necessary. Henceforth the reproEpochs of Incredulity and Belief

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duction of the principle requires nothing more than the presence of living microbes, which, having absorbed a sufficient quantity of it, liberate new amounts of the same agent.”98 Given the similarities in and alignment of so many arguments, it may come as a surprise to those looking at these ideas years later that there does not seem to have been much direct communication between the opponents of the viral theories of the bacteriophages and of the tumor viruses. Even Gratia and Murphy, who would have overlapped when the former worked at Rockefeller, do not appear to have exchanged ideas, or at least, not any that I could find in the available records. Murphy, on his own, would later attempt to draw parallels between the sarcoma agent, the bacteriophages, and what was called the “transforming principle” of certain pneumonia-­causing bacteria being studied at the Rockefeller.99 But he does not seem to have had any sort of sustained dialogue with Bordet, Gratia, or other prominent opponents of the virus theory of bacteriophage. Looking over the vast literature and discussions on the issues of tumor viruses and bacteriophages, then, it would seem that the long drawn-­out resistance to Rous and d’Herelle’s ideas lay not so much in a quarrel with the idea of what a virus was—which, for much of this period was still quite vague—as it did in the idea that the culprits responsible for these phenomena were infective living agents.

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3WHAT WAS A VIRUS? En résumé, tout virus est un microbe. —Louis Pasteur, 1890 We do not know just what a filterable virus really is. —Earl B. McKinley, 1932

At the heart of the discussion recounted in this tale is the “virus,” the very concept of which was a matter of considerable uncertainty for much of the late nineteenth and early twentieth centuries. It remained in flux, confirming Ilana Löwy’s claims that scientific terms frequently undergo changes, or “variances,” in their meaning, without a conscious recognition of the changes by the very scientists who use these terms.1 Against this backdrop, it is easy enough to understand why Peyton Rous and Félix d’Herelle faced such strong opposition to their ideas that the causative agents of chicken sarcomas and of bacteriophagy/ transmissible lysis were these indefinable “viruses.” Of course such an explanation would be an oversimplification. After all, as discussed in previous chapters, both men had their fair share of supporters who were well respected within the scientific community. Furthermore, objections to notions of phage or tumor agents as viruses were often aimed at ideas about the nature of these entities, which transcended mere terminology. D’Herelle would seize on this uncertainty to argue for the weakness in his opponents’ views, claiming, for instance, that the arguments of those contesting the viral identity of bacteriophages

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were “but a repetition of those which have been raised against the living nature of each ultra-­filterable virus.”2 And the cancer pathologist James Ewing would remark in a letter to Rous some years later, “It all settles down to the question, what is a virus?”3 The shifting sands of the answer to this question formed the backdrop for most of the time that the viral identities of individual examples such as the bacteriophage and tumor viruses were under debate.

Terms of Engagement On the surface of things, virology—­t he study of viruses—­might be considered to have begun with the discoveries of the first-­known viruses in the late nineteenth century. Such a characterization gets complicated, however, when we consider the definitions of different terms that were used. For instance, although it was a concept in flux throughout the early part of the twentieth century, the word virus had entered the English-­language medical lexicon centuries ago, imported from Latin as a word for poison or venom.4 Perhaps the best-­known reference, at least to historians of science and medicine, is in Edward Jenner’s famous exposition on the prevention of smallpox in 1898.5 Virus appears multiple times in this treatise, variously representing the “morbid matter” of the disease—the material from the pustules of the pox—or the disease agent itself, insofar as such things were understood at the time. While the meaning had narrowed by the end of the nineteenth century to signify contagious disease agents that were “small and living,” the term virus was still quite nonspecific.6 In Louis Pasteur’s description, for instance, it appears to be a generic shorthand term for “disease agents” that were microbial in nature. By the end of the first decade of the twentieth century, however, the word accompanied by the qualifier filterable had become something somewhat more specific, clearly differentiated from the bacteria and defined by most of the research community as “microorganisms which will pass through filters, the pores of which are too small to give passage to ordinary bacteria.”7 Compared to virus, the term virology, is a much later entry in the annals of science. The earliest usage cited by the Oxford English Dictionary dates to the 1930s, but the term was not widely used until the 1950s, the period in which scientists such as the Australian researcher Frank Macfarlane Burnet—­well known for his work on different viruses—­situated its birth as an independent science. Defining viruses as “microorganisms, potentially capable of producing disease, which are smaller than bacteria [and] capable of growth only within the sub-

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stance of living cells of a susceptible host,” he reasoned that the science of virology could be “regarded essentially as the unfolding of the implications of that definition.”8 Whereas it may be true that the concept of unifying studies of different sorts of viruses into one common branch of study did not really emerge until the 1950s, it is equally true that investigations of the sort Burnet was referring to had been in existence for many decades by then. The Dutch plant virologist Lute Bos, for example, argued, “There is no doubt that conceptually virology was conceived in 1898,” which was the first time that someone isolated a filterable but invisible—­t hat is to say, ultramicroscopic—­disease agent. But, he added, “The development of the new discipline remained embryonic until its actual birth, in 1935, when viruses became subject to isolation and study in vitro.”9 Bacteriologists or microbiologists were not the only scientists to work with viruses in their investigations. There were others, from areas as diverse as plant pathology and human disease, who encountered viruses, but these researchers did not focus their efforts toward understanding their nature. Even Rous, best remembered now for his discovery of the sarcoma virus and his later decades of work on the Shope papilloma virus, was not a virologist either in his own estimation or in the eyes of his contemporaries. More than two decades after his discovery of the sarcoma agent—­which he believed was a virus—­he would write to tell his fellow New Yorker Ewing in the course of an exchange on tumor etiology, “I am coming to know about viruses secondarily and that may be to some extent the case with yourself.”10 Coy as that remark might seem from someone who would go on to win a Nobel Prize for his discovery, Rous’s self-­characterization was, in fact, reinforced years later by his colleague Thomas Rivers: “Rous has always been a lot more interested in what his virus did than in what it was. To my mind, he is not a virologist; he is still a pathologist.”11 This remark by Rivers—­who was nearly a decade younger than Rous both in age and his career—­casts an interesting light on the shifting boundaries among and within various medical disciplines in the early half of the twentieth century. At the turn of the century, when the Rockefeller Institute was being established, for instance, medical microbiology—­t he study of infectious diseases—­was subsumed within pathology. The examination of specimens (for example, blood or urine) from patients suffering from infectious diseases was a routine part of a pathologist’s duties, and research projects often originated from such materials. Simon Flexner, Rous, and James B. Murphy, for example, What Was a Virus?

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were all trained as pathologists and while some of their projects stemmed from studies of infectious diseases, others did not. Whereas Flexner was well regarded in his time for his work on such infectious bacterial diseases as dysentery and meningitis, he had hired both Rous and Murphy to work on problems to do with cancer, which is not an infectious disease by any means. Furthermore, regardless of the status of the debate over the viral nature of chicken sarcomas, both men also tackled problems that had no links to contagious diseases—­Murphy on the role of lymphocytes in cancer resistance, and Rous on blood chemistry. Although microbiology remained (and indeed remains) a part of the pathologist’s duties especially in the clinical domain, there was a definite shift during that time in the research foci or its different subdisciplines. Thus, by the time a younger generation of pathologists—­for example, Rivers, Christopher Andrewes, and Burnet—­had begun their training, the research specializations were more clearly demarcated, not only between microbiologists and those pathologists studying other, noninfectious types of diseases—­for example, chronic conditions such as cancer and inflammations—­but also between microbiologists who studied infections caused by different categories of microbes; for example, bacteria and protozoan parasites. Rivers, Andrewes, and Burnet were, in fact, part of that cohort of microbiologists who went yet another step further to become first generation of bona fide virologists, who made viruses the main, if not exclusive, object of their investigations. Although these labels—­virology and virologists—­would not come into common use until the 1950s, Rivers had already made a case for creating the discipline by the late 1920s: “Although knowledge concerning the exact nature of filterable viruses is lacking, the idea is gaining favor that these infectious agents present many problems which warrant special consideration. An expression of this idea is reflected in the fact that investigators in certain universities are realizing that these disease producing agents deserve more consideration from bacteriologists and pathologists than they now receive, and that separate departments should be established for their study and for the instruction of students.”12 Rivers’s preface reflects a shift within the medical scientific community from the prevalent notion that, save for their size, “viruses were no different from bacteria.”13 This earlier view of viruses was quite natural, given that the earliest discoveries in the 1880s and 1890s were discovered in the context of the newly emerging science of microbiology—­at first synonymous with bacteriology—­and pathology. Following in the wake of fun-

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damental work in the 1860s and 1870s by Pasteur and Robert Koch, who convincingly demonstrated a causal relationship between specific infectious diseases and specific bacterial species, most scientists investigating infectious diseases tried to pinpoint the bacterial culprits for these diseases.14 While many were successful in their quests, others were not. Quite often, the reasons, as we know now, lay in the fact that the agents were not bacteria (or what we now call bacteria) at all.

Discoveries of and Early Debates over the “First” Viruses No history of viruses would be complete without a mention of the tobacco mosaic disease, the earliest known example of a viral infection to be investigated in a laboratory, which the German American biochemist Heinz Fraenkel-­Conrat once aptly characterized as “almost always the first” virus.15 The tobacco mosaic virus (TMV) was the first virus that was “obtained in pure enough form to be analyzed in a centrifuge”; the first to be prepared and viewed in crystalline form, among the first to be visualized under the electron microscope, and “the first biological particle to be studied in detail.”16 As Angela Creager has shown, TMV has functioned as a key model in virology, both as representative—­in that knowledge gleaned about it could be generalized to other viruses—­and exemplar, showing how other viruses might be studied. Here I focus on the ways in which the early conceptions and debates about the nature of TMV set the stage for way in which debates over the identity and nature of other viruses were played out. In 1879, a group of colonial farmers from tobacco plantations in the Dutch East Indies, who were worried about a strange disease affecting their tobacco crops, brought the matter to the attention of Adolf Mayer, the director of the Agricultural Experimental Station at Wageningen in the Netherlands. Mayer was quick to realize that this disease was something new, and based on the principal symptom, gave it the name “mosaic disease.”17 An agricultural chemist and chemical technologist who trained in Germany during same period that Koch’s ideas about the bacteriological etiology of infectious diseases, Mayer was naturally steeped in these ideas, and attempted to apply these principles to his new find. His earliest results seemed promising as he found that the “the juice from diseased plants obtained by grinding was a certain infectious substance for healthy plants.”18 But he was unable to isolate or culture any bacteria from the infectious material, and in addition found that the infectivity of the plant extract—the “juice”—diminished signifiWhat Was a Virus?

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cantly when passed through double layers of filter paper. Interpreting this decreased infectivity as a sign that the filters retained the disease agents despite his inability to detect them under a microscope or to culture them, he concluded that the mosaic disease was caused by some infectious bacteria, even though “the infectious forms are not isolated nor are their form and mode of life known.”19 A few years later, a Russian student in Saint Petersburg named Dmitri Ivanovsky revisited Mayer’s experiments on the tobacco mosaic disease, armed with some improved bacteriological filters that had been developed in the interim.20 He obtained very similar results in all but one key respect: “The sap of leaves attacked by the mosaic disease retains its infectious qualities even after filtration through Chamberland filter-­candles.”21 Like Mayer, Ivanovsky, too, was unable to isolate or grow any specific disease-­inducing bacteria from either the infective material (filtrate) or from the material held back by the filters. But rather than ignore these results, he factored them into his interpretation of experimental results. Drawing parallels to disease mechanisms very recently demonstrated by Pasteur Institute researchers Émile Roux and Alexandre Yersin in the case of diphtheria, Ivanovsky suggested that his experimental results were “explained most simply by the assumption of a toxin secreted by the bacteria present, which is dissolved in the filtered sap.”22 Ivanovsky is generally recognized as the first person to have isolated what we would identify as a virus today, although the jury remains out on whether he understood the significance of his work or has a rightful claim to founding the field of virology. In 1898 in the Netherlands at the Polytechnical School in Delft, Dutch microbiologist Martinus Beijerinck, a former colleague of Mayer’s from Wagingen, reported the findings of his investigations on the tobacco mosaic disease, first at a meeting of the Dutch Royal Academy and subsequently in a pair of nearly identical reports in Dutch and German.23 He corroborated Mayer and Ivanovsky’s evidence of transmissibility but, like the latter, also showed that the plant extract retained its infectivity even after filtration of the material through Chamberland filters. He made no mention of Ivanovsky’s work, an omission that was noted by the latter who in his doctoral dissertation, remarked that Beijerinck “does not know that I had already established this fact a long time ago.”24 Unlike the later controversy over bacteriophage, however, the priority issue was nipped in the bud, for Beijerinck graciously acknowledged Ivanovsky’s earlier discoveries “with pleasure.”25 But, in a manner much like d’Herelle on the bacteriophage some decades later,

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Beijerinck interpreted the results of his experiments quite differently from his forebears and contemporaries. Where, for instance, Ivanovsky saw the diffusible substance as an enzyme—a ferment—produced by some bacteria, Beijerinck thought that the diffusible substance itself was living. The main evidence on which Beijerinck rested his inference was the seeming ability of the substance—which he called a virus in the general sense of the word as an infective agent—to reproduce. In his paper he reported: “The amount of virus which is sufficient to infect a large number of leaves is quite small. It is then possible to obtain material from these diseased leaves which can be used to infect unlimited numbers of new plants. It is therefore quite clear that the virus is reproducing within the plant.” Not content with merely expressing his belief, however, Beijerinck also designed a series of experiments to prove his point that the infectious principle was the living/reproducible diffusible substance. First he spread material from diseased plants on the surface of a thick layer of agar and then incubated the plates for some days to allow the material to diffuse into the agar medium. After washing the surface of the plate, he infected healthy new plants with material extracted from the deeper layers of agar, taking great care to avoid contamination from the upper layers. By his reasoning, a particulate virus would have been unable to diffuse through the pores of the solidified agar. “The deep layers of the agar would therefore not become virulent. But a water soluble virus ought to be able to penetrate to a certain depth in the agar plate,” he predicted. When the material from the deeper layers did indeed prove infective—­evidenced by its ability to induce symptoms of the original disease when injected into healthy new tobacco plants—­ Beijerinck felt confident in stating, “We were dealing here with a disease which was caused by a contagium which was not a contagium fixum in the usual sense of the words.”26 He called the agent a contagium vivum fluidum, Latin for “living liquid contagion.” Although the direct translation of fluidum in English is “liquid” or “fluid,” Beijerinck in his early papers appears to have used the descriptors “dissolved state” interchangeably with “liquid state,” which terminology indicates that he thought of the tobacco mosaic agent or virus as a soluble substance.27 With his contagium vivum fluidum, Beijerinck introduced into infectious disease etiology a brand new entity, conceptually different from any known disease agent at the time: one that was neither corpuscular or particulate like the bacteria, nor yet a soluble product of cells, such as toxins or enzymes. It was not an easy idea for his contemporaries to What Was a Virus?

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grasp or accept. For example, the first time he presented his paper the botanist and geneticist Hugo de Vries questioned him on the inclusion of vivum in the label, to which Beijerinck responded that he “considered the ability to reproduce to be the major characteristic of life.”28 In considering the formulation and reception of Beijerinck’s ideas, it would also be wise to step back and remember that in the 1880s, when Mayer first put forward his ideas, “the attribution of plant diseases to bacteria was itself rather novel.”29 Beijerinck’s further spin, suggesting that these bacteria were not bacteria at all but some other, entirely new type of living agent would have seemed to most of his contemporaries to be piling novelty upon novelty. Consequently, his conception was either criticized by scientists or ignored altogether as being too outlandish. In contrast to de Vries, Ivanovsky agreed with Beijerinck about the living nature of the agent. For him it was the fluidum aspect of the label that was troublesome; such a characterization he wrote in his next paper, was “unnecessary” and “completely untenable.”30 According to him, the experiments showing “the persistence of infectivity of the filtered sap [could] only be explained by the assumption that the microbe produces resting forms,” which he furthermore took to be a sign that the contagium was able to “multiply in the artificial media.”31 Although Ivanovsky ended his paper on a somewhat cautious note, acknowledging the need for further investigations on the question of the artificial cultivation of the mosaic agent, he was categorical in his opinion that Beijerinck was wrong about its soluble nature. In his doctoral dissertation he had declared: “We see that there is not a single fact which supports the hypothesis on the soluble character of the infectious agent of mosaic disease. On the contrary, the experiment with the diffusion into agar and especially the fractionated filtration clearly indicates that we are dealing with a contagium fixum.”32 Beijerinck’s evidence did little to change Ivanovsky’s mind. Meanwhile, even before Beijerinck had replicated Mayer’s work, a pair of German investigators, Friedrich Loeffler and Paul Frosch, former students of the famed Koch, reported results similar to Mayer and Ivanovsky’s based on their investigations into the causative agent of foot-­ and-­mouth disease of livestock. Although they did not introduce any new labels for the agent, or even use the term virus in their paper, they came to a conclusion about the nature of the agent that at first take seems to resemble Beijerinck’s notion in that it was living and so tiny as to be filterable. Contrary to the Dutch microbiologist, however, they

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did not believe that this filterable agent was a soluble or fluid entity, and instead held that it was particulate or corpuscular in nature, only smaller than the limits of visibility: [The] activity of the filtrate is not due to the presence in it of a soluble substance, but due to the presence of a causal agent capable of reproducing. This agent must then be obviously so small that the pores of a filter which will hold back the smallest bacterium will still allow it to pass. The smallest bacterium presently known is the influenza bacillus. . . . If the supposed causal agent of foot-­and-­mouth disease was only 1/10 or even 1/5 as large as this, which really does not seem impossible, then this agent would not be resolved in our microscope. . . . This would explain very simply why it has been impossible to see the causal agent in the lymph under the microscope, even after the most extensive search.33

Beijerinck would explicitly disagree with this last aspect of their interpretation, claiming that “it was not to be expected that a substance like the virus, which does not diffuse easily[,] would flow through in a diluted form at the beginning of the filtration process yet without being composed of corpuscular parts of corpuscular parts because of this behavior” and emphasizing in a footnote that he therefore could not “agree with the conclusion of Mr. Loeffler as regards the corpuscular nature of the virus of the foot and mouth disease.”34 Indeed, save for the fact that they made no claims about the cultivation of the virus on artificial media, Loeffler and Frosch’s notion of the infective agent seems more akin to that of Ivanovsky’s vision of the tobacco mosaic disease. In effect they initiated a third line of thinking about ultramicroscopic and ultrafilterable disease agents, one that shared a few features with both Ivanovsky’s and Beijerinck’s views, and yet was not completely like either. As will be discussed later, these early ideas about the nature of viruses would set patterns of thought and experimental approaches about the nature of viruses and diseases that would echo in debates on these matters for many decades. More immediately, the discoveries of the tobacco mosaic and foot-­ and-­mouth disease viruses marked the beginning of a period in the history of microbiology and infectious pathology when the terms filterable and ultrafilterable viruses entered the scientific literature. Filtration techniques—­which allowed the separation of particulate substances from liquid—­merit at least a passing mention in any history of virology because, as the pathologist Fred Murphy recently observed, they “lay at What Was a Virus?

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the center of the development of virology yet nearly disappeared from the scene a few decades afterward.”35 It may be considered as the first of many technologies, which, as Creager has rightly stressed, were as important as the conceptual advances “that shaped the meaning of the category ‘filterable virus.’”36 Indeed, the discovery of the first viruses, by different individuals or groups—Mayer, Ivanovsky, Beijerinck, or Loeffler and Frosch—occurred very soon after the development, by using unglazed porcelain, of special filters whose pore size was smaller than the diameter of any known bacteria (as evidenced by their retention of all bacteria when a liquid known to be infected was passed through these filters).37 It was only after the development of such “ultrafiltration” techniques—­so dubbed to distinguish it from filteration through coarser media—­t hat it became possible to detect the existence of entities that were smaller than the limits of resolution afforded by the microscopes that had been developed at that time.38 Or to borrow the observation of Émile Roux, who had succeeded Pasteur as the director of the Pasteur Institute, “Until 1898, invisible microbes were nothing but figments of imagination; the work of the past four years have given them a reality.”39 With specific reference to the viruses in this book, it was the separation of the sarcoma agent from tumor tissue by filtration that stimulated Rous to think that the agent could be a virus.40 Despite such a significant role, however, filtration has received short shrift in accounts of the history of virology and more generally, microbiology. Part of the reason for this oversight may well be that the use of the label was often no more than a “convenient form of expression as in certain cases it would otherwise be necessary to state ‘specific causes unknown.’”41 Rivers, for example, was critical of this terminology, and once even commented that the phrase filterable viruses was “misleading and confusing.”42 But he too used the term for his 1928 edited volume on the subject.43 Years later, when asked, he justified his choice by saying, “In the early days of virology, filterability was rather important because it was the only way workers had of differentiating the so-­called viruses from bacteria.”44 Another reason for filtration’s low profile in the annals of virology may have been “the apparent simplicity” of the technique itself. As microbiologist Stuart Mudd, author of the chapter on filtration in Rivers’s book, remarked, this simplicity posed a “pitfall, leading usually to incomplete recording of essential data and frequently to unwarranted conclusions.” In fact, in addition to proving useful in separating viruses from bacteria, ultrafiltration was, according to Mudd, “perhaps the best method we have at present for approximat-

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ing the size of ultramicroscopic viruses.”45 The measurements were based on passing various virus-­containing suspensions through a series of “ultrafilters of graded porosity,” and determining the limits at which different viruses were able to pass through. In his interviews with Saul Benison in the 1960s, Rivers would reiterate Mudd’s earlier observations about the centrality of ultrafiltration for virology in the 1920s and 1930s.46 Still another adjective or descriptor of the viruses that came to be widely used in the wake of the early discoveries of the agents of tobacco mosaic and foot-­and-­mouth disease is ultramicroscopic. Like ultrafilterable, the term was a reference to the extremely small size of the viruses, the limit in this case being defined by the resolving power of microscopes rather than pore size of the filters. The term was used quite widely, specifically with reference to the subject at hand, such as in the title of the paper in which Twort first reported his findings on glassy transformation, “An Investigation on the Nature of Ultra-­ Microscopic Viruses.”47 Given the undisputed centrality of microscopy to both the founding and development of microbiology and its offshoots, it has not been ignored in histories of these subjects. But during the early decades of virus research, microscopes did not play much of a role, save for confirming the extremely tiny size—invisibility, in fact—of the entities causing the infections.48

Early Intellectual Contexts for Conceptualizing Viruses That the discoveries of the first viruses took place in the context of the recently established science of bacteriology might seem so self-­ evident as to not bear discussion, but I would argue that parsing through these origins is important for a better understanding of how bacteria and viruses eventually came to be seen as separate. Both concepts are derivatives of a common idea of “a living contagion,” the basic elements of which doctrine, according to the pioneering British historian of medicine Charles Singer, may be traced back to antiquity. “Among the works of the classical writers of Rome and Greece we find a distinct tradition of creatures, minute and even ‘invisible,’ bringing disease from marshy land,” he wrote, in his classic treatise on the history of contagion, which, “like many other scientific ideas, appeared more than once only to disappear again.”49 In addition to classical antiquity he identified two more distinct historical periods when this notion was revived: once during the late seventeenth and early eighteenth centuries and What Was a Virus?

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most recently, again in the late nineteenth century—­what he identified in 1913 as the modern period. Singer’s ideas about the origins and revival of the contagion theory were echoed (though not cited) in a much more comprehensive analysis on the epistemology and sociology of scientific knowledge by the Polish microbiologist and philosopher of science Ludwik Fleck some decades later, in his magisterial The Genesis and Development of a Scientific Fact, first published in German in 1935, wherein he claimed: “Very clear ideas about tiny visible and living agents as causative agents of diseases were expressed long before the advent of the modern theory of infection and even before the invention of the microscope.” Rather more specifically than Singer he named names from both antiquity and the “modern” era, comparing, for example, the writings of the Roman scholar Varro from the first century BCE to his own near-­contemporary the German microbiologist Carl Flügge.50 In a later article he elaborated still further on this idea, arguing: The whole of modern knowledge of infection and infectious diseases originates in very ancient beliefs in an analogy between putrefaction and disease, and in small “animalcules” as the cause of both. The idea may be found in Greece and Rome (Hippocrates, M. T. Varro), during the Renaissance (Fracastorius, 1546), in the 17th century (Kircher, 1658; Leeuwenhoek, ab. 1680), in Pasteur’s first papers. . . . All these “ingenious intuitions” which existed before any empirical proof, and stemmed from an old pre-­scientific Denkstil, acted throughout the ages as a propelling force for a host of discoveries. It is doubtful whether our knowledge of infectious diseases would have made such progress without these “intuitions.”51

Neither Singer nor Fleck mentioned Beijerinck or his ideas in their discussions of the history of the idea of contagion, in all likelihood because at the time his ideas were very much under the radar. But his conceptualization of the contagium vivum fluidum fits so well into the accounts of the history the idea of contagion that it almost serves as an independent confirmation of Fleck’s theories. Fleck’s main thesis, which was brought to the attention of the Anglophone scientific community via Thomas Kuhn’s famous The Structure of Scientific Revolutions (1962), was that all scientific knowledge is constructed. For Fleck, “scientific ‘facts’ [did] not exist ‘out there’ in nature waiting to be discovered.” Rather, he felt, they emerged as the final result of a social process.52 In his explication of this social process, he in-

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troduced two concepts—­proto-­ideas and thought styles—­t hat I shall briefly attempt to explain here, as they help in better understanding the antecedents and influences on the conceptualization of viruses. According to Fleck, “many very solidly established scientific facts are undeniably linked, in their development, to prescientific, somewhat hazy, related proto-­ideas, even though such links cannot be substantiated.” Whereas a proto-­idea may not be judged outside the context of the time of its existence as right or wrong, he argued that regardless, there was no doubt that a fact developed “step by step from this hazy proto-­idea, which is neither right nor wrong.” Each step or iteration of the fact was propagated by a thought collective, “a community of persons mutually exchanging ideas or maintaining intellectual interaction,” which, by implication, “also provides the special ‘carrier’ for the historical development of any field of thought, as well as for the given stock of knowledge. This we have designated thought style.” That is to say, that as proto-­ideas move through history, they are constantly reconsidered and reinterpreted by new thought collectives.53 The thought style—­akin to the references made in the fine arts to “style” as cultural patterns—­may be rather more familiar to Anglophone historians of science and medicine than is the proto-­idea, for it has been applied more widely, both as a way of emphasizing the role of continuity and tradition in science and conversely also to explain often competing approaches of doing science. Fleck defined it in the sciences as a constraint on the way of thinking of a scientist, and “even more; it is the entirety of intellectual preparedness or readiness for one particular way of seeing and acting and no other.” According to him any scientific fact was in fact dependent on or constructed by the thought style of the scientist, which was shared by other members of the research community or thought collective and furthermore was the driving force that both determined “the direction of research and connected it with a specific tradition.” In other words, the thought styles and collectives—­ Denkstil and Denkkollectiv in the original German—­determined both “what can and must be considered as a scientific problem, and how this problem is to be dealt with.”54 At the same time, however, any given thought style is far from static and is inclusive of more issues than a set of shared opinions; it is possible, therefore, for several thought styles to coexist at once. Moreover, a single researcher may subscribe to more than one style at any time, depending on the sum total of his or her experiences. Interestingly, then, even as he argued for the social nature of knowledge production, Fleck gave considerable agency to the indiWhat Was a Virus?

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vidual even in a collective: “It is in the mind of the individual where different thought styles mix—­either in a fruitful and enriching way or in a silent and solitary way.”55 Beijerinck’s mind appears to have been fertile ground for such a mixing of thought styles and ideas, evidenced by the name he gave to the tobacco mosaic agent: contagium vivum fluidum. His characterization of the virus in these specific terms was no accident but a deliberate way of situating his concept within the ferment of ideas of the times—­not only bacteriology but also protoplasmic theory.56 By the time Beijerinck began to work on the tobacco mosaic disease, the historically vague proto-­idea of contagion had taken on a definite incarnation of a living corpuscular (or particulate) agent of infectious disease—­t he contagium vivum fixum—­probably the best known and most numerous of which were bacteria, although single-­celled fungi such as yeasts as well as protozoans also fit the description. As seen in earlier discussions, Beijerinck and Ivanovsky used the label for bacteria quite matter-­of-­factly and without explanation, although they shortened it by taking for granted its vivum component, or living nature.57 It was in order to key his ideas to this existing iteration of the contagion proto-­idea that I believe Beijerinck chose his label, simultaneously making claims for its similarity to known agents of disease—­t hat is, the infectious and living contagium vivum—­and differences in its nature—­namely fluidum, soluble or fluid, as opposed to corpuscular, or fixum. Beijerinck would elaborate upon the possibility of a more fluid, noncellular form in more detail in a 1913 address to the Royal Netherlands Academy of Sciences: “The concept of life—­if one considers metabolism and proliferation as its essential characters—­is not inseparably linked up with that of structure; the criteria of life, as we find it in its most primitive form, are also compatible with the fluid state. . . . In its most primitive form, life is [not] bound to the cell.”58 Evident in this admittedly vague description is a clear alignment with the idea that life resided in the rather amorphous protoplasm—­which Thomas Huxley had dubbed as the physical basis of life—­rather than in the structured cell.59 In his analysis of the development of the virus concept through the case history of TMV, Ton van Helvoort built a persuasive case for an early demarcation between two divergent thought styles in the conceptualization of the mosaic agent, which would last for many decades: one a “bacteriological” style, which saw the agent as “living” and organized, and the other a “chemical” style, which thought of the agent as

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an inert chemical.60 The contagium fixum concept may be clearly and unproblematically situated within a corpuscular or particulate thought style of disease etiology; not just Mayer and Ivanovsky’s characterization of TMV but also Loeffler and Frosch’s description of the agent of foot-­and-­mouth disease are examples of this way of thinking. At the same time, however, Ivanovsky might be said to represent the chemical viewpoint, for although he believed that the tobacco mosaic virus was a particulate bacterium, he attributed the disease symptoms to a soluble product—­enzyme or toxin—­produced by the causative bacterium. Beijerinck’s unique twist—­or “ingenious intuition,” to borrow Fleck’s terminology—­confounded the neat categorization of the contagium vivum fluidum into either existing style. At first glance at least, it appears to be a “hybrid” that straddled them both.61 Whereas a comparison between his ideas and those of Ivanovsky show clear alignments to the opposing bacteriological and chemical thought styles respectively, matters get more complicated when Beijerinck’s virus is considered against the ideas of Loeffler and Frosch. A comparison of their ideas about the physical form nature of the agent—­soluble vs. particulate—­ might appear to move the contagium vivum fluidum from a bacteriological to a chemical entity. But such a characterization is clearly at odds with Beijerinck’s unequivocal belief in the living nature of the disease agent, which idea the German scientists also agreed with. In this latter comparison, therefore, a consideration of the bacteriological versus chemical thought styles is less meaningful. It might be more fruitful to consider the differences in light of an alternate pair of thought styles—­ one that distinguishes between particulate and soluble entities, more akin to the difference in a pair of styles recently identified by a group of French philosophers analyzing conceptions of heredity.62 In this alternative framework, Beijerinck’s virus might actually seem to fit in better with Ivanovsky’s idea that the disease symptoms were produced by a bacterial toxin; that is, a soluble entity. But such a categorization is not entirely satisfactory either, for it ignores the fundamental point of difference between the two TMV researchers—­t he living nature of the symptom-­causing agent. In the late nineteenth century then, the contagium vivum fluidum would appear, like the proto-­ideas from which it derived, too hazy for most scientists to grapple with, for although Beijerinck was quite precise about the nature and abilities—­activity—­of TMV, neither he nor his contemporaries had either the technological tools nor the knowledge base to further test his ideas. The contagium vivum fluidum did not fit What Was a Virus?

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comfortably into anyone of existing thought style, and rather than being used as a way to reconcile those styles, it was ignored as an outlier for several decades. Eventually, though, the very properties that made it a conundrum in Beijerinck’s time would facilitate the recognition of viruses as different from other invisible pathogens.

The Fate and Historical Significance of Beijerinck’s Contagium Vivum Fluidum The trajectory of research and thinking on the tobacco mosaic disease and TMV—­t hat is to say, the specific virus—­turned out to be quite different from that of the concept of virus embodied by the contagium vivum fluidum. That the two were linked at all in Beijerinck’s original conception appears to have been an accident of circumstance, a matter of historical contingency. It is clear, for instance, in the disagreements between Beijerinck and Loeffler and Frosch that the specific disease and agent were not matters of pressing concern to either; their fundamental disagreement was about whether the agent (virus) of either was soluble (as per Beijerinck) or particulate (the Germans). Specifically with regards to the tobacco disease, interest in the subject in Europe seems to have flagged by the turn of century. Likely in part due to the lack of the experimental means to test his work, and certainly also because he turned his attention to other matters of more pressing concern to him in his academic position, Beijerinck neither pursued further investigations on this disease nor attempted to defend his ideas regarding the contagium vivum fluidum. After completing his doctorate, Ivanovsky, too, abandoned work on this subject and turned his attention to the study of photosynthesis. But the force of his arguments against the idea of contagium vivum fluidum was such that it had persuaded the examiners of his doctoral thesis that he had “refuted” Beijerinck’s hypothesis, which they furthermore deemed as “a sad chapter in the annals of contemporary science.”63 Meanwhile, the breakthroughs and insights of neither researcher had any practical value for controlling the infections in the plants, and faced with massive losses, the colonial farmers ceased their large-­scale cultivation of tobacco.64 The lack of both commercial and academic interests seems to have stalled further research on the tobacco mosaic disease and its agent, at least in Europe. There was a 1902 report by plant physiologist Albert Woods at the US Department of Agriculture. But the author appears to have regarded the disease as a physiological problem of the plants and made little, if any, progress toward under-

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standing the nature of the disease agent.65 A table listing milestones in TMV research, for instance, jumps directly from Beijerinck’s discoveries at the turn of the century to a cluster of reports in 1928/1929 from three independent laboratories in the United States.66 All three of these research groups did seminal and important work on different aspects of understanding the tobacco mosaic virus, work that not only established the cause of the disease but also provided “the key elements to study and define the nature of a virus” more generally.67 But although they cited Ivanovsky and his discoveries, none of them, in these publications at least, so much as mentioned Beijerinck or his ideas about the contagium vivum fluidum. It is likely that Beijerinck’s ideas about viruses was overlooked because of the rather narrow and specialized dissemination of his ideas. At least at first, few people beyond the relatively small intellectual community consisting primarily of Dutch-­ and German-­speaking microbiologists, and very likely only those who focused on plant and agricultural diseases, learned about his work on the tobacco mosaic virus. When he discussed the sarcoma agent of chickens in 1912, for instance, Rous offered as evidence in support of the agent’s living nature, experiments that showed that a very small amount of sarcoma agent would “give rise to a growth from which numerous others may be started, each yielding the agent in abundance.”68 But although his reasoning and arguments were very similar to those of Beijerinck’s from little over decade earlier, Rous did not cite these earlier ideas. D’Herelle, who would state his belief with even less concession or equivocation—­claiming that his observations that the invisible microbe “grows in the lysed culture of Shiga bacillus because a trace of this liquid, placed in a new culture . . . reproduced that same phenomenon with the same intensity” were “visible evidence that the antagonistic action [bacterial lysis] is produced by a living germ”—­seems to have similarly overlooked Beijerinck’s precedence in this matter at first.69 But when he was awarded prestigious Leeuwenhoek Medal by the Royal Netherlands Academy of Sciences in 1925, d’Herelle made up for this oversight by paying special tribute to the senior Dutch microbiologist’s ideas: “There has been much discussion on Beijerinck’s conception [of the virus] but I do not think that we have yet understood its true profundity. All of biology was, and indeed is still, based on the fundamental hypothesis that the unit of living matter is the cell. Beijerinck was the first to free himself from this dogma and in fact, proclaim that life is not [solely] the result of cellular organization, but is derived from anWhat Was a Virus?

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other phenomenon, which can only reside in the physicochemical composition of a proteinaceous micelle.”70 Given d’Herelle’s own stance on the nature of the bacteriophage as viruses, it is not very surprising that Beijerinck’s ideas about life resonated with him. Furthermore, it is very likely that his tribute to the septuagenarian Beijerinck was prompted by the source of the award—­ by the same society to which Beijerinck had presented his contagium vivum fluidum more than a quarter of a century earlier. Beijerinck—­an enthusiastic proponent of the bacteriophage, which he considered to be a confirmation of his contagium vivum fluidum theory—­was likely tremendously influential in the society.71 Despite his glowing words on this occasion, d’Herelle did not allude to Beijerinck when he published a more detailed essay on the subject a few years later, which leads me to regard his 1925 remarks as an incidental and anomalous reference rather than as a harbinger of widespread recognition of Beijerinck.72 Into the early 1930s, therefore, the Dutch plant virologist Lute Bos’s assessment that “in terms of spin-­off and follow-­up” there was no direct legacy from Beijerinck, would appear to have held true.73 But by the end of the decade there were definite signs of a turn in the tide of opinion, as the literature shows an increasing number of virologists awakening to the importance of the ideas embedded in the contagium vivum fluidum. In a 1938 address on the nature of viruses to the New York Academy of Sciences, for example, Wendell Stanley opened with a reprise of the investigations of both Ivanovsky and Beijerinck, but emphasized the fact that it was the latter “who first recognized the true significance of the results and the fact that viruses differ from bacteria.”74 His colleague the renowned virologist Thomas Rivers would reiterate these views. Toward the end of his life, in an oral history interview with the historian Saul Benison, Rivers recounted the details of a longstanding disagreement with his colleague (and for a time, Stanley’s supervisor), the plant pathologist and virologist Louis Kunkel, over whether it was Beijerinck or Ivanovsky who deserved the title of “the father of virology”: “My own choice for that honor was [Beijerinck]. There is no doubt that Iwanowsky made the first observations; the difference for me lay in the fact that, while Iwanowsky always believed that the agent that went through the filter was a little bacterium, Beijerinck realized that it was a new agent and put his neck out by calling it a living contagious fluid.”75 To this day, there is yet another school of thought that passes over both Ivanovsky and Beijerinck, to give the credit for the first discovery

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of the virus to Loeffler and Frosch. According to the immunologist and virologist Jean Witz, for example, it was Loeffler who found that the “agent of foot-­and-­mouth disease was too large to pass through these very fine grain filters [and] concluded that viruses are particles not liquids and therefore came closest to the modern concept of a virus.”76 I confess to a greater sympathy for the view that Beijerinck’s claim of the contagium’s fluidity is a matter of semantics, especially because he used the term interchangeably with solubility. Both Loeffler and Frosch as well as Ivanovsky were right in the literal sense that the contagium responsible for the disease was particulate, but “in a different sense than the organismal corpuscularity” that they seemed to have in mind.77 Also tipping the balance in favor of ceding priority to Beijerinck are his novel ideas about the nature of multiplication of the mosaic agent, his hinting at obligate parasitism, which to date remains the single most important criterion by which viruses are distinguished from other disease agents. The shift in attitudes toward Beijerinck and his contagium vivum fluidum led Bos to remark on a “striking similarity between Beijerinck’s fate in virology and that of Mendel in genetics.”78 The story of Gregor Mendel—­t he monk who discovered the laws of heredity through his experiments on pea plants, but the implications of whose work for genetics remained unrealized until after they were revived by three researchers independently of one another—­is arguably the most written-­ about rediscovery saga of twentieth-­century biology.79 Whereas, as historian Robert Olby affirmed, Mendel is almost universally and without dispute “considered the father of the discipline of genetics,” he was equally surely not “the person who institutionalized genetics.”80 Something very close might be said of Beijerinck’s claim to the title of the “father” of virology, and the actual formation of the discipline. As a quick aside, it is irresistible to wonder what Beijerinck himself might have made of this comparison to Mendel, because from his remarks to a colleague it is evident that he saw the earlier rediscovery as a missed opportunity for himself: “I had rediscovered the Mendelian laws, five years before Hugo De Vries,” he claimed, but also admitted that he had failed to follow up more thoroughly.81 Returning to the comparison itself, a common feature is that both men died without seeing their ideas fully take hold. But I am inclined to agree with those virologists who think that unlike Mendel vis-­à-­vis genes, Beijerinck’s description of the contagium vivum fluidum demonstrates that he “had a clear notion already of the close association between virus and host metabolism.”82 What Was a Virus?

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Another significant difference between the two cases is that no single person claimed credit for rediscovering Beijerinck’s ideas in such direct terms as Carl Correns did in the case of Mendel: “In my hybridization experiments with varieties of maize and peas. . . . I thought that I had found something new. But then I convinced myself that the Abbot Gregor Mendel in Brünn, had, during the sixties, not only obtained the same result through extensive experiments with peas . . . but had also given exactly the same explanation, as far as that was possible in 1866.”83 In contrast to such an explicit rediscovery, Beijerinck’s revival appears to have taken place gradually and without fanfare over the 1930s. But in both cases, the scientists who “rediscovered” Mendel and Beijerinck—­or claimed them as founding figures of genetics and virology, respectively—­did so, in large part, to apply the unique claims of these men toward projecting a specific image and delineating the borders of their own nascent disciplines. A notable similarity between Mendel and Beijerinck is that the early reception of their ideas was fraught with instances of misunderstandings over the implications of their experiments and ideas. Historians Robert Olby and Peter Gautrey, for instance, analyzed eleven separate instances before 1900 in which Mendel’s name was mentioned in the context of plant hybridization, but concluded that none of the writers “really understood Mendel’s theory.”84 A survey of references to Beijerinck’s virus for nearly three decades after the time of his discovery reveals a similar cognitive gap. In a 1914 review on the tobacco mosaic disease, for instance, the virologist H. A. Allard would list all the details of Beijerinck’s idea—­“He assumed that the virus must be an unorganized material, fluidlike in its nature, and capable of symbiotic growth in the presence of living cells”—­but then dismissed the idea on the grounds that it was too difficult to conclude “just what Beijerinck wished to convey by these vague and indefinite terms.”85 Helen Purdy, one of the first scientists to revive interest in TMV, listed the contagium vivum fluidum among one of several extant hypotheses regarding the nature of mosaic agents, but her subsequent conclusion, that it and other ideas “would be superseded by the parasitic theory, if the virus could be multiplied in vitro,” indicates that she too interpreted Beijerinck’s idea incorrectly.86 As for the timing of the rediscovery—­or perhaps more accurately the reawakening—­of Beijerinck and his ideas, I would venture to suggest that the resolution of many of the paradoxes that had been inherent in Beijerinck’s claims in 1898 played a major part. As indicated ear-

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lier, by the 1930s such investigators as Rivers were discontented with the definition of viruses in negative terms. By tethering their virus concept to Beijerinck’s contagium vivum fluidum and the host-­dependent nature of its replication, he and other virologists of his ilk were able to construct a narrative of their discipline that was consistent with preference for a “positive characterization of the viruses, one emphasizing the intimate relationship that exists between them and their host cells.”87 Beijerinck’s emphasis on the unique nature of the reproduction of the tobacco mosaic agent as being intimately linked to that of the host cell was precisely the type of idea that had immense heuristic value for understanding obligate parasitism of viruses, which turned out to be such a fundamental aspect of their nature. Consequently it is no surprise that his ideas resonated with the new virologists.

Other Contexts for Considering Viruses Before going on to examine the mutual impact of the changing definition of viruses on the research and ideas about the bacteriophages and the tumor viruses in greater detail, I will dwell briefly on some of the other developments in the later nineteenth and early twentieth century that had a significant impact on discussions and debates. An often unspoken theme undergirding the various debates was the nature of life itself. The question of what made something living was not simple, and the boundary between living and nonliving things remains fuzzy to the present day. In fact, the question “What is life?” might well have been, as the historian Lily Kay once observed, historically “deemed unproductive by most practitioners of life science.”88 Kay is right, of course, insofar as actually investigating the question is concerned, but I would argue that every biologist or life scientist; that is, every scientist engaged in investigating living beings, has some idea, whether or not it is consciously articulated, of what life is. Or as Kay put it in more pragmatic terms, different researchers or research schools have “privileged different scientific representations of life,” which in turn determines their various choices including their choice of experimental systems and methods, theoretical frameworks, and manner of interpreting results. This influence may be seen to have been all-­pervasive; the fundamental issue at the heart of virtually all of the disagreements discussed so far—­Beijerinck and Ivanovsky; Rous and Murphy; or d’Herelle and Bordet—­was whether or not the agent or entity in question was living. Viruses in general and bacteriophages in particular were often the What Was a Virus?

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focal point for discussions about the nature of life, not only in the early days following their discovery but also long after their chemical makeup came to be known. Indeed, the medical historian Scott Podolsky has argued in his thoughtful analysis of the role of viruses in theorizing about the life’s origins that ever since its first formulation, the concept of the virus became “inextricably interwoven with questions concerning the nature of life” in part because it “could be utilized metaphorically, as a conceptual shorthand for a definition of life itself.”89 Beijerinck’s arguments about his contagium vivum fluidum exemplify such metaphorical uses of the viruses. Confounding the issue of the chemical nature of life, especially in the late nineteenth century and early decades of the twentieth, was that enzymes—­often suggested as the alternative for the bacteriophages and tumor agents as well—­were often regarded in a very similar light. For example, in the late 1890s, just around the time that Beijerinck was launching his theories of the TMV agent as living, a new idea was taking hold: that of the “enzyme theory of life,” which saw life as a “self-­ regulating dynamic equilibrium of [a] system of catalytic reactions,” and would give rise to the discipline of biochemistry in the next few decades.90 D’Herelle later took advantage of such currents of thought to imply that arguments over whether or not bacteriophages were enzymes were perhaps moot when he contended that the bacteriophage was a “living colloidal micell,” the micell defined as aggregate of molecules—­which included enzymes—­in colloidal form.91 And still later the enzyme biochemist John Northrop would hark back to similarities in the controversy of the time over the nature of viruses and those over enzymes in the late nineteenth century when he proposed: “Some filterable viruses are probably enzymes which possess the property of forming themselves under proper conditions.”92

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4ROMANCING THE PHAGE At present I believe—­rather open mindedly—­in the theory that the phage is a living bug that lives at the expense of bacteria. —Frank Macfarlane Burnet to Linda Druce, 1926 Lets talk about bacteriophage—­its my second great interest at present—­your only great rival for my affection is the correct term I believe. —Frank Macfarlane Burnet to Linda Druce, 1927

Although the intellectual pathways toward understanding the nature of the Rous sarcoma viruses and the bacteriophages maintained a high degree of conceptual parallels, they proceeded along somewhat different timelines after the initial phases of discovery and debate. Whereas RSV was discovered some five years earlier than the bacteriophage, it was the research on the latter that was more consistently productive in the decade or so immediately following the two discoveries, while new research on tumor virology underwent a period of dormancy, at least in terms of new findings or ideas about it. According to Hansjürgen Raettig, from the mid-­1920s until 1941 output on the bacteriophages continued at a more or less consistent rate of 100–150 publications per year.1 Hence this chapter picks up where chapter 2 left off, with the bacteriophage caught in the crossfire of a long standoff between Félix d’Herelle and Jules Bordet. Near-­invisible creatures that infect bacteria that in turn cause rather nasty intestinal infections such as dysentery in humans, bacteriophages are not likely to signify romance to most people. But they seemed to have done so for Frank Macfarlane Burnet, a twenty-­

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something Australian doctoral student in Britain in the mid-­1920s. So all-­consuming was his interest in them that for nearly two years, from 1925 to 1927, while in England working on his doctorate, Burnet would fill his letters to his fiancée, Linda Druce, back home in Melbourne, with the details of his experiments and ideas. In one of these letters toward the end of this period, he would claim, with no small amount of bravado, “I shall soon consider that there are only 2 Frenchmen and 5 Germans who know more about phage than I do.”2 His diaries from an earlier period also contain many references to his work on bacteriophages, and decades later in his memoirs he recalled the bacteriophages fondly, describing them as “a fascinating new fairy tale of science” in the 1920s.3 This chapter delves into some of the details of the fairy-­tale years of Burnet’s bacteriophage research from the mid-­1920s through the end of the 1930s, during which time he coaxed out their secrets and revealed them to the world of science. Why focus on a young Australian, who until the beginning of this chapter has been mentioned only in passing, and who, furthermore, was responsible for a very small fraction of the bacteriophage publications during the period covered herein?4 Indeed, most readers who recognize Burnet’s name are, in fact, more likely to do so in the contexts of immunology or animal virology (or both) rather than the bacteriophages. Burnet was a rather unusual biologist in his time for combining meticulous laboratory work with an epidemiological interpretation of the results. A recipient of the 1960 Nobel Prize in Physiology and Medicine for his contributions toward understanding immunological tolerance, research conducted near the end of his long career, Burnet had also been nominated on at least two earlier occasions for his contributions to understanding various infectious viruses.5 In a commemorative essay, Peter Doherty, another Australian who won a Nobel Prize for his contributions to immunology, remarked: “He was a superb research investigator during the virology phase of his career. It would not have surprised anyone if he had shared a Nobel Prize for his influenza work.”6 The main reason for considering Burnet’s small body of work on the bacteriophages here, however, is that role it played in rescuing d’Herelle’s idea about its viral identity. Just as Peyton Rous believed that his viral theory of the sarcoma agent was “saved by an Englishman” (William Gye),7 d’Herelle’s idea might be regarded as being rescued by an Australian, at roughly the same time. As seen in chapters 1 and 2, bacteriophage research had been fraught with controversy since

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Fig. 4.1 Frank  Macfarlane Burnet working at his bench, circa 1930s. 1986.0107.00056, Frank Macfarlane Burnet Collection, University of Melbourne Archives, .

d’Herelle had announced his discovery, and even the most basic facts about them were matters of strong contention. Burnet’s contributions, though small in number, were valuable, for through his steady work Romancing the Phage

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and measured writings, he played a key role in advancing the state of knowledge about them. For all its apparent conceit, then, Burnet’s boast to his fiancée about his standing in the world of bacteriophage research was a fair portrayal of the status of the field at the time. His contributions were recognized in his own day, albeit by that small group of researchers interested in bacteriophages. Little over a year after he had written that letter to his fiancée, for example, he received an invitation from the Medical Research Council (the governing body for allocating funds for medical research in England) to contribute a chapter on bacteriophages for a new reference series on bacteriology.8 As he reported to his fiancée, “I was delighted because when you are asked to write a section of an authoritative work by H.M. Government someone evidently thinks you more than an enthusiastic youngster playing with test tubes.”9 Despite the steady research activity on the subject in a small but select number of research laboratories, primarily in Europe and North America, there had been no consensus or resolution on the fundamental matters such as bacteriophage identity or behavior, save for a strong opposition to d’Herelle’s ideas. There is some evidence that by the later 1920s and early 1930s a larger number of researchers were willing to accept that he had some aspects right. In his essay on the bacteriophages in Thomas Rivers’s 1928 volume titled Filterable Viruses, for example, Rockefeller Institute scientist Jacques Bronfenbrenner would concede that despite opposition from various quarters, d’Herelle’s belief that bacteriophagy was “a disease of bacteria produced by an autonomous, ultramicroscopic, corpuscular virus is sufficient reason for including it in a book on filterable viruses.” But he also added, “Many of the author’s ideas are not in agreement with those of d’Herelle,” displaying a reluctance to align himself entirely with d’Herelle’s view, an attitude that was quite widespread among the few but dispersed bacteriophage researchers at the time.10 Something new was needed, in the form of data, evidence, or ideas to break the deadlock. That something came in the person of Burnet, whose fresh “outsider” perspectives would bring the first clues to resolving some of the most contentious debates and disagreements over the nature of the bacteriophages.

Encounter and First Forays The earliest reference to the bacteriophages in Burnet’s extant writings is to be found in one of the personal diaries that he maintained as a young man.11 While a medical resident in pathology at Melbourne

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Fig 4.2 (a-­b) Recording the bacteriophage: A. Cover of diary maintained by Burnet from 1922 to1924, with inset (B) showing his entry of January 30, 1924, containing his first mention of the bacteriophages. Photographs courtesy of University of Melbourne Archives, Frank Macfarlane Burnet Collection, 1986.0107, unit 8, file 02/007. Copyright Macfarlane Burnet Estate.

Hospital, he routinely encountered the full gamut of common diseases of the day, dysentery and typhoid fever among them, and the medical specimens from patients—­blood, pus, sputum, urine, stools—­were often the starting points for investigations. The outcome of one of these experiments excited the following entry on January 30, 1924: “Today I’m in the best of spirits with the world. I have isolated a bacteriophage that is behaving very nicely indeed and probably today was the first occasion on which any man in Australia ever saw the curious worm eaten appearance of a bacteriophage culture on agar. The—­alone I did it—­feeling is nice and it is pleasant to be tackling something new. I have more imagination than is good for a pure scientist and at the moment rosy colored visions of the possibilities in the bacteriophage are opening before me.” Exciting to be sure, but the entry raises the question of how a young medical resident with little or no research experience at the time hit on the bacteriophage—­a rather new discovery made halfway across the world from him—­as an object worthy of laboratory investigation, without any real guidance from more experienced physicians or researchers. The records are sparse on detail, but later, in his memoirs, Burnet .

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would positively identify d’Herelle, rather than any notoriety that the bacteriophage had gained due to the various debates on matters of priority or identity, as his main influence: “Very soon after I had started work in the laboratory as the resident pathologist of the Melbourne Hospital a curiously exciting book came into my hands, I forget by what circumstance. It was the English translation of d’Herelle’s Le Bactériophage.”12 Elsewhere he also referred to the importance of his having read this book at the time, remarking: “I clearly remember observing a culture from urine on a agar slope of uniform lawn of B coli with four large bacteriophage plaques. This was only about a month or so after I had produced typhoid phage. . . . I wondered whether if I had seen those plaques before reading d’Herelle I would have realized that there was something worth studying before me.”13 In addition to these direct statements, one can find, peppered throughout the diaries from the period, little notes communicating his excitement and interest in the possibilities that the bacteriophage was opening up for him. Barely a month after his first mention of bacteriophage isolation, for instance, there is another entry that reads: “A few months ago I elaborated a system of immunity with one wide generalization. Kellaway at least did not sneer at it. That generalization seemed to cover all but one mysterious gap and now as I study the bacteriophage I seem to feel that the filling of the gap is close at hand.”14 Unfortunately, there are no further records about this particular line of investigation, but Burnet was evidently successful in certain investigative endeavors on the bacteriophage, and in the next two years was able to publish a couple of independent reports on his findings.15 Although he also published papers on a few other topics during this period, it was his experiments on the bacteriophages that yielded some of the most promising and exciting results and held his sustained interest. Of his early research endeavors, they were the only subject that he took with him, when the following year (1926) he went to the United Kingdom to work on his PhD as was customary at the time for Australians wishing to gain research experience.16 Burnet’s early bibliography reveals a steady trickle of publications on bacteriophage for the next decade or so, while most of the other topics he had also started to investigate in 1924–25 fell by the wayside. Although not explicitly stated in the titles, the debate over the nature of the bacteriophage—­namely whether it was an external particulate organism that infected bacteria, or a ferment (enzyme) of bacterial origins—­was a steering theme in Burnet’s earliest publications on bac-

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teriophages. Right from the beginning, he appears to have been sympathetic to d’Herelle’s view that they were viral in nature. Although in his first paper he tried to examine the feasibility of both extrinsic and intrinsic origins for bacterial lysis, he found it difficult to accept theories of a “purely bacterial origin” on the grounds of what he termed as “the phenomena of facilitation.” To him, the observation, borne out through experiments, that the power of a bacteriophage to lyse bacteria increased upon serial passaging, was more “readily explicable if phage be a living organism capable of variation but difficult to understand if it is a normal internal constituent of the bacillus.”17 Although he conceded that the difficulty was “not insuperable,” and that bacterial lysis might be induced by enzymes, Burnet’s explanation nevertheless relied on describing the bacteriophages in particulate terms. In his second paper, which appeared in tandem with the first, he was even more forthcoming in his dissatisfaction for the idea of bacteriophage as enzyme and his support for d’Herelle’s point of view. He clearly declared his stance in the very first sentence, noting, “It is a fundamental point in d’Hérelle’s argument in favour of the particulate organismal nature of the bacteriophage that if equal amounts of a strong Shiga phage are plated with variable numbers of sensitive bacilli an equal number of ‘taches vièrges’ or clear plaques appear on incubation.”18 Burnet’s first two papers on the bacteriophage were part of a cohort of publications on a mixed bag of topics that had likely aroused his interest as he came across them in the course of his work as a resident pathologist in Australia. It was really in England that his work began to take a more defined direction, which in turn crystallized his ideas about bacteriophage nature. Bacteriophages very quickly became the primary focus of his doctoral thesis, and for the two years that he would spend in England, they were virtually the only topic on which he conducted research. As various personal and published papers from this period reveal, his alignment with d’Herelle’s bacteriophagy was not one of unconditional acceptance but rather a critical vision that he continued to test and refine over the next several years, depending equally on experimental evidence and his own argumentative reasoning. His state of mind is well demonstrated in his various remarks about bacteriophages sprinkled throughout his letters to his fiancée. They display the inner dialogue of a scientist willing and active in changing his mind while he juggled different concepts, arguments, and evidence and tried to fit them together into a cohesive concept. On one occasion he wrote to Romancing the Phage

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tell her: “You are in for a dissertation on the hundred and fifty-­t hird theory of bacteriophage and at least the fourth that the present author has been willing to place his undying faith on (for at least a week).”19

Insights into Bacteriophage Multiplication Alone of all d’Herelle’s claims, the observation that the number of bacteriophage plaques was dependent on the number or concentration of bacteriophage particles inoculated into a bacterial suspension, and independent of the population of the bacteria in that suspension, had stood uncontested, even by the likes of André Gratia, who otherwise opposed him at every turn. This premise undergirded d’Herelle’s interpretation for yet another result observed early on in the course of his investigations, on which there was, as Burnet noted, “practical unanimity” in the bacteriophage research community; namely, the fairly consistent pattern to the increase (or growth) of bacteriophages.20 When inoculated into a broth culture of growing bacteria, there was an initial lag or period of latency followed by a sudden increase of bacteriophage concentration in the culture. After this initial increase, the tube would undergo successive cycles of plateaus (lags) and exponential increases of bacteriophage, until virtually all the bacteria in the culture disappeared. D’Herelle’s interpretation of these observations was that the first lag or latent phase corresponded to the entry of the bacteriophages into their host cells wherein they replicated or multiplied. The sudden burst or increase in concentration of bacteriophage that followed this latent period was therefore explained as the outcome of the release of new bacteriophages into the medium as they induced bacterial lysis.21 Burnet noted, however, that aside from opposing d’Herelle, not one of his opponents had ever produced any evidence that actually refuted his ideas. Nearly a dozen years since he had first announced his discovery, d’Herelle remained the sole person to have produced “the only direct evidence that has been published to substantiate the theory of step-­like increases.” But even more significant, Burnet argued, were the implications of the stepwise growth of bacteriophages: d’Herelle’s “work showed the increase by sudden steps quite clearly, but since it has been neither confirmed nor refuted by later authors, its importance for the interpretation of lysis had not been appreciated by the opponents of d’Herelle’s theories.”22 Thus Burnet’s first order of business in a report on bacteriophage growth was to tackle these gaps head on. D’Herelle’s basic technique to track bacteriophage growth over time had been to introduce a small quantity of known concentration of

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bacteriophage into a tube containing a broth culture of actively growing susceptible bacteria and gauge phage concentration in this culture by withdrawing drops at different points of time.23 The main drawback of this approach was the high possibility for sampling error. Because d’Herelle grew the bacteriophage in a single tube of bacterial culture, there was no certainty that the contents of any single drop of suspension that he drew out at different time intervals were identical to the contents of the rest of the tube. A further problem was that this approach assumed complete synchronicity of the life cycles of all the phages in the solution, a highly unlikely occurrence. In his paper Burnet introduced several modifications to d’Herelle’s method. Rather than follow the growth in a single tube, for instance, he set up the experiment by mixing a drop of bacteriophage suspension into a known quantity of bacterial culture and distributing this suspension in a series of capillary tubes, each containing the same volume of culture. At fixed intervals over the course of several hours, he sampled ten of these capillary tubes, in effect creating multiple snapshots of the population of bacteriophage at each time interval. In a conversation with his biographer many years later, Burnet described his modified experimental protocol and the meaning of the outcome: Phage multiplication in bacteria was constrained for 20–30 min. D’Herelle thought this was the case because there is a sudden burst [of phage in test tube with culture]. But I did it by making a limit dilution of phage, adding an actively growing culture to it . . . and then as rapidly as I could, putting them into capillary tubes . . . 20–30 within first two or three min and seal them rapidly in flame, put them at 37°C and then, at intervals break them out to a seeded plate. And incubate these to count plaques. At first one . . . , two . . . , one . . . , zero . . . , one . . . , and then Bang! A 100, 70, 80, 100, 100, 100. . . . This indicated some liberation or bursting of the phage.24

The method went a long way in removing the randomness of d’Herelle’s sampling method. Burnet introduced further rigor to the experimental system by testing the protocol on two different types of bacteriophages specific for two different bacterial species. The consistency of the two sets of results led him to conclude, with perfect justification, that, in concordance with d’Herelle’s results: “Phage increase in broth is in the early stages at least due to the liberation at [bacterial] lysis of [phage] particles that have multiplied in or on a sensitive bacterium.” Romancing the Phage

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For all its usefulness in improving d’Herelle’s methodology and verifying his results, the greater value of Burnet’s paper lay in the fact that he was the first researcher who went beyond mere criticism of theories of bacteriophage other than his own, and actually considered the implications of the actual results for each extant theory. He accomplished this task by first setting up, in his introduction, a series of if-­t hen predictions of the outcome of his experiment for each of the theories. For instance, he pointed out that if Bordet’s idea that the cause of bacterial lysis was an enzyme or some other “dysfunction of the bacterial cell,” then the increase in bacteriophage “should take place steadily rather than in sudden steps.”25 The German researcher Robert Doerr’s idea that the lytic agent was a growth hormone whose activity had somehow run amok, eliciting an uncontrolled growth—­followed by lysis—­of the bacterial cells was also refuted for the same reason.26 Oskar Bail, another German scientist who contributed significantly toward developing schemes for bacteriophage classification by demonstrating the existence of different types, had disagreed with both d’Herelle and Bordet on the nature of the bacteriophage.27 Bail’s alternative theory was that the bacteriophages descended from the “generative substance,” or hereditary material, of their bacterial hosts and therefore that each particle represented “a definite unit of the chromatin hereditary substance (Erbmasse) of the bacterium lysed.”28 But for this theory to hold, Burnet argued, both Burnet’s and d’Herelle’s experiments should have yielded evidence of “a steady, unitary increase” of bacteriophage due to the release of one particle from each bacterial cell lysed. But such an outcome was clearly not the case. One of the few researchers who agreed with d’Herelle that the bacteriophage was a “living rather than a nonliving factor; that it possesses a corpuscular form rather than existing as a substance in solution,” was the American Philip Hadley.29 Unlike d’Herelle, however, Hadley’s model considered phages to be one phase in the life cycle of a bacterial cell itself, which, if true, would also produce unitary growth patterns inconsistent with those actually observed. Bronfenbrenner, whose first publications on bacteriophages appeared in 1925, believed that the lytic principle in bacteriophage was a minute particle adsorbed to bacterial fragments and argued for considering bacteriophage separately from bacterial lysis rather than trying to ascribe a causal link between the two.30 While Burnet conceded that Bronfenbrenner’s “description of the mechanism of the actual lysis is supported by adequate experimental evidence and is almost certainly correct,” he pointed out that the

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growth patterns predicted by this model implied that the first stages of bacteriophage multiplication were not discontinuous as actually observed.31 To this day the introductory section of Burnet’s paper on bacteriophage growth serves as an exemplar of clear and masterful scientific reasoning—­specifically the hypothetico-­deductive method—­and the rhetorical power of evidence and argument.32 By comparing each of the predictions against the actual experimental outcomes he was able to systematically refute each alternative theory of bacteriophagy by virtue of a single experiment. In its own time, this paper on multiplication did not make much of a splash in the bacteriophage research community. Certainly Burnet’s idea never drew the fierce opposition faced by d’Herelle, but whether it was due to the difference in personalities of the two men or the nature of Burnet’s logic, is not possible to judge fairly on the basis of the available historical evidence. Bordet, who was undoubtedly d’Herelle’s fiercest detractor, for example, did not engage with Burnet’s work even though he continued to dispute the viral theory of phage.33 J. C. G. Ledingham, who had served nominally as one of Burnet’s doctoral advisors, would admit as late as 1933 that “great admiration and respect” for Burnet’s experimental work on the bacteriophages, although he was still “agnostic” on the matter of the viral nature of bacteriophages. “Probably [Burnet] would agree that the reason why it is so difficult to get unanimity on this fundamental matter is because crucial experiments are hard to devise and consequently the inferences drawn will be inevitably coloured by previously held conceptions of the process of phage action,” he suggested.34 Just a few years later there appeared a report from the laboratory of physicist-­turned-­molecular-­biologist Max Delbrück, describing a new set of elegantly designed experiments that convincingly demonstrated the single-­burst growth pattern of bacteriophages.35 This paper has justly achieved the status of a classic because of its elegant style and the fact that it offered a standardized technique for culturing bacteriophages, which could be applied to study virus growth in the laboratory under well-­defined conditions. It heralded an explosion in bacteriophage research, which for decades would be dominated by the work of its principal author, Delbrück, and his collaborators, who came to be known as the American Phage Group. One of the later exponents of this group, the pioneer American electron microscopist Thomas Anderson, would recollect that, in the 1940s, when he had just embarked on his doctoral research, this paper formed “a little green island of logic in Romancing the Phage

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the mud-­flats of conflicting reports, groundless speculations, and heated but pointless polemics that surrounded the Twort-­d’Herelle phenomenon.”36 But aside from offering considerable refinements on the techniques of both d’Herelle and Burnet—­whose paper was admittedly conservative in scope, being limited to the first stages of bacteriophage multiplication—­I contend that Emory Ellis and Delbrück’s publication did not offer much that was new by way of ideas about the nature of the bacteriophages.37 To them the fact that phages were viruses was a given, and the technique they described was a means to an end—­t hat of studying viruses. For Burnet, in contrast, the demonstration of single-­burst growth was the end in itself, primarily as evidence that the bacteriophage was a particulate virus. Indeed, the opening lines of Ellis and Delbrück’s paper immediately dispel the notion that they played any role in resolving the conflict over phage identity, for they explicitly call the bacteriophages viruses: “Certain large protein molecules (viruses) possess the property of multiplying within living organisms. This process . . . is exemplified in the multiplication of bacteriophage in the presence of susceptible bacteria.”38 Neither in these lines nor anywhere else in this or the succeeding papers did Delbrück (with or without Ellis) bring up so much as a whisper of the implications of their results for the discussion about bacteriophages as viruses.39 They even cited Burnet’s paper, but made absolutely no mention of its theoretical contributions. Reading their papers side by side in the present day, however, it seems evident that it was a growing acceptance in the wider scientific community of the evidence and arguments presented by Burnet in 1929 that enabled Ellis and Delbrück to make the assumptions about bacteriophage with relatively little opposition in 1939. Consequently, I would argue that it was Burnet, rather than Delbrück and Ellis, who actually came closer to fulfilling the role in the history of bacteriophage research that Anderson assigned to the latter.

Burnet and the Problem of Lysogeny The controversy over bacteriophage identity was not the only matter that Ellis and Delbrück disregarded. There was an even larger elephant in the room, which they ignored altogether: the phenomenon of lysogeny—­until then, the most formidable obstacle by far in the way of achieving consensus on the issue of bacteriophage as virus. For Bordet and others, lysogeny was the best weapon against d’Herelle because it

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was the one phenomenon that could not be explained adequately by the viral theory. Controversies over the meaning and mechanism of the phenomenon were still very much in the picture in the late 1930s, and yet Ellis and Delbrück did not even acknowledge the existence of lysogeny in their papers, much less explain the controversy over it. Their reasons for omitting any mention of it were consistent with the larger context of Delbrück’s broader agenda for working on bacteriophages in the first place. For him the bacterial viruses were a means to a different end entirely, that of finding a good model system for studying gene duplication. Drosophila (fruit flies) and even the tobacco mosaic virus had proven too complicated for the type of analyses that he had envisioned, and he had begun to collaborate with Ellis specifically to see if the bacteriophage would serve as the “right organism for the job.” But, as William Summers has argued, the prerequisite for Delbrück’s use of the bacteriophage as the model would have been an acceptance of the notion of the bacteriophage as an exogenous virus. Had he given more than a passing consideration to any theory suggesting “that the bacteriophage phenomenon resulted from activation of a latent lytic enzyme, the study of phage would have been irrelevant to the problem at hand.”40 On the other hand, Burnet, for whom the bacteriophages were the main objective and not a tool, did not avoid lysogeny any more than he had the implications of the results of the growth experiments. Rather, he tackled it head on, albeit in a separate paper published toward the end of that same year.41 Although this paper would be the first time he gave public voice to his ideas about lysogeny, it is evident from other sources that he was aware of the debates and had developed his own ideas on the matter for some time. In his chapter on the bacteriophages for the Medical Research Council reference series, for example, he paid considerable attention to discussing theories and the work of other researchers on the topic throughout the chapter in addition to dedicating a section titled “The Spontaneous Development of Phage from Bacterial Cultures” to the subject.42 He was more guarded with his own ideas and does not appear to have shared them with anyone except his fiancée, at least in writing. As he explained to her on a chilly November day: I came across an interesting little difficulty in my work the other day by finding definitely what I had begun to suspect[,] that the bug I had been using in most of my phage experiments had a power to

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produce phage spontaneously. At present I believe—­rather open mindedly—­in the theory that the phage is a living bug that lives at the expense of bacteria. If so my stock culture which is in every respect an apparently healthy and thriving lot of individuals, has the disease in a latent form like a man with diphtheria bacilli in his throat that don’t quite give him dip but are very capable of infecting the more normal people that he comes into contact with.43

It is evident from this explanation that Burnet saw no conflict between the notion that the phage was an autonomous virus—­a “living bug,” in his words—­and the phenomenon of lysogeny, which he presented as a latent bacteriophage infection of the bacterial host. By using the analogy to asymptomatic carriers spreading infections, he was tapping into a subject familiar and hence easily understood by a much wider audience than medical researchers. Druce, for example, was a schoolteacher. Although by this time the apocryphal story of Typhoid Mary, the asymptomatic hospital cook who passed the disease to several patients who subsequently died, was well known in Australia as well, Burnet anchored his analogy in diphtheria, probably because more people around him had direct experience with it. When he got to formally publishing the results of his investigations on lysogeny, Burnet would use somewhat different analogies and metaphors, but the underlying mechanism that he described—­framed as a contest between the pathogens (bacteriophages), and the apparently healthy carriers (in this case the resistant, non-­plaque-­forming bacteria)—­remained at the heart of his understanding and explanation of lysogeny. At first glance, Burnet’s description of lysogeny might not seem so different from d’Herelle’s idea that lysogeny represented some sort of disrupted symbiosis between the bacteriophage and its host bacterium. But there were important differences between the ideas and approaches of the two men. For instance, Burnet, unlike d’Herelle, gave due credence to Bordet’s claims for the authenticity of the lysogeny phenomenon and also did not dismiss it as a result of mixed cultures of bacteria with different levels of resistance to bacteriophage. Rather, as he had in his paper on growth, Burnet began by articulating the knottiest aspects of the controversy around lysogeny and considering the implications of the phenomenon for understanding the bigger picture—­ bacteriophagy—­in its entirety. As he rightly recognized, the “most disputed points in the current controversies as to the nature of the bacteriophage are probably (1) the possibility of procuring phage from

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normal bacteria, and (2) the status of permanently lysogenic bacteria.” Therefore, his first step was to determine that lysogeny was a genuine phenomenon, at least in some species of bacteria that he called the “permanently lysogenic bacteria.” To this end he followed the growth and plaque formation of seven different strains of intestinal bacteria through six generations to confirm the fact that the cultures were purified and free of extrinsic bacteriophage. As he reported: “The strains tested . . . include practically every variant type that has ever been described amongst the Salmonella group. Every single colony that has been tested has shown definite lysogenic activity.” The rigor and comprehensiveness of this survey enabled him to declare with confidence: “It is evident that the lysogenic function in this case is characteristic of every cell in the culture, and presumably is intimately related to some of the most permanent of the cell’s activities.”44 Burnet’s position in these statements might come across to some as a retraction of his stance—­published scant months earlier—­on the viral identity of bacteriophages. But to him, there was no inconsistency because, as he elaborated in his article, he considered that the liberation of phage by the lysogenic bacteria occurred by “some totally different process from that occurring in ordinary lysis [classical bacteriophagy].” Among the different lines of evidence to support this belief was the finding that following the spontaneous lysis of bacteria from a lysogenic culture, the lytic principle did not produce its effects on the bacteria strain from which it originated—­t hat is, the lysogen—­and could propagate only at the expense of susceptible nonlysogenic bacterial strains. Even more persuasive to Burnet were the results of experiments designed to track the increase in numbers of bacteriophages within different types of host cells. By this time it was possible to demonstrate the increase in phage numbers within bacterial cells by using such means as mechanical agitation or enzymes or unrelated phages to lyse the bacteria. The basic principle of the approach might be better understood by considering an analogy to bursting a water balloon with a pinprick to demonstrate the presence of the water within, rather than waiting for the balloon to burst from the buildup of water pressure inside it. As both Burnet and d’Herelle had demonstrated independently, the lysis of a bacterial cell after a certain period of incubation following infection with a bacteriophage resulted in the liberation of several new bacteriophages. Such a finding was easily explained in terms of bacteriophage multiplication within the bacterium. If, Burnet argued, bacteriophage particles were “preexistent within the lysogenic bacteria, Romancing the Phage

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one might expect them to be liberated when lysis by some unrelated phage occurred.” But the results of a comparison of the number of phages released by a lysogen when allowed to grow naturally and lyse spontaneously versus those lysed by extrinsic means showed a dramatic difference in the latter. A control run used a nonlysogenic strain of bacteria, which showed no evidence of containing any bacteriophages. Based on these results, Burnet felt perfectly justified in concluding that the two phenomena were quite different. “The difficulty of reconciling these two aspects of bacteriophage phenomena has been responsible for all the current controversy on the intimate nature of phage, whether it is an independent parasite or a pathologically altered constituent of normal bacteria,” he wrote. “In our view both these contentions have been completely proved and the current attitudes on both sides of regarding them as irreconcilable alternatives is quite unjustified.” In Burnet’s opinion, therefore, the findings about the “permanence of the lysogenic character [made it necessary] to assume the presence of bacteriophage or its anlage in every cell of the culture, i.e., it is part of the hereditary constitution of the strain.”45 In effect, then, his claim was that during lysogeny, the bacteriophage virus was becoming a gene! Although he did not use this term in this first paper, he did so explicitly in later publications, describing the bacteriophage as a “gene re-­introduced into the genetic make-­up of the organism.”46 Before considering the rather startling implications of his idea that viruses could be genes, a brief aside is called for to explain Burnet’s use of the German word anlage instead of any English translation of the word to describe one of his central arguments in a paper written for an Australian, English-­speaking, audience. Although the word anlage is not in common use any longer, in the early twentieth century, biologists, and especially embryologists, used it as a matter of course to describe what the Oxford English Dictionary defines as “the rudimentary basis of an organ or organism.” The word can also be understood in the sense of “potential and specific agent,” the meaning of which can be “traced back in genetics, to what Mendel himself designated as ‘anlage’: the specific power of sexual cells to produce individuals identical [to themselves].”47 Burnet had most likely learned the term during his medical studies, and although he was discussing asexual bacteria rather than sexual organisms in the 1929 paper on lysogeny, anlage can be seen to have retained something of the Mendelian sense of the concept as the “potential and specific agent,” within the bacterial cell, which enabled it to periodically produce bacteriophages. The explanation in

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the discussion section of the paper certainly indicates Burnet’s understanding of the word in terms of a rudiment that required additional activation to furnish actual phage particles: “All bacteria of this permanently lysogenic type contain in their hereditary constitution a unit potentially capable of liberating phage. In a proportion . . . phage is actually present within the bacteria, and can be liberated by treatment with distilled water. Phage may also be liberated, presumably from bacteria of this group, during the normal processes of growth. The evidence further suggests that unless the activation of the hereditable anlage takes place spontaneously, disruption of the cell by any means will not liberate phage.”48

Lysogeny’s French Connection From today’s perspective Burnet’s hypothesis that lysogeny was a latent viral infection of bacteria, during which phase the bacteriophage assumed an alternate form—­t hat of a gene integrated into the genome of its host—­seems to have offered a simple and elegant solution to the seeming paradox that the phenomenon had posed. But for those closer to his own times, his explanation invoking anlage might have seemed more ambiguous, as evidenced in the following assessment of their work by the French microbiologist André Lwoff: “Burnet and McKie spoke of the activation or liberation of the anlage. But activation and liberation may correspond to two completely different types of processes: unmasking of a pre-­existing latent virus or development of a phage from something which is not a phage.”49 But this critique of the anlage idea was itself a retrospective remark, made more than two decades after Burnet and his colleague Margot McKie published their paper. With one notable exception, the paper seems to have gone largely unnoticed by contemporaries. This exception was the French scientist Eugène Wollman, who along with his wife, Élisabeth Wollman, at the Pasteur Institute in Paris, produced, next to d’Herelle himself, the most sustained output on the subject of bacteriophages, until the advent of the American Phage Group. In fact, Eugène Wollman holds precedence over Burnet in the matter of attempting to reconcile the views of his Parisian and Belgian colleagues, for he published his first ideas on the subject in 1920.50 In doing so he brought bacteriophage research back to its original home at the Pasteur Institute. There is an exquisite irony in this turn of events, for in effecting this move, Wollman laid the groundwork for the eventual, definitive resolution of lysogeny, the very phenomenon that had Romancing the Phage

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Fig 4.3 Eugène and Élisabeth Wollman, circa 1905. Copyright Institut Pasteur/Musée Pasteur.

been touted as the strongest evidence against bacteriophagy by Bordet and others of his ilk. The resolution itself would not be reached until many years after the Wollmans’ tragic demise at the hands of the Nazis. But their involvement was no less direct for all that, because they were responsible for introducing the problem of lysogeny to their young colleague Lwoff, widely recognized as the person to satisfactorily and decisively explain the phenomenon in molecular terms. The Wollmans, meanwhile, devoted more than a decade and a half toward attempting to clarify—­t hrough both experiment and theory—­t he various problems posed by the bacteriophages.51 During this time, Eugène Wollman also began a manuscript titled “Le bactériophage et le problème des ultravirus,” which unfortunately was not completed or published. A detailed analysis of this entire corpus of work is to be found in the French histo-

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rian Charles Galperin’s intricate and comprehensive history of understanding lysogeny and in a recent overview by Jean Gayon and Richard M. Burian, both of which, like most of Wollman’s own papers on the topic, are in French.52 Although a full translation and reprisal of these various analyses is neither possible nor even necessary in this book, I will revisit some of the salient concepts and arguments introduced by the Wollmans as an interesting point of comparison for Burnet’s ideas. Right from the start, Wollman seemed to agree with Bordet’s basic idea that lysogeny was “a hereditary trait that was acquired by infection.”53 But the two men seem to have had somewhat different ideas about heredity, at least in the context of bacteriophagy. Bordet, for instance, thought of heredity in strictly physiological terms; to him it was a “regulation which prolongs itself in the course of cell division.” Therefore, he did not consider either bacterial heredity nor lysogenic power as being “associated with specific particles endowed with genetic continuity.”54 Wollman, on the other hand, had a more conventional view of heredity and offered what Galperin has characterized as a Mendelian interpretation of lysogeny.55 In Wollman’s view the lysis of the bacteria was the result of a variation, whose determining element could be “transmitted not only from mother-­cell to daughter-­cell (ordinary heredity) but also, through an external medium, from affected cell to normal cell (paraheredity).”56 In this explanation, the former scenario equated with lysogeny while the latter with classical bacteriophagy. But it should also be mentioned that Wollman considered the external factors or bacteriophages to be “infectious processes of non-­microbial nature.” Burnet stressed the same distinction between classical bacteriophagy and lysogeny in his 1929 paper, and although he did not cite the Wollmans at all in this paper, he did in fact give their work credit for the genetic theory of lysogeny on at least one later instance, albeit without any specific citation: “According to Wollmann’s hypothesis the distinction between the two alternatives would disappear, the phage being regarded as a gene re-­introduced into the genetic makeup of the organism.”57 Wollman, in his turn, was complimentary about Burnet’s contributions toward understanding the relationship between bacteriophagy and lysogeny, crediting him as the person “to whom we owe the most complete study of this question,” and specifically citing his demonstration that “all cultures of B. paratyphosus C, examined . . . were lysogenic and the bacteriophages from all these cultures were identical.”58 Interestingly, however, he too neglected to cite any specific paRomancing the Phage

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pers. But it seems evident from these explanations that both men had conceived—­t he Wollmans in more explicit terms than Burnet—­of the existence of what Lwoff later described as “an alternation infectious → not infectious → . . . in the life cycle of bacteriophage.”59 As indicated earlier, these ideas about bacteriophage lysogeny did not attract much attention or followup from a broader audience at the time they were published. The American Phage Group, though aware of the work of the Wollmans and Burnet, had ignored lysogeny in their publications and were outright dismissive about it in private. Elie Wollman, the son of Eugène and Élisabeth—­who as a young man had fortunately escaped their fate at the hands of the Nazis by moving in 1940 from Paris to Lyon and later Toulouse (a free zone during the war)—­ who would also become a student of the American Phage Group, recalled an occasion when “looking into a bibliographical index, at Caltech [in Delbrück’s laboratory], I came across a reference to one of my parents’ papers.” To their conclusion that bacteriophage “entered a noninfectious intracellular phase after infection,” the comment on the “index card referring to this paper, was ‘Nonsense.’”60 Although the younger Wollman did not identify the author of this note, the remark was quite possibly made by Delbrück, notorious for his impatience with ideas that could not backed up with convincing evidence. More broadly, too, according to Elie Wollman, the leaders of the American Phage Group “swept away the facts and interpretations accumulated by their predecessors over twenty years and started anew. . . . I found myself admiring their revolutionary progress, and at the same time surprised by the carelessness with which they treated historical matters.” Matters were compounded by the fact that at the time Burnet and Eugène Wollman put forward their ideas, little was known about the chemical composition of either bacteriophages or genes. Indeed, the very status of bacteria as true “genetic organisms” was still a matter of dispute at the time, because they neither possessed true chromosomes nor exhibited Mendelian patterns of inheritance due to the lack of “the morphological apparatus associated with the genetics of sexual reproduction.”61 The prevailing belief at the time, as summed up by bacteriologist Joseph Arkwright, was that bacteria possessed no chromosomes, since none had “been observed and genes are purely hypothetical.”62 Thus there was an inferential gap between the proposal of bacteriophage as gene and the canonical knowledge at the time. Even if scientists had accepted the idea of the bacteriophage as a genetic rudiment or anlage, they did not have the experimental or con-

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ceptual tools to test the concept or link it to the known facts about the nature of either bacteriophages or heredity. DNA was an obscure chemical—­t he sticky viscous stuff found in pus from wounds—­still fifteen years away from being considered, much less recognized, as the material basis of genes.63 Still farther away were the discoveries regarding its structure and mechanisms of action.64 Consequently, as the biochemist and immunologist Melvin Cohn would remark many years later, Burnet’s “insight into lysogeny slept for twenty years.”65 I will leave the sleeping beauty that is lysogeny to rest for the time being and return to Burnet and the bacteriophage.

Bacteriophage in the Golden Age of Virology The “golden age of virology” is a phrase directly borrowed from Burnet’s characterization of the period from 1932 to 1933, although he added the caveat that virology was “a word, by the way, that we never used.” Upon completing his doctoral work in England in 1927, Burnet had returned to Australia, taking up a position as assistant director at the Walter and Eliza Hall Institute of Medical Research. Although he would later recall this time as a period when the bacteriophages represented “a second string activity,” those years actually accounted for nearly half his total output on the bacteriophage. Then in 1932 he was invited by Sir Henry Dale, director of the National Institute of Medical Research (NIMR), to undertake a fellowship primarily intended to finance studies of animal viruses. But upon his arrival he found himself in labs adjacent to those of Christopher Andrewes and the physical chemist William Elford, who, as he quickly learned, were “deeply involved in phage work and using several of my phages in the first studies of the size of virus particles. So I slipped comfortably into association and collaboration with them on a variety of topics. Phage was still nominally a secondary topic but I am sure it had the major place in my enthusiasms and I believe that the phage work at Hampstead probably represents the peak of my scientific achievement.”66 Andrewes, the British virus researcher who played a key if often behind-­t he-­scenes role in this book’s tale, was by the 1930s well entrenched in the British medical research establishment. Trained in medicine like his father before him—­he was the son of the eminent British pathologist Sir Frederick Andrewes—­Christopher had joined the staff of the NIMR in 1927, having already cut his teeth working with viruses: first as a medical student working with the British medical researcher Mervyn Gordon on the vaccinia and mumps viruses, and later in 1925 Romancing the Phage

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with Gye, specifically investigating the filterability of the Rous sarcoma agent from different sources.67 Elford, who joined the NIMR around the same time as Andrewes, had come from a somewhat different background; he had studied physical chemistry and, in fact, had worked in the industrial sector doing research for a soap manufacturing company. His entry to virus research came about through his being asked to develop ultrafiltration methods for J. E. Barnard, whose work in ultraviolet microscopy had led to his interest in viruses. Elford’s most significant contribution was the development of graded collodion membranes for measuring the size of small particles.68 Elford and others went on to use these membranes, in combination with ultracentrifugation techniques—­also developed by him—­toward the measurement of sizes of a large number of viruses being studied at the NIMR in the 1930s, including both the bacteriophages and the Rous sarcoma virus.69 Like Burnet, Andrewes would later recall this time at the NIMR as “an exciting period in the early days of virology with discoveries coming in thick and fast, and my colleagues seem, in retrospect, to have been more colorful than those of today.”70 Burnet evidently made an impression on Andrewes, for not only does his name appear in the roster of names and achievements in Andrewes’s review article but he also merited the following mention in one of the many exchanges between Andrewes and Peyton Rous: “We have Burnet working with us on phage. He’s a very bright spark.”71 In a description of the relationship between these two men, a colleague later recollected that Andrewes “admired [Burnet’s] scientific powers. They both felt that bacteriophages could be regarded as genuine viruses and that there were many practical advantages in using them as a model to answer basic questions about the properties and behaviour of viruses in general.”72 Perhaps the most lasting impact of any of Burnet’s work and publications on bacteriophage from this second time in England was a review article published in 1934, which went on to become a “classic” in the field.73 The microbial geneticist Joshua Lederberg, for example, has credited this article with inspiring him to take up the study of bacteriophages.74 After his return to Australia in 1933 (this time for good), Burnet continued to work on the bacteriophages for a few years longer, even as his interests turned increasingly toward animal viruses. He ceased work on the bacteriophages altogether in 1937, but not before publishing, as a penultimate parting shot, a paper about bacteriophage-­ induced mutations and lysogeny.75 This paper, which presented “one of the earliest and clearest cases of mutation in a bacteriophage,” was in-

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cluded many years later by Lederberg in a compendium of foundational papers in microbial genetics.76 But at the time he did the work, Burnet almost didn’t submit the paper because the research, he confessed, “seemed almost too trivial for publication.”77 Another visiting bacteriophage researcher who elicited Andrewes’s regard was Martin Schlesinger, a young Hungarian from the laboratory of the German chemist Heinrich Bechhold. In one of his letters to Rous, Andrewes commented: “He’s a bright lad, has views on the origins of viruses from cells of higher animals or plants. I argue with him.”78 Unfortunately, scarcely anything else is known about Schlesinger except as can be gleaned from his publications and passing references by others who interacted with him or cited his work. Another reference to Schlesinger, coincidentally in another letter to Rous, from Gye rather than Andrewes, bore news of his tragic death: “Do you remember a little refugee Jew named Schlesinger who used to work with Dale’s labs with Andrewes and others? He . . . committed suicide last March. . . . I was very upset. He was an extraordinarily able and cultured man. I liked him very much and gave him every opportunity. His mind gave way. It was a blow to us all.”79 In the short interval that he worked, however, Schlesinger was immensely productive and contributed significantly to the physicochemical characterization of viruses. Like Elford, he appears to have come to virus research through his work on colloids and ultrafiltration, and he made a mark with his acumen for precise quantitative experiments. Specifically with respect to the bacteriophages, the Swiss molecular biologist Eduard Kellenberger recounted: “Seven years before the fundamental papers of Max Delbrück on the experimentation with bacteriophages, M. Schlesinger in Germany carried out very similar, equally excellent experiments. Nobody took note of them so that Max Delbrück rediscovered them later. He quoted and discussed Schlesinger’s results, related to adsorption of phage particles to bacteria. I share the opinion of [Gunther] Stent that Schlesinger was the most notable forerunner of the ‘strategy of experimental phage work’ introduced later through the strong personal influence of Delbrück.”80 Also while at the NIMR, Schlesinger became the first person “to show that a phage contained DNA as a major constituent.”81 As was the case with Burnet’s work on lysogeny, however, the significance of this finding was lost at the time because virtually nothing was known about the role of nucleic acids in biology. Assessing the bacteriophage work of this period many years later, Romancing the Phage

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Gunther Stent would name Schlesinger and Burnet as “two notable exceptions” to the bacteriophage researchers of the 1930s, a group that he otherwise described as “extremely opinionated men, whose motivation for the design and interpretation of experiments must have derived more from the desire to vindicate intuition than from finding out, step by step, what things are really like.” But, he went on to observe, “Schlesinger’s premature death ended his work, and Burnet turned from bacteriophages to focus his attention on animal viruses. Since none of their collaborators or disciples continued on this truly pioneering work in exact experimentation on bacterial viruses, the continuity of ‘modern’ phage research really dates only from 1938, when Max Delbrück took up work in this field.”82 Although Delbrück did in fact discuss Schlesinger’s work at some length in 1940, he overlooked it and other advances of that period in his later writings about bacteriophage research, where he tended to highlight the work of the American Phage Group. One occasion he even claimed that the research on bacteriophages “after a fitful history during its first twenty years, had all but died out in the 1930s.”83 In an interview with his biographer many years later, Burnet would allude to this attitude by Delbrück with some acerbity: Andrewes, Elford and I did a lot of work on phage and we did bring it into pretty good shape. But it didn’t go on in the English language literature because of Delbrück and his group who started working on it immediately after the War. [Our work] was all before the War. And they [Delbrück et al.] isolated a little group of viruses and laid down the law that “we’ll work on no other viruses than these and we’ll do it properly.” And they did it properly and made no reference at all to the work that had been done previously.84

Burnet also offered an equally candid, if less pointed, assessment of the work in his memoirs, remarking: “In 1932–33, the work on phage that Andrewes, Elford and I were doing at the National Institute for Medical Research was undoubtedly far ahead of anything else in the world. . . . It is true that what we did failed to initiate continuing progress, but it had its influence.85 While the idea that bacteriophages were indeed autonomous viruses was by this time fairly widespread and also, gaining gradual acceptance, it was not a universally accepted fact. Part of the reason was fuzziness of the concept of virus itself. This issue is exemplified, for example, in a debate that took place in the early 1940s between Del-

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brück and the biochemist John Northrop, which focused on the matter of how bacteriophages were formed.86 A contemporary of Rous and Murphy at the Rockefeller, who worked primarily at the laboratories in Princeton, Northrop had, in the 1930s, successfully purified the enzymes pepsin, trypsin, and chymotrypsin in their crystalline form and thereby shown that they were proteins.87 When in the mid-­1930s he turned his attention to bacteriophages, he naturally compared them to the enzymes. Conceding that the apparent ability of the “bacteriophage of Twort and d’Herelle,” to multiply—­albeit only in the presence of living bacteria—­had led many scientists to consider bacteriophages to be living, his own findings that the enzymes “pepsin and trypsin, under the proper conditions, also increase,” led him to favor Bordet’s explanation “assuming the autocatalytic production of phage from a normal cell constituent.”88 Despite such occasional detractors, the idea that bacteriophages were viruses became so ingrained in the scientific community over the next decade that Burnet could remark in 1959: “I suspect that, for most American workers under 40, virus unqualified means bacterial virus. The animal viruses have lost their prototype status and must be specifically qualified as such. Which adds another devious turn to the strange etymological history of the good old Latin word ‘virus.’”89 The broader question of the nature of viruses themselves, meanwhile, remained in flux for some decades longer.

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5REAWAKENINGS

THE VIRAL ETIOLOGY OF TUMORS

Might it not be advisable to differentiate [fowl tumor agents] from the heterogenous group of viruses by applying to them a distinctive name, calling them, for example, transmissible mutagens[?] —James B. Murphy, 1931 The theoretical objections to the parasitic theory of cancer are no longer tenable in view of the facts of the filtrable fowl tumours. —Christopher H. Andrewes, 1934

In contrast to bacteriophagy—­which was a specific, brand-­new phenomenon that Frederick Twort and Felix d’Herelle stumbled upon in the course of their investigations of other problems—­ Peyton Rous’s claims of his discovery of a filterable cause of avian sarcomas was but one development in the much broader terrain of tumor etiology, which had a much longer history. Consequently, the understanding of the nature of the sarcoma agent and, more generally, the viral etiology of tumors proceeded in a somewhat more punctuated fashion, with phases of visible activity—­namely, new findings or evidence that appeared to favor one or the other idea about the nature of the agent—­ and those of relative dormancy, especially in the years between Rous’s discovery and the mid-­to late 1930s, when the development of sophisticated new techniques and instruments ushered in a new era of virology.

The First Dormancy Readers may have noticed in the accounts of Rous’s discovery and the reception to his ideas that the first lull in chicken sarcoma research

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followed rather quickly in the wake of the discovery of the filterable cause of chicken, and in fact, lasted a good deal longer than the first spurt of research. Rous himself stopped working on the tumor etiology problem in 1915, “pinched and parched mentally,” in his own words, from repeated failures, in his attempts both to grow the sarcoma agent and to find mammalian tumors transmissible through cell-­free extracts.1 And although his colleague and closest collaborator James B. Murphy would emerge as the person who maintained the continuity of avian tumor research at the Rockefeller, he too did not immediately pursue investigations on the subject. In fact, at the time that Rous switched research topics, Murphy also nearly quit—­not only cancer research but also the Rockefeller itself—­which would have resulted in the end of tumor research at the institute. But fate intervened yet again in the affairs of cancer research at the Rockefeller. This time around it was in the guise of a $200,000 endowment to the Rockefeller from a businessman named Henry Rutherford, for the express purpose of investigating such aspects of cancer as its nature, etiology, prevention, and treatment. Since Rous had already committed to other projects, Simon Flexner naturally requested Murphy—­by then the only other person at the Rockefeller other than Rous or himself who had anything to do tumor research—­to head the laboratory for cancer research.2 Ironically, then, it would fall to one of the most staunch disbelievers of the virus theory of tumor etiology to lead one of the most active inquiries into the nature of the chicken sarcoma agent and produce some of the highest and most consistent output anywhere of experimental work on the subject. Murphy, like Rous, had trained in medicine at Johns Hopkins University, and within a year of completing his studies there he opted to accept a staff position at the Rockefeller specifically to work on the chicken sarcoma problem with Rous. Apparently he was influenced in his decision by the advice of his anatomy teacher at Hopkins, the physician and medical researcher Florence Sabin (who herself joined the Rockefeller in 1925).3 Rous, for his part, “was eager to draw [Murphy] into the work impending, and the later records attest to his active share in it.”4 In less than a couple of months into his arrival at the Rockefeller, Murphy had proven his mettle, successfully developing two useful techniques that greatly improved the efficiency of tumor research. One achievement was the successful propagation of chicken sarcomas by growing the tumors on the membranes of fertilized eggs.5 This method was a huge advance because the growth of the tumors in Reawakenings

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the embryos was not species specific in the same way as it was in adult birds. Thus, this technique ensured that researchers could maintain a ready supply of the tumor agent in the laboratory and would not have to depend on the rather unreliable availability of affected birds for their research.6 Of more immediate importance to characterizing the chicken tumor agent was Murphy’s success in transmitting—­for possibly the first time anywhere—­t he sarcoma to new birds using material from tumor tissue that had been frozen and dried under vacuum, and then reconstituted in solution when required.7 The freeze-­drying technique, called lyophilization, was immensely useful because, unlike the tumor tissue—­which lost its tumor-­producing activity when stored in the laboratory for more than a few days—­t he freeze-­dried material preserved the tumorigenic capacity indefinitely. Despite Murphy’s efficiency, his results with the lyophilized material were unfortunately not confirmed until after Rous’s second paper on the chicken sarcomas had already gone to press. Consequently, although Murphy was not named as an author, Rous took pains to acknowledge the newcomer’s contribution. As he stressed to Flexner, he made sure to give Murphy the “entire credit for the transmission of the tumor by means of dried powder material” by appending a footnote at the end of the paper, reporting: “Later work in this laboratory, by Dr. James B. Murphy, has demonstrated that the tumor can be transmitted by means of the dried and powdered neoplastic tissue, kept at room temperature for many days. The tumors resulting from its injection do not appear for several weeks.”8 There is no evidence in the laboratory reports and publications to emerge from Murphy’s laboratory during the first decade of his role as head of cancer research to suggest that there was any progress or indeed work on the nature of the chicken sarcoma agent. Perhaps, like Rous, Murphy was also discouraged by the lack of much progress on the matter and did not immediately continue investigations into the matter. Most certainly, and quite unlike Rous, he did not believe he had anything to prove—­he was, after all, in accord with the consensus view. Meanwhile, however, he pursued other problems such as species and tissue specificity of transplanted tumors, which led to his seminal investigations into the central role of lymphocytes in the rejection of tumor grafts, what he referred to as tumor resistance.9 Over the course of a decade he published a series of articles on his lymphocyte work, mostly in the Journal of Experimental Medicine, and in 1926 summarized these experiments in a monograph.10 Such deep immersion into the cellular

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mechanisms of tumor resistance and rejection entrenched him in a physiological thought style and likely strengthened his opposition to the viral theories of tumor causation, in much the same way that Jules Bordet’s immersion in immunology had influenced his thinking about bacteriophagy.11 In fact, the specifics of Murphy’s claims about lymphocytes resonate strongly with the first ideas put forward by Bordet and Mihai Ciuca about the role of leukocytes induced by immune reactions to the dysentery bacilli in the mediation of bacterial lysis.12 Meanwhile, despite the lull within the Rockefeller on chicken sarcoma research, the virus theory of tumor causation did not die out. It simmered quietly in other places, not unlike the lava of a dormant volcano bubbling under the earth’s crust, ready to erupt into the open at any moment. Though not actively engaged in research on the topic of the chicken sarcoma, Rous played no small role in maintaining this underground activity, both materially—­by providing sarcoma material that he maintained in his laboratory to researchers in different parts of the world—­and intellectually—­by keeping the virus theory alive through correspondence and conversations. Especially notable in this regard were his exchanges—­beginning in the early 1920s and lasting for most of the rest of their lives—­with two men in England: the virologist Christopher Andrewes and the cancer investigator William Gye. Rous styled the three of them as the “three musketeers” of the viral theory, and it would not be an exaggeration to claim that these correspondences provided him with a vital lifeline to the topic during his long hiatus from active work in the field.13 Whereas Rous’s friendship with Andrewes began while the latter spent two years (1923–1925) working at the Rheumatic Fever Service at the Rockefeller as part of his training as a pathologist following his medical studies, it was a request from Gye for the chicken sarcoma material in 1923 that led to their friendship. Of the many requests that Rous received and responded to over the decades, this one undoubtedly had the most impact on the fate of cancer virology. Gye, who joined the National Institute of Medical Research (NIMR) in 1919 and who had a background in chemistry, had begun work on the Rous sarcoma agent “expecting to find it chemical in character, not a virus,” but he was very soon convinced otherwise.14 Describing his work in his first letter requesting Rous for new material, he said: My own work, which began last spring time, has been up to a point surprisingly successful—which keeps me on the lookout for a hu-

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miliating disaster. I am now able to cultivate an organism regularly from tumour filtrates. It passes through a fine candle . . . and is not definitely distinguishable in stained filters. I mean by this that the granular background which can be seen in many filter preparations is similar in appearance to the minute particles which I believe to be the organism. . . . Impression preparations of the colonies stained with Giemsa’s stain show as a mass of particles too small to be distinctly resolved by the best microscope we possess.15

Rous immediately and enthusiastically responded to Gye’s request: “Everything you tell about the cultivation of the agent causing the chicken sarcoma has an intense interest for me who strove nearly two years at that job, years rendered the more unprofitable through my inborn lack of aptitude for bacteriology. People talked then as now of a ‘chemical’ agent causing the sarcoma and, presumably, other causes. . . . I am rejoiced that you have already got this far.16 Two years after this exchange, Gye published the results and interpretations of his experiments in the Lancet, alongside an article by another NIMR colleague, Joseph Barnard, on the microscopy of the sarcoma agent, which explosively reawakened the decade-­long dormant theory that the cause of the chicken sarcoma might be a virus.

The First Eruption Gye appears to have been the first person to explicitly call the sarcoma agent a virus in no uncertain terms in his 1925 paper. Opening with a recap of Rous’s 1911 discovery and ideas about the nature of the agent, as well as the history of the “parasitic hypothesis” of cancer, he explained: “The problem which remained when Rous concluded his work on these tumours was the determination of the nature of the filterable agent. Rous and his collaborators brought forward strong evidence in favour of [the sarcoma agent] being a filterable virus—­a living but extremely small microbe. . . . Rous used the term ‘agent’ rather than ‘virus,’ since the final proof that the agent is animate its cultivation outside the body was missing.”17 Gye went on to detail various experiments, the results of which he believed offered positive evidence that the agent was indeed a living parasite. He reported experiments that confirmed earlier findings; for instance, he found that the time taken for a new tumor to develop from cell-­free filtrates of the original tumor was proportional to the dose or amount of material used, and confirmed the destruction or inactivation

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of the tumor-­inducing ability of the filtrate with chloroform. In addition, based on evidence from “spinning experiments,” or, high-­speed centrifugation, he argued that the tumor-­producing filtrate consisted of an essential factor for this purpose, which was “particulate and  .  .  . therefore probably a virus.” He also reported the transmissibility of certain mice sarcomas by cell-­free filtrates, something that had proven elusive to Rous. But undoubtedly the most important support for the virus theory in Gye’s estimation was the “proof of multiplication” of the agent. Such proof or evidence he obtained by inoculating cell-­free tumor filtrates into a broth medium consisting of pieces of chicken embryo as well as serum from rabbit’s blood. The incubated material showed increased tumor-­producing activity upon incubation, peaking at around the fourth or fifth day, and could be subcultured into a new series. This finding seemed to clinch the evidence in favor of the idea that it was a virus that was causing the sarcoma. Gye’s paper, which was heralded with much fanfare on both sides of the Atlantic, certainly helped the viral theory of tumor etiology overcome its status as “the Cinderella of oncology.”18 But his paper was far from the definitive evidence he claimed it was, mainly because his specific ideas about the causative mechanism for avian sarcoma—­t hat in addition to a virus there was also a second unstable, chemical factor produced by the host cell19—­were erroneous. Indeed, he faced a great deal of criticism for these ideas from many quarters. Even Rous, who maintained an extremely warm and cordial relationship with him throughout their lives, would admit to Andrewes in a private letter after Gye’s death, “Between ourselves his work on the specific factor got me into a jam when visiting England in 1926–7, so many people . . . expected me to endorse it, as in conscience proved impossible.”20 When and where Gye did play a more tangible role was during the dormant phase of tumor virology, as part of a behind-­t he-­scenes communications network that kept the theory alive.21 Meanwhile, even before American scientists responded publicly to Gye’s paper, it had succeeded in arousing the interest of the Rockefeller men, as evidenced by letters that crisscrossed between Flexner, Rous, and Murphy soon after the news of its publication reached American shores. Flexner, who was in New York at the time, wrote to both Rous and Murphy, perhaps with one eye toward reigniting their interest in renewing the Rockefeller’s flagging investigations on tumor etiology. As it happened, Flexner’s letter reached Rous in the same batch of mail as the proofs of the article directly from Gye: “You can imagine the Reawakenings

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eagerness with which I opened both [letters], right in the middle of our village street,” he wrote to tell Flexner almost immediately. But although he expressed “great hope that someone will prove the findings with the chicken tumor to be examples of a general rule,” and his belief that the Rockefeller should “search actively into the possibilities opened by Gye’s work,” Rous begged off pursuing the work himself. “You are correct to assume that I wish to remain on the sidelines,” he wrote. “But I greatly hope you will let Murphy do the work, or rather appoint him to it so definitely that there can be no collision within the Institute.” In a postscript, he added, wryly, “All this harking back to the work of 1911 and 1912 has made me feel like Rip Van Winkle emerging from the forest.”22 Although likely less in the know about the specifics, and certainly not invested in seeing Gye proved right, Murphy nevertheless appears to have welcomed the news from England. He responded to Flexner just as promptly and enthusiastically as Rous had, albeit with a touch less panache: The papers here have been full of English cancer work but I have been unable to make heads or tails of it. In any case I am tremendously interested and would be very glad to take it up. I have already sent for a copy of the Lancet. . . . [I]f this is not a slip in the English work it is the most important thing of the moment for investigation. Would you think it best for me to go over to England in the Fall and get more first hand information? . . . Your letter has given me the first idea of what it is about. I gathered from the last account in the paper of today that the work had been confined to sarcoma and that the report had not been well received at the [British Medical Association]. I wonder if this is true.23

This missive marks the beginning of an involvement with the issue of the sarcoma agent that would play a dominant role in Murphy’s research career for many years to come. Unlike Wood, Murphy did not immediately react to the announcement in print. Neither did he question the authenticity of Gye’s findings as Wood implied in his New Republic article. Indeed, Murphy appears to have valued the data furnished by Gye, even going so far as to identify it as one of three “outstanding contributions” of the past year, in a response to a request from the Nobel Prize committee for nominations of candidates to consider for the 1926 prize in the physiology or medicine category. But he stopped short of making an actual nomination, saying, “I

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don’t feel justified in nominating unqualifiedly either of the three men mentioned.”24 That Murphy even considered Gye worthy of his list quite remarkable, because he appears to have written the recommendation letter to the Nobel committee mere weeks after publicly, and very thoroughly, contesting the selfsame work at the annual meeting of the American Zoological Society. “Doctor Murphy Doubts Cancer Isolation,” ran the headlines of a December 31, 1925, writeup in the New York Times. Unfettered by the type of inhibition that would have held him back when Rous still headed the tumor lab, Murphy was finally able to expound at length on his opposition to the idea of the cancer virus. He reserved most of his criticisms, and the strongest, for Gye’s interpretation of the results rather than the experiments themselves. Most significantly, he disputed Gye’s notion that the only possible explanation for the multiplication of the tumor agent and its ability to be passaged through multiple animals without losing its ability to induce tumors was that it was a living virus. The multiplication of the agent was not enough, in his estimation, as evidence to show that it was a living virus: “A few years ago this deduction would have been considered justified, but now we know of the existence of certain agents more closely allied to the enzyme group which give just as good evidence of multiplication as does this agent of the chicken tumor,” he argued. As a supporting example, he added, “You are all no doubt familiar with the bacteriophage which gives every evidence of increasing when brought in contact with bacteria—yet there is very strong reason for believing that this is not a living, formed agent.”25 As a quick aside, Murphy’s reference to the bacteriophage here supports this book’s overarching argument, that there was more—­much more—­to the similarities in ideas about tumor viruses and bacteriophages than mere coincidence. Murphy offered two main reasons to justify his alternative interpretation of Gye’s data as indicative of a cellular substance rather than a virus: first, the high degree of specificity of the hosts in which the tumors were transplanted successfully; and second, the type of tumor induced in the new animal. Bolstering these objections was his observation that the susceptibility of a chicken toward a particular type of tumor seemed to be inherited over multiple generations: “Is this to be assumed as an inherited susceptibility to an infection which is always present?” he asked. Stressing his view that there was inadequate information to formulate a viable theory, Murphy concluded his talk with the remark, “May I suggest as my belief, that when the cause of cancer Reawakenings

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is discovered, it will be found to be some agent or force most intimately associated with mechanism of the cell and will be a discovery of equal importance to those studying normal cell phenomena and to the cancer investigator.”

Data, Disbelief, and Alternative Interpretations One justifiable charge that a modern reader might level against Murphy’s criticisms of Gye’s ideas at the American Zoological Society meeting—­which were published the following year—­is that he relied entirely on argument, supplying no new experimental data of his own. And while it is true that this first critique was grounded completely on interpretive differences, records show that he soon backed up his initial objection with a steady output, until the 1940s, of both experimental reports and commentaries on the topic of the chicken sarcoma agent. Murphy’s first order of experimental business was to apply Gye’s methods himself in order to “examine the experimental proof” on which Gye based his viral theories. He ran additional controls in which the media were supplemented with uninfected cultures of noncancerous embryonic tissue or placental cells instead of the “so-­called cultures of malignant tissues” that Gye had reported.26 Although he was able to corroborate Gye’s result that tumor filtrates treated with chloroform were indeed inactivated, he found that their reactivation was just as effective in the control cases. In Murphy’s view, such a result eliminated “the necessity of assuming a cultivated living organism in the interpretation of Gye’s results.”27 But rather than stopping there, he used the opportunity to attempt to further characterize the intrinsic cellular factor that be believed triggered the formation of sarcoma tumors. According to the report he submitted to the scientific board at the Rockefeller in April 1927, one of the first things he did at the time was to undertake a comparison of “the properties of the causative agent of the chicken tumor group with the filterable viruses on the one hand and enzyme-­like agents on the other.”28 Following a series of experiments chemically analyzing fractions of tumor-­causing material from chicken sarcomas, he found that the active agent seemed to consist mainly of a nucleoprotein.29 He also compared the resistance of the sarcoma agent to ultraviolet radiation with that of bacteria and viruses, and found that whereas bacteria and viruses were “killed by hundreds of units of ultra-­ violet energy” the tumor agent was inactivated only with substantially higher doses of radiations; “thousands or tens of thousands of units. The character of the curve plotted for the chicken tumour agent is fun-

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Fig. 5.1. American cancer researcher James B. Murphy. Print by Rockefeller University, courtesy of Rockefeller Archive Center, Rockefeller University records, Graphic Services, Photographs, Portraits—­Prints, box 13, folder: Murphy, James B.

damentally different from that obtained for bacteria and viruses, the latter two being similar.” To him these different data seemed to strongly suggest that the agent had an “enzyme-­like nature.”30 In the context of the driving premise of this book that the histories of thinking about tumor agents and bacteriophages ran similar courses, Murphy’s idea that the nature of the tumor agent resembled an enzyme inevitably reminds one of Bordet’s ideas about the bacteriophage. In Reawakenings

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true Plutarchian style, a comparison of the two throws into relief not only the similarities but also important differences between them. As mentioned already, among the many and striking similarities is the fact that their common opposition to the notion of an extrinsic agent as well as the alternative theories they had suggested stemmed from their work in immunology. But the differences between their thinking and work were no less marked or meaningful. Indeed, points of departure are especially interesting because they may have had some bearing on why the ideas of the one (Murphy) carried currency for a much longer period than the other (Bordet). At the time of the most contentious debates in their respective arenas, Murphy and Bordet were at very different stages of their careers. In the 1920s, when he entered the debates over the nature of bacteriophagy, Bordet was already a well-­established scientist who had won a Nobel Prize for his immunological work. Indeed, readers may recall that his foray into investigating bacteriophage had been instigated by his umbrage at Félix d’Herelle’s perceived challenge to his authority in immunology. Other than his discovery of the first lysogenic bacteria, he did very little laboratory work on bacteriophages. Most of his opposition to the viral theory was based on his vast experience as a bacteriologist and immunologist, and likely also, to his antagonism to d’Herelle. It is not clear if Bordet ever responded directly to, or indeed even commented on, the evidence in favor of the viral theory offered by investigators other than d’Herelle—­Frank Macfarlane Burnet, for example. Furthermore, little or none of the data to emerge from Bordet’s group was brought to bear upon the question of the viral identity of bacteriophages. In contrast to Bordet, Murphy was yet in the early stages of his career when he first began work on the chicken sarcoma, and despite his personal opinions he never publicly contradicted Rous about the nature of the causative agent. Moreover, his disbelief of the theory was regularly buttressed by interpretation of data produced in his own laboratory over the course of some decades.31 Murphy’s notion about the enzyme-­like nature of the sarcoma agent found favor with other cancer researchers opposed to virus theories, particularly James Ewing. “I will take the liberty of stating that the theory of enzyme action has been adopted by you throughout your work on the chicken sarcoma,” Ewing wrote to tell Murphy, who responded with gratitude for the older researcher’s offer “to put a statement in your discussion about my theory in regard to the chicken tumor.” But, conscientious as ever, he also warned Ewing, “It hardly seems

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justified, however, [because] I have very little direct supporting evidence.”32 Indeed, the lack of evidence led Murphy himself to eventually discard the idea that the sarcoma agents were enzymes because, “unlike enzymes, the tumor agents essentially reproduce themselves and can be perpetuated by passage in vivo.” In an explanatory footnote to this statement he stressed that in his original reference he had “used the term ‘enzyme-­like’ only to emphasize the conception that the agents were produced by cells. No closer likeness was suggested or considered.”33 To Ewing as well, he stressed the difference in terminology: “I have rather avoided the statement that the chicken tumor agent belongs to the enzyme group. I have used enzyme-­like only to indicate that it is an active product of the cell . . . that the agents are of cellular origin.”34 But Ewing remained enthusiastic about the enzyme theory, and years after Murphy himself had set aside the idea, would claim in a lecture about tumor etiology: “That the active agent in chicken sarcomas is a chemical substance resembling the class of enzymes is strongly indicated, if not definitely proven, by the investigations of Murphy and his associates.”35 Whereas he changed his stance toward the enzyme-­like nature of the sarcoma agents Murphy remained steadfast in his adherence to the idea that it was a product of the host cell, or, as he would later put it, belonging “to the field of biochemistry rather than to that of bacteriology.”36 At first he justified his stance on the basis of “two striking facts that indicated to my mind that we had to deal with endogenous chemical substances rather than with extrinsic living viruses; and these were the high degree of selectivity [of the tumor agents] and the specificity of the types produced.”37 As time went on, his lab gathered increasing amounts of data using new biochemical and biophysical techniques, which reinforced his belief that the sarcoma agent was not a virus.38 A number of people worked for or with Murphy on the chicken sarcoma problem over the years, but there are two who merit at least a brief mention at this stage, due their engagement with the issues most pertinent to the history narrated here. One was Francesc Duran-­ Reynals from Catalan (Spain) who may have been the first person to move directly from bacteriophage to avian sarcoma research.39 The second, the Belgian Albert Claude, already discussed briefly in chapter 2, would go on to win the Nobel Prize in Physiology or Medicine in 1974, together with two other researchers for discoveries “concerning the structural and functional organization of the cell,” which were rooted in Reawakenings

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chicken sarcoma work.40 Both men were proponents of viral theories of cancer, which would lead to some rather contentious exchanges with Murphy, but oddly enough, despite their overlapping tenures in his laboratory, they do not appear to have collaborated or joined forces in any way. Duran-­Reynals had first encountered and become interested in bacteriophages and their applications to therapy against typhoid fever sometime in 1922 during his military service in Barcelona, and upon receiving a grant to train for a research career in 1925, chose to go to the Pasteur Institute, where he wanted to gain experience in working with bacteriophages under Eugène Wollman. Much like Burnet, Duran-­ Reynals had, right from the start, accepted d’Herelle’s hypothesis that the bacteriophage was a virus. At the same time, however, he also agreed with parts of the opposing view that the phenomenon of bacteriophagy was an autolysis generated from within the bacteria. This view ran counter to the ideas of Wollman, whose notion was that the bacteriophage was a product of bacterial mutation. After just one year at the Pasteur, Duran-­Reynals applied to transfer to the Rockefeller to complete the remaining two years of his grant.41 In his application he explicitly invoked Alexis Carrel’s recent ideas about the analogies between bacteriophagy and avian sarcomas as justification for his proposal to move: “Recent research . . . carried out by Professor Alexis Carrel of the Rockefeller Institute in New York has revealed the unsuspected relationship that the problem of cancer (in a newly-­discovered aspect which he summarizes from the studies of Peyton Rous) with that of bacteriophagy. In the critical review of the question which I published . . . I attempt to establish the analogies clearly. Moreover, in the past month I have been studying Rous’ chicken sarcoma virus experimentally [at the Pasteur Institute] and will continue working on this problem until the end of my fellowship.”42 Upon his arrival at the Rockefeller, Duran-­Reynals began in Carrel’s laboratory as originally planned, but very soon switched to Murphy’s lab, which was more engaged in investigating the type of questions about the chicken sarcoma agent that interested him. Within his first year he had accomplished enough to be included as a coauthor of a paper on the topic.43 A tangential physiological discovery made during his initial investigations of the transmissibility of the sarcoma agent led to a project on “spreading factors,” which became one of his main pursuits for many years to come. In 1932 Duran-­Reynals returned to Spain with the intention of establishing a research institute in Barcelona, but

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was prevented from doing so because of the revolution. Although he managed to obtain a reappointment at the Rockefeller in 1937, he did not find a congenial home where he could pursue attempts to prove the virus theory of cancer, and in 1938 he moved to Yale where he remained till the end of his life. While at the Rockefeller Duran-­Reynals had deferred to Murphy’s preference for the label “chicken tumor agent,” but once at Yale it did not take him long to start calling the causative agent a “virus.” Soon after his move he wrote to Murphy with drafts of papers he was preparing for publication with a note about this change of heart: “I think it is only fair for me to explain frankly the reasons why I have adopted, concerning the agent of Ch. T. 1, a point of view which I know is not in agreement with yours. During the last few years I have been more and more inclined to think of the agent as a typical filterable virus.”44 Needless to say, Murphy did not take kindly to this decision, and wrote back to say so: “You must have been quite well aware what my reaction to your letter would be. . . . Your action will be taken as a deliberate slap in the face for me and even those who will take satisfaction in your ignoring my point of view, will certainly not admire you for the manner in which you have done it.”45 Perhaps to appease his former mentor Duran-­ Reynals did not change the terminology in his 1939 papers, but from 1940 onward he began to use the term virus in all of his publications and for the rest of his career remained “one of the most tenacious advocates of the viral theory of cancer.”46 Albert Claude’s trajectory in sarcoma research was somewhat different from Duran-­Reynals’s, although his beginnings at Rockefeller—­ as a European medical student interested in the problem—­were quite similar. Soon after graduating from medical school in Belgium, he wrote directly to Simon Flexner, with a proposition, “hand-­written, in poor English  .  .  . to isolate and determine by chemical and biochemical means the constitution of the Rous chicken tumor I agent.”47 His application was accepted and upon his arrival he was placed in Murphy’s laboratory, where the latter assigned him with the task of consolidating evidence “that the ‘chicken tumor I agent’ was not a ‘virus,’ an exogenous parasitic microorganism, but rather an endogenous product of the tumor cell itself.”48 Claude had a long, nearly two-­decade tenure in the Murphy laboratory, during which period he published a succession of papers sometimes on his own, and often with Murphy and others in the laboratory, on the purification and physicochemical characterization of the tumor Reawakenings

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agent, using increasingly sophisticated methods and new cutting-­edge instrumentation, notably the ultracentrifuge and electron microscope.49 Unlike Duran-­Reynals, he did not publicly name the sarcoma agent as a virus and simply called it an “agent” for a much longer period, which may have contributed to his longer tenure in the Murphy lab. But eventually he too had run-­ins with Murphy on the issue, as is evident from a rather testy letter that accompanied some revisions of a draft of a report by Claude on the electron microscopic work on how tumor cells infected the sarcoma agent, accusing him of being “resentful and disloyal to our group.”50 Murphy certainly had reason for his ire, for in the concluding sentence of this paper Claude, perhaps for the first time, openly declared his opinion that the “chicken tumor viruses appear to possess properties commonly attributed to viruses.”51 Whatever their later relationship, however, the steady output of data on the physicochemical and antigenic properties of the sarcoma agent that emerged from the laboratory during the period Claude worked in Murphy’s lab consolidated Murphy’s views on its nature as being endogenously derived from the host cells and not a virus. He argued that the increased knowledge about properties of the sarcoma agent revealed “certain apparently fundamental differences between this group and the animal viruses [that] make it seem unlikely that they belong to the same order.”52 Even the property of multiplication or self-­perpetuation, which he conceded had been conclusively established, did not seem to him adequate reason for believing that the tumor agent was a living parasite or virus, as Rous, Gye, and others had claimed since the beginning. Murphy was by no means the only person to quarrel with the idea that the fact of multiplication was an index for the living nature of a substance; Bordet, one may recall, made very similar arguments against the idea of bacteriophage as virus; and closer to home, the biochemist John Northrop would soon be arguing in favor of considering the possibility for “the autocatalytic production of phage from a normal cell constituent,” in a manner similar to the increase of enzymes such as pepsin and trypsin.53 Meanwhile, Murphy would draw on even newer discoveries by other colleagues at the Rockefeller to support his argument that multiplication alone was not definitive evidence that the sarcoma agent was a virus. At a 1931 meeting of the Association of American Physicians, he argued that even considering the capability of the sarcoma agent for multiplication, “there are agents which probably furnish a closer analogy to the tumor agents than do

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the typical viruses, namely those recently demonstrated . . . as responsible for the specific pneumococcus types.”54 Murphy was alluding here to bacterial transformation, which has since earned a permanent place in the history of modern biology as the system through which the role of DNA as the material basis of heredity was discovered. The phenomenon had been first described in 1920s, by Fred Griffith, an English physician and bacteriologist, while he was investigating an outbreak of pneumonia caused by bacteria of the genus Pneumococcus. Griffith had found that there were several types or strains of pneumococci—­often from the same patient—­t hat were identical to one another in all respects except in their serological or antigenic specificity. This finding had obvious practical implications for the diagnosis and immunological therapies for pneumonia but also, as Griffith pointed out, significance for understanding the epidemiology of the disease.55 It was during these in vitro investigations of these bacteria that he discovered the phenomenon of transformation: the ability of dead pathogenic strains of the bacteria to transfer their virulence (ability to cause disease) to living but harmless strains.56 Quickly realizing the significance of Griffith’s findings and its potential applications for dealing with pneumonia, at the time a very serious problem especially among soldiers at war, Rockefeller microbiologist Oswald Avery began to search for the biochemical basis of the variety in serological types and as well as transformation, with a view to managing the disease. Eventually, in 1944, this investigation led to the discovery of DNA as what was dubbed the “transforming principle” for its ability to transform one type of bacterial strain to another.57 In the early 1930s, the Avery lab had succeeded in demonstrating the transforming effect in vitro and also in extracting an active principle capable of effecting the transformation of an avirulent bacterial type to a virulent strain.58 Working in close proximity to Avery, Murphy was privy to the work even before it was published, and he drew on the group’s findings not only as an analogy for the tumor agent but also to propose a new and distinct category by which to identify them: Here an agent present in a specific type of pneumococcus has the power to transform the undifferentiated pneumococcus into the specific form. Thus we have a group of agents, products of the specialized cells, capable of conferring the peculiar type quality to undifferentiated cells of the same species which, in turn, may produce the active factor and transmit this to their descendants. If it be ten-

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tatively admitted that the agents of fowl tumors and those conferring specificity on the pneumococcus belong to a similar group, might it not be advisable to differentiate them from the heterogenous group of viruses by applying to them a distinctive name, calling them, for example, transmissible mutagens. Thus we would emphasize the principal characteristic, the induction of permanent cell mutations.59

Etymologically the term mutagen means something that generates a mutation, which, as is evident from his description, is the sense in which Murphy was using the term. But although he claimed it was “probable that the chicken tumor agents have a closer analogy to the mutagens than to the viruses,” he never explained exactly why he thought viruses could not be mutagens or what set the transmissible mutagens apart as a separate category of entities.60 His vagueness might be one reason why his transmissible mutagen concept does not appear to have gained much traction in the scientific community. Even Ewing, Murphy’s staunchest supporter in his opposition to the viral theory, did not bring it up as a possible explanation in his many and impassioned arguments against the viral theory of cancer. Most scientists mentioned the label treated it as just that, sometimes setting it aside in quotes and at other times not, but did not give it more attention. Virtually the only one of Murphy’s contemporaries who appears to have engaged with his transmissible mutagen concept more than cursorily was Andrewes, who appears to have learned about it even before it first appeared in print. In April 1931, shortly before the talk was published, Andrewes had requested a copy of another recent paper published in Science, to which Murphy had responded with not only the requested item but also a confidential copy of “a slightly dressed up edition” of the talk, adding by way of explanation: “Avery has some important new data on the pneumococcus. I think he undoubtedly has a cell-­free extract and can easily induce the transformation into specific types in the test tube. As this material has not been published it had to be left out of my paper. Avery and [Rufus] Cole are inclined to be enthusiastic about ‘mutagen,’ but Rivers is scornful.”61 Andrewes’s response to Murphy began on a very congenial and sympathetic note: “I was particularly interested in the observations about mutagen and the analogies you drew between that and the fowl tumor agent, because the same thing has struck me very forcibly. Of

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Fig. 5.2. British virologist Christopher Andrewes, who had differing ideas about the viral etiology of cancer, specifically that of the chicken sarcoma tumor. Photograph courtesy of the Wellcome Collection, CC BY.

course I didn’t know that Avery had isolated an extract which would do the trick; . . . I agree with you that there is an obvious analogy between mutagen & tumor agent (and also phage); time will show if it’s a superficial analogy or a real one. I shall watch eagerly for full details.” But Reawakenings

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Andrewes was critical of the proposal that these entities were separate category from the viruses and went on to say: I don’t agree with you in [forcing] a sharp dividing line at the point x in the series below. z Mutagen

y Phage

z

x Tumour agent

y

Other viruses

Pleuropneumonia

Bacteria

x

If you look at other properties of the tumour agent (sensitivity to other types of rays, to antiseptics, etc) you can draw just as good a dividing line at y or at z or anywhere else you like according to the criteria you pick on. I’m much more sympathetic with [Arthur] Boycott’s view that there is no very sharp dividing line anywhere in the series, not even between live things and dead things.62

How Murphy responded to Andrewes’s comments is not known, for there are no further references to the matter in the letters from the period that are in the archives of their correspondence, but the papers he published after this exchange suggest that he did not give much weight to Andrewes’s reasoning.63 Andrewes did not immediately react either, but some years later, he sharply and publicly criticized the idea: “Murphy seeks to separate off from the other viruses certain agents to which he would apply the name ‘transmissible mutagens.’ This category would include, I gather, the fowl-­tumour agents, such an agent as that causing change of type amongst pneumococci and possibly also plant mosaic viruses and bacteriophages. The characteristics of a transmissible mutagen are, first, ability to direct the specific differentiation of the cell into an abnormal channel, and secondly, of course, transmissibility.”64 The transmissibility of the sarcoma agent being universally agreed upon, in Andrewes’s estimation there was no reason why viruses or any living infectious being could not act as a mutagen—­t hat is to say, as a substance that induced its host cell grow abnormally and cause a tumor. One factor that may have played a significant role in fanning the flames of uncertainty about the various entities under discussion—­ specific ones such as the sarcoma agents, bacteriophages, and the pneumococcal transforming principle as well as less defined things such as viruses, enzymes, or genes—­was the fact that investigators at this time did not have much idea of the correlations between their chemical makeup and function. For instance, even though Northrop

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Fig. 5.3. Excerpt of a letter from Andrewes to Murphy written on May 22, 1931, detailing a particular interpretive difference. James B. Murphy collection, Folder Andrewes, C.H. (1930–1932). Courtesy of the American Philosophical Society.

had established that pepsin, trypsin, and other enzymes were proteins, and knew that the bacteriophages, in contrast, were nucleoproteins, he had no idea about the significance of this difference. Consequently he would argue that “some filterable viruses are probably enzymes.”65 Such views were neither uncommon nor, in fact, new. Much earlier in the twentieth century, for instance, Beijerinck had noted the parallels between some enzymes and his tobacco mosaic virus and argued that enzymes were themselves “partly living protoplasm,” although he did not believe the converse, that “living protoplasm must not be considered as a simple mixture of enzymes.”66 And little over a decade after Murphy and Andrewes’s exchange, Oswald Avery, on the eve of publishing his seminal paper about the chemical identity of the transforming principle, would, in a private letter to his brother, speculate: “If we are right, and of course that is not yet proven, then it means that nucleic acids [are] functionally active substances . . . and that by means of a known chemical substance it is possible to induce predictable and hereditary changes in cells. . . . After innumerable transfers without further addition of the inducing agent, the same active and specific transforming substance can be recovered far in excess of the amount originally used to induce the reaction. Sounds like a virus—­may be a gene.”67 This comment recalls various other comparisons and analogies cited in different parts of the book, including Burnet’s idea from a decade Reawakenings

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earlier that lysogeny could be considered as a virus becoming a gene. Of course at that time virtually nothing was known about the chemistry of either entity, whereas Avery was clearly mapping his speculations on to specific molecules. Also, although the first part of this description may be taken to mean that Avery would have had no quarrel with Murphy’s characterization of the transforming principle as a transmissible mutagen—­in his published paper he did, in fact, mention it specifically, as well as the papilloma virus in analogy68—­t he latter speculation about viruses and genes surely would have caused Murphy distress and Andrewes some pleasure or vindication. But in the early 1930s Avery’s speculations were yet a decade away and were, moreover, only privately expressed. Murphy, meanwhile, does not appear to have given Andrewes’s objections to his views much credence or attempted to resolve their differences. As he remarked rather acerbically some time later in a letter to Ewing: “As far as my own work is concerned I gave up trying to discuss it with Andrewes long ago.”69 In his mind, at least, the issue—­ or nonissue—­of the viral involvement in tumor causation was quite settled.

Rousing Van Winkle Whereas new work on the chicken sarcomas declined for a time in the mid-­1930s when Claude’s research took off on a tangent toward cell biology, theories of viral etiology of tumors did not undergo a similar eclipse. One reason was that after a twenty-­year hiatus—­albeit not a silent one—­Rous reentered the tumor virus fray as an active researcher in the field. This time around it was not the chicken tumor that roused him but rather a new discovery by his friend and junior colleague the virologist Richard Shope. In 1932, Shope, who had made a name for himself with the discoveries, in quick succession, of swine influenza and rabbit fibromas, published a pair of papers reporting the discovery of a tumor-­like condition (warts) in wild cottontail rabbits that could be transmitted specifically to other rabbits—­both wild and domestic—­but not to any other animals, through cell-­free filtrates of the tumor tissue, which was found to contain a virus.70 He also found that although the warts progressed and became malignant tumors (papillomas) in many of the tame rabbits, they did not similarly change in the wild cottontail rabbits. Busy with work on his earlier discoveries, and being “more of a field virologist than a dedicated pathologist,” Shope offered the papilloma material to Rous, who was only too pleased to accept.71 As Rous later explained to Andrewes:

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When I quit the tumor problem in 1915, only two of its aspects had any drawing power for me: a) the make-­up and nature of the chicken tumor virus, an undertaking for which I had neither the knowledge nor technique; . . . b) the virus causation of tumors in general. To this I would have gladly returned at any time if opportunity arose. It didn’t arise, but was given by that great fellow Dick [Shope], who knew of my long hankerings. Immediately after reporting upon his newly discovered papilloma virus, its character and scope [and] of the growths it induced, he offered both to me to do with as I wished, fancy free. . . . That’s really how I came to take off again into the cancer problem.72

The rabbit papilloma system proved useful to Rous in addressing many of the issues that had besieged him in his decades-­earlier work on the chicken sarcoma viruses. For one, the fact that the papilloma was a mammalian tumor virus addressed one of the main objections that such cancer researchers as Ewing had leveled against the chicken sarcoma studies: “This disease is clearly sui generis, and observations upon it may not be transferred safely to any other.”73 Until Shope’s discovery of the rabbit papilloma agent, all efforts to demonstrate the presence of causative agents in the recognized mammalian tumors had been unsuccessful. But with a transmissible mammalian tumor system in hand Rous could begin to try and “determine whether a growth known to be caused by a virus . . . namely the Shope rabbit papilloma[,] possesses the immediate characters and the potentialities of a tumor.” In the third and final paper of the series, the authors concluded with a fair measure of confidence that Shope’s rabbit papilloma was indeed a virus-­induced growth that possessed the “immediate characters whereby tumors are recognized.” The papillomas differed from other known tumors in that the incidence was “that of an infectious process, and from other mammalian tumors in that its cause has been demonstrated.” Consequently, Rous argued, “The morphology and behavior of the generality of tumors can no longer be taken to exclude the possibility that these are produced by extraneous, living entities.”74 Rous’s return to cancer research was greeted with enthusiasm by his friends and colleagues. One of the earliest people to comment was his former director Simon Flexner, who, upon reading the proofs of his first papers on rabbit papillomas, wrote to tell Rous: “I have taken the greatest pleasure in reading the three papers on the Shope papilloma. A splendid piece of work: thorough, ingenious, illuminating. . . . I conReawakenings

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gratulate you.” He particularly praised Rous’s general discussion from the third paper of the series: “I see only good to come of it. It’s provocative and not excessive in any way.” Indeed, Flexner’s one criticism, a “question of principle only,” was that the wording in the opening paragraph of the first paper of the series seemed “ambiguous. If the Shope papilloma is a tumor, then the first sentence is of doubtful validity.”75 In the absence of the original proofs, it is difficult to know the details of the ambiguity, but it would seem from Flexner’s comments that Rous—­ possibly in deference to the sensibilities of scientists of Ewing’s ilk—­ had probably hedged on the matter of labeling the papillomas as true tumors. Another person who was understandably enthusiastic in his praise of Rous’s early publications was Andrewes, with whom Rous has been in constant communication with details of his progress on the papilloma work. A short time before the papers were submitted for publication, Andrewes had written to congratulate Rous on his successful experiments. “Will the pundits think up a good reason for not calling it a true tumor? Or has the virus metamorphosed into a transmissible mutagen yet?” he asked provocatively, and later, after the papers were published, exuberantly claimed that Rous had “done for the papilloma what you did for fowl sarcoma—­proved up to the hilt that it is a veritable tumor.”76 Flexner and Andrewes were by no means alone in their assessment of Rous’s work; by the following year he had already received an invitation from prestigious Harvey Society to a deliver a lecture on the topic of cancer etiology, news that he shared with Andrewes in one of the many missives that crossed the Atlantic in that period: “May it please you to know that in early Dec. I’m giving a Harvey Lecture with the title “The Virus Tumors and the Tumor Problem.” It will be richly seasoned with Andrewes. . . . I shall have a go at what the tumor problem now seems to be; and it most decidedly seems to be a virus problem. In providing new carcinogenic agents one merely sends the tissue in a cab to the railroad station. There’s a virus at the throttle of every cab that steams out.”77

Interpretive Differences Come to a Head The cancer research community was not uniform in its reception of Rous’s comeback and his revival of the viral theory of cancer, however. Despite Andrewes’s hope that “I suspect that this time your fellow pathologists will prove wiser & won’t try to wriggle away from facts,”

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Murphy and Ewing were not persuaded in the slightest by this round of data and interpretations.78 Following a meeting where one of Rous’s reports on the Shope papillomas had been discussed, for example, Murphy reported to Ewing: “[Eugene] Opie surprised everyone when [he said] that Rous has now shown that mammalian tumors were caused by a virus.”79 In his reply a few days later, Ewing offered his take on the situation: “[Rous] now admits that the difference between the exciting agent of a cancer, and the factors which maintain malignant growth. This is the first acknowledgement of the sort that I have seen from him or any other virus exponent.  .  .  . Of course, the admission knocks the bottom out of the virus theory of malignancy.”80 Ewing’s line of argument here was entirely in keeping with his long-­held view that the origin and propagation of tumors were separate problems. “One concerns the exciting factors, the presence of which initiates the tumour process, while the other relates to the nature of the tumour process itself. The former may be called the causal genesis, the latter the formal genesis,” he had proposed at the same international conference where Murphy had presented his data and argued against the virus theory.81 He would reiterate this view in a lecture delivered shortly thereafter at the University of Toronto, with specific reference to agents of the chicken sarcomas and rabbit papillomas, which he evidently saw as formal and causal agents respectively: “In the chicken sarcomas the filterable agent appears to be essentially connected with maintaining the malignant process, but is not the original cause of the disease, while in the rabbit papilloma the virus originates the papilloma but is not concerned with the malignant process.” Not content to limit himself thus, he also presented strong arguments against virtually any statement that even carried a whiff of the suggestion that viruses could be involved in cancer etiology. For instance, he claimed that when a tumor-­like process was “really initiated by a virus, as seems to be the case with human warts and the papillomas . . . these diseases are infectious and contagious, and they are not cancer.”82 Andrewes, who had requested and received a reprint of this lecture, reacted quite predictably in his turn. As he described it to Rous, “Poor Ewing! I read with moist eyes the lecture he gave in Canada. A most heroic defense of a crumbling citadel! But when he tried to maintain that your Shope cancers were not cancers but something sui generis, methought his shells were duds!”83 Unable to “resist having a shot at the citadel myself,” Andrewes had written to Ewing with a heavily marked-­up copy of the article, together with a letter expressing “apoloReawakenings

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gies for some comments I should like to make.”84 These comments had amounted to an itemized list of points on which Andrewes disagreed with Ewing’s interpretation, most of them leveled against Murphy’s ideas: “The evidence which has apparently convinced you . . . seem[s] based less on naked facts than on Murphy’s interpretation of the facts (with which I and many others profoundly disagree),” he charged. For example, he said with reference to Ewing’s argument that the fowl tumor agents were capable of surviving much harsher chemical treatment than those withstood by typical animal viruses: “I know of no vigorous treatment which fowl tumour viruses withstand which other viruses won’t. (Another vision of the truth through Murphy’s spectacles).”85 Ewing shared this letter with both Murphy and Rous along with a request for their opinions on various points: “I am sending you a copy of a letter  .  .  . which Dr. Andrewes has honored me with,” he wrote, understandably disgruntled at receiving such a critique from a virtual stranger.86 Murphy responded by dismissing Andrewes’s critique, comparing his arguments to “a high school debating match [with] no merit.” He praised Ewing for the “clarity and directness” of the latter’s response to Andrewes, which he also felt was “fresh.”87 Rous may have shared Murphy’s opinion on the tone of Andrewes’s letter, for in private he took his younger friend to task: “The first I know of your attack on Ewing,—­for it can be termed nothing less,—­was when he sent a copy of your letter accompanied by a rather crusty note asking what I had to say to it. There was reason for him to be crusty. . . . His opinions don’t delineate Ewing. He is a completely honest man deformed by bias, a wholly sincere sectarian savant.”88 Aside from acknowledging Andrewes’s “characteristic breeziness,” however, Rous seems to have agreed with Andrewes on most of the salient points of the critique. “Before reading Andrewes’ queries carefully it seemed best to re-­read your paper and mark down the points of objection which had occurred to me. On comparing this list with Andrewes’ they check closely,” he told Ewing. In the same letter he also attempted to solicit Ewing’s empathy as an outsider to virology, with the observation about Andrewes’s expertise in the field, adding, “I am coming to know about viruses secondarily and that may be to some extent the case with yourself.” Ewing evidently did not agree; “I thought Andrewes’ letter was very restrained,” he responded. “Your own is much more subtle. I presume it all settles down to the question, what is a virus?”89 Ewing’s question, although presented just to Rous, and somewhat rhetorical in nature, actually hit the nail on the head, and brings this

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discussion back to the problem at the heart of these debates. The various ways in they conceived of viruses and differentiated them from other entities that prevented the various sides from truly understanding their opponents’ viewpoint. Paramount in Murphy’s view, for instance, was that viruses were capable of autonomous living, whereas the various entities that he classified as the transmissible mutagens were “inanimate substances (chemicals).”90 Meanwhile, Ewing, as evidenced by his remarks belonged to an old school of thinking that saw viruses strictly as agents of infectious diseases, a category of disease from which cancers were emphatically excluded. Andrewes, in contrast, was impatient with such distinctions, which he saw as splitting hairs. While he unequivocally agreed that tumors were not infectious, he also stressed that diseases of “parasitic origin need not be demonstrably infectious.”91 Andrewes also disagreed with those who felt that viruses could not be present asymptomatically in normal cells, which arguments ran along very similar lines to the those that Bordet had raised in considering lysogeny as evidence against the possibility that bacteriophages were viruses. Whereas, for example, the pathologist Boycott had contended: “If one postulates a normal virus occurring in normal cells, one had better call it something other than a virus,” the idea was far from inconceivable to Andrewes.92 “Why not call it a virus if, in fowls, it has the same properties as other viruses? Many symbiotic intracellular organisms are known throughout the animal and vegetable kingdoms. It would be strange if viruses never acted as symbionts,” he said, declaring in public an idea he had floated to Rous: “I keep brooding along the lines that the cancer virus is a normal symbiont of the cells of all of us and only declares itself when the controlling mechanism gets out of gear.”93 In a subsequent letter he explained, “The reason I suggested that the virus might be a normal inhabitant of the cell was this: in its disease-­producing state I cannot conceive of it as other than an intracellular parasite; I therefore find it hard to believe that in its harmless (? saprophytic) condition it should be extracellular.”94 Although cautious at first—­“Your view that the cancer virus is a normal symbiont of the cells of all of us is more daring than I have dared,” he wrote95—­Rous not only warmed up to the idea but eventually embraced it completely, and even included it in his Harvey lecture, supplemented with analogies and example: “Cancer does not spring full-­ blown from normal cells but develops as the result of gradual and often long-­continued changes: The changes induced by all the various carReawakenings

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cinogenic agents may be of a sort to urge a symbiotic virus or viruses to pathogenic activity. A mere dietary error will bring out a crop of fever blisters on the skin of a man in whom herpes virus has lain latent. . . . Virus-­induced rabbit papillomas . . . can be made to keep on growing while untreated growths are retrogressing in the same animal.”96 Andrewes himself developed his notion of symbiosis further, albeit in private at first. Just around the time that Rous was to deliver his Harvey lecture, Andrewes shared with him some ideas, “rather woolly for public demonstration,” in the form of a private “Christmas fairy story for oncologists.”97 Combining elements of metaphor, analogy, satire, and sarcasm, the story was a vehicle in which Andrewes laid out his speculations about the mechanisms by which viruses could cause tumors. Beginning seemingly innocuously with known facts about viruses and their relationships with their hosts and couched in the time-­ honored language and style of all good fairy tales, the story quickly moves on to present then novel ideas about the behavior of viruses once they had entered the cells of said their hosts: “Once upon a time there was a family of viruses, much like other viruses. Some of them liked mutton, others poultry. Some of them multiplied wantonly in the cells they inhabited, and smashed up their homes; others, more restrained, practiced a magic which caused their homes to proliferate. But after a while most of the homes got broken up and the viruses had to seek new ones in new hosts. These new homes they penetrated by means of the powerful tusks in their upper jaws.”98 The story goes on to propose the development of a “feudal” relationship between the host cells and viruses where the hosts “made terms with the enemy and said ‘Come right in and live in our cells. But you must practice birth-­control and leave the furniture alone. If you do that you may come even into the inner sanctum of our germ plasm.’ . . . In this feudal existence most of the viruses lost all ambition [and] after a few generations the race was quite tuskless.” In addition to the idea of symbiosis as the mechanism, this scenario contains a second, truly revolutionary idea, fully articulated for the first time: that the site of symbiosis between a virus and its host cell might be the host cell’s “germ plasm.” The idea might ring a bell to some readers, as it did to this author, for here is yet another parallel drawn from the world of bacteriophages; specifically, Burnet’s model for lysogeny described in 1929, where he had suggested that the bacteriophage virus became a part of the hereditary constitution of the bacterial cell.99 Substitute the words “hereditary constitution” for “germ

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plasm” in the fairy tale and Andrewes’s account of the feudal relationship between the viruses of mutton or poultry with their host cells is a very close reproduction of Burnet’s proposal in a different host-virus system. It is not surprising that Andrewes was familiar with Burnet’s work; after all, the two men had worked in close proximity at Hampstead Heath, where each would have been privy to the work of the other. But why, then, had he waited nearly half a dozen years before borrowing the idea? Although it is not possible to say for sure, there are clues in his description of the “gene theory” of tumor causation in his 1934 lectures: “‘Gene’ is the term used by geneticists to indicate the unit in the chromosome which carries a hereditarily transmissible character. It has been suggested [that] a gene might become wild, loose from the chromosome which is its natural environment, and imprint its character on fresh cells with which it came into contact: such a theory has been put forward to explain the nature of viruses in general, including bacteriophages as well as those of tumour agents.” But until then there had been no suggestion that a gene could act as an antigen in the way it was known that a tumor virus could in its host animal. Consequently, Andrewes had concluded, “In our present state of knowledge it seems wiser on the whole to confine our speculations to less fantastic channels.”100 Of course, we know from the fairy story that he did wander over to fantastic channels in private, but a few years later he went public with many of those some ideas in the decidedly less fantastic form of a lecture about virus latency delivered before the Royal Society of Medicine. Interestingly, although this lecture was primarily about the relevance of latent viruses to cancer, Andrewes devoted a section to the lysogenic bacteria likening their mechanisms of host-­parasite relationships to those of certain “indigenous” animal viruses that were set apart from other animal viruses by a combination of three main properties: their ability to cause “symptomless” infections that were not self-­limited, their capacity for persisting indefinitely in their host, and their tendency to usually infect young host animals. Lysogenic bacteria, he said, afforded an excellent example of indigenous viruses of bacteria, because here too the virus was “handed down in the germ plasm” of the host bacterium.101 Rous was a fitting recipient of Andrewes’s fairytale and he wrote appreciatively to tell him: “I’ve shown it to sundry and it may well get to all in the course of time as it acquires historical value which may very Reawakenings

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well hap[pen]. I’ve sent a copy to Gasser who is in the process of understanding virus problems and he says he wants to read it again for his own uses.”102 But Rous’s description of the reactions to his Harvey lecture—­in which he championed not only the latent virus idea but several others—­shows that Andrewes was indeed correct in surmising that many oncologists, of Ewing’s stripe for example, were not ready for such ideas: The Harvey Lecture proved far from a joke. I never laboured more for what is called a “definitive pronouncement.” You know that at the end of such a lecture it is the custom for someone to arise, being bound thereto beforehand, and say pleasant things of the lecturer. This time it was Ewing, and casting my [eyes] about I knew it was sure to be he and was prepared for what came as the audience were not. . . . For Ewing’s praise turned out to be the most ardent and lop-­ sided damnation. . . . The outcome has been a lively discussion and some partisans, which are what our view needs, at least over here.103

Although Rous’s eloquent and vividly detailed description of the incident to Andrewes was filled with humor, he was not quite as even-­ tempered about it elsewhere. To Gye, who, upon hearing of Ewing’s “crusty” reaction to Andrewes, had offered to play peacemaker—­“I should hate anybody from England to be unjust to Ewing . . . would you tell him from me that Andrewes was writing a mixture of fun & serious comment?”—­Rous had replied a trifle testily: “Your remarks on the subject of Ewing find me somewhat disposed to let him have it. He has become fanatically active in the attempt to keep the tumor problem in limbo.”104 Andrewes had come to much the same conclusion as Rous, albeit more colorfully in his story: “pathologists, who thought that all viruses had tusks, studied the neoplasms and grunted into their beards ‘The parasitic hypothesis of cancer is dead.’”105 Although unnamed, it is not unreasonable given the tenor of their exchanges that Murphy and Ewing were who Andrewes had in mind as these pathologists or the oncologists for whom he had purportedly written the story. But Rous and Andrewes need not have worried about the future of theories of the viral etiology of tumors, for Rous’s Harvey lecture turned out to represent their final eruption from dormancy. A chronology of discoveries related to tumor viruses offered by Jacob Furth and Donald Metcalf a couple of decades later, for instance, shows that there was a significant rise in discoveries more explicitly relating tumors and virus-

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es with relatively little controversy after the mid-­1930s; more than two-­ thirds of the twenty-­five examples of viral tumors listed in their table were discovered after 1938.106 Although the viral theories would continue to face naysayers for some years to come, such voices became less broadly influential, as new information would pour in about the nature of viruses, genes, and even smaller units of life.

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6WHAT VIRUSES BECAME

NEW VISIONS FROM NEW TOOLS

I presume it all settles down to the question, what is a virus. —James Ewing, 1935 Viruses should be considered as viruses because viruses are viruses. —André Lwoff, 1957

Debates over the nature of viruses continued to provide the backdrop for discussions about bacteriophages and tumor viruses well into the 1950s, and even beyond, in the latter instance. For a majority of bacteriophage researchers, the issue of the identity of phages as viruses had been largely resolved by the late 1930s, with the glaring exception of temperate phages, bacteriophages that underwent lysogeny. Research in this last field experienced an eclipse of nearly two decades before being revived in the mid-­to late 1950s, as I will discuss in the final chapter. Meanwhile, within the cancer research community—­which was larger and considerably more disciplinarily diverse—­t he possible involvement of viruses in tumor etiology remained a topic of dissension. One big hurdle in resolving this issue was, of course, that the concept of viruses continued to be in flux. Until there was a wider consensus about what viruses were, such debates would always remain in play. But that consensus was not to be hurried, as evidenced by the following observation by cancer researchers Jacob Furth and Donald Metcalf as late as 1958: “The concept of viruses varies with the discipline of the investigators and the kind of viruses they studied. Some

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microbiologists (as [Frank Macfarlane] Burnet) believe that viruses are microorganisms, some chemists (as [Wendell] Stanley) that they are molecules. Both views are doubted by some investigators ([Frederick] Bawden and [Norman] Pirie, [André] Lwoff). Most geneticists tend to consider viruses as replicating agents of cellular origin. All of these views have merit and could conceivably be true.”1 Furth and Metcalf’s comments—­including the examples they cited—­are a near echo of those made by the French molecular biologist André Lwoff just a few months prior in the very paper in which he furnished what is generally held to be the first articulation of the modern definition of the virus: “The man in the street generally considers viruses as the dangerous agents of infectious diseases. . . . Some virologists are convinced that viruses are micro-­organisms. This view is expressed in Sir Macfarlane Burnet’s book Virus as Organism. Other virologists, like Wendell Stanley, feel that viruses should be considered as molecules. A third class is represented by F. C. Bawden and N. W. Pirie who write, ‘statements that viruses are small organisms should be regarded with as much suspicion as statements that they are simply molecules.’”2 Although these statements might, at first glance, make it appear as though virus research had hardly changed since the early part of the century, the truth was quite the opposite. The main difference, I contend, between the status of research at the beginning and end of the period covered in this chapter, is that whereas the uncertainty until the 1930s was due to a lack of direct knowledge about the viruses as a separate group of beings, the confusion on the issue by the 1950s stemmed from too much information from too many different quarters. A quick survey of the literature on viruses from the early twentieth century reveals that virtually all publications originated from studies of their pathological effects—­not just on humans and animals, but also on plants and even, according to many, bacteria. As Thomas Rivers, quoting from the Bible, told his friend Ernest Goodpasture circa 1923, “You shall know them by their deeds.”3 Bacteriophages were detected by their effects on their bacterial hosts, whether or not one considered these effects pathological, or for that matter, considered phages to be viruses. By the late 1930s, on the other hand, Lwoff and Furth and Metcalf had culled examples of viruses from diverse and unconnected disciplines; not only infectious disease and plant pathology but also physical chemistry, biophysics, molecular biology, and genetics. It is hardly surprising, then, that the picture of viruses seemed complicated. Like the six blind men of an ancient Indian parable who What Viruses Became

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described an elephant in six different ways according to the part that each could feel—­t he tusk, trunk, ears, and so on—­each of the aforementioned disciplines offered a partial description about the unseen virus without necessarily considering how the parts made up the larger whole. Only after this information was integrated properly could one conceive of defining viruses in a way that satisfied all the different parties. In calling on this parable, I should make clear that in my mind, it takes the reality of the elephant as a given. In a similar vein, I should add, Lwoff’s opening gambit in his paper, stating that “viruses are viruses” makes a similar assumption about the reality of viruses. In a way, this chapter is an account of the realization of a prediction made in the early 1930s by the American pathologist and bacteriologist Earl B. McKinley, that for any real progress in virology to be made the “greatest need is for working tools, new ideas and methods of approach.”4 Without such tools, virus research was, as the British biochemist Frederick Bawden in the 1930s lamented, “like trying to find a black cat in a dark cellar, with no certain knowledge that the cat was there.”5 At the same time, however, this chapter may also be viewed as an account of the unfolding of events in the two decades or so between James Ewing musing in 1935, “I presume it all settles down to the question, what is a virus,” and André Lwoff’s remark in 1957 that “viruses should be considered as viruses because viruses are viruses.” Ewing’s question, to Rous, is a fairly accurate representation of the multiple views about viruses at the time. Virtually all of specific points raised in Rivers’s complaint about the characterization of viruses in terms of negatives—­invisibility, inability to be retained by filters, and inability to be cultured in artificial media—­concerned issues that could be resolved only through better instruments and laboratory techniques rather than any major conceptual advances. Indeed, until that period, the only technology to have made any significant impact on this field had been ultrafiltration. Although undeniably vital to the discovery of the first viruses, the scope of the technique on its own was rather limited. Virologist Karen-­Beth Scholthof tellingly equated advances in the knowledge about viruses with “seeing” them; until some sort of visualization became possible, she said, the virus “was outside the bounds of understanding. We couldn’t see it.”6 Arguably, the instrument that wrought the most dramatic change in terms of understanding viruses and filled precisely that gap identified by Scholthof—­visualization—­was the electron microscope. Invent-

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ed and developed over the 1930s, this instrument enabled scientists to create and see images of the viruses and thus revealed a world of creatures far more diverse in both size and variety than had been surmised earlier. But even before it was invented and deployed for the purposes of visualizing biological objects, other techniques had opened windows, allowing scientists to “see” viruses either indirectly or partially; for example, as lesions in their hosts, as crystals, or in terms of their component molecules.7 The further understanding of the structure and functioning of these component molecules through advances in molecular biology and genetics enabled scientists to think about, if not actually see, how each component performed functions that were earlier understood only through the indirect effects on their host cells. The development of techniques for the controlled propagation of viruses in the laboratory further expanded the horizons of what virus researchers could see: for one, they could directly observe their effects in specific cells and tissues. Virtually every one of these individual advances has garnered significant historical interest, and much has been written in considerable depth about different discoveries, instruments, and techniques by historians and scientists alike. Here I consider only the bearing of each toward the emerging modern virus concept as it impinged on the unfolding of parallel histories of the bacteriophages and tumor viruses.

Ultrafiltration, Whirligigs, and Whatnots It is no coincidence that the discoveries of the first viruses in the late nineteenth century—­by Dmitri Ivanovsky, Martinus Beijerinck, and Friedrich Loeffler—­followed so quickly on the heels of the development of bacteriological filters. As discussed in chapter 3, not only did the discoverers find their viruses due to the inability of these creatures to be retained by the filters but they also forwarded specific—­albeit widely varying—­speculations about the nature of the disease agent on the basis of information gleaned from filtration experiments.8 All of them, for instance, agreed that the active principle in causing the disease—­whether it was the actual organism (as per Beijerinck or Loeffler and Frosch) or a product thereof (according to Ivanovsky)—­was smaller than the lower limit determined by the pore size of the filters. In this early phase, the invisibility of these particles did not allow for further conclusions about their size. In the decades following the discovery of the first viruses, however, the improvement of the filters

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themselves, as well as a growing knowledge about the property of differential filterability of particles, became the basis for a technique to estimate the size of viruses. It was the British physical chemist William Elford, at the National Institute of Medical Research, who devised a technique to estimate the size of different particles. The method was based on the ability of different particles to be retained when passed through a series of collodion membrane filters of graded pore sizes, collodion being far more measurable than the earlier earthenware filters.9 Within a few years of developing this technique, Elford, working with other colleagues, notably Christopher Andrewes, had applied it toward estimating the sizes of different viruses, including both bacteriophages and avian sarcoma viruses.10 Though not a coauthor, Burnet, who worked for a time in the neighboring laboratory, was acknowledged as the source of most of their bacteriophage strains. It is obvious from their remarks that Elford and Andrewes felt that their data were consistent with Burnet’s belief that bacteriophages were a population of viruses and not all representatives of a single species: “It would be as reasonable to talk of the size of ‘a bacterium,’” they wrote.11 As for the sarcoma agent, given Andrewes’s involvement, it is perhaps not that surprising that the viral identity of Rous sarcoma virus was treated as a given. But Andrewes and Elford also gave their opponents a voice by opening their paper with the claim that determining the size of a fowl tumor agent was “of obvious importance in relation to the controversy” over the issue of its viral identity.12 A striking example of the vindication of Elford and Andrewes’s projections in this regard was an endorsement from the Japanese biologist Waro Nakahara, who spent the first years following his doctoral research, 1918–1925, working in the laboratory of James B. Murphy, who, as has been established, was one of the firmest opponents of the viral theories of tumor causation. One might expect that under this influence, Nakahara too would have been skeptical, and so he was until his use of Elford’s techniques persuaded him otherwise. Reminiscing about his career in cancer research toward the end of his life, he wrote: “My initial attitude toward the Rous virus was overly critical, but time and labor eventually nullified all my suspicions, and in the end the size of the viral particles was determined to be about 0.07 µm. This last work was done . . . by means of ultrafiltration through Elford’s ‘radocol’ membranes, which had been developed just a year or two before. This value determined by us for the first time remains unaltered.”13 So copious was Elford’s output

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of ultrafiltration data on viruses that Andrewes would later recall the pathologist Arthur Boycott inquiring, “Has not the time come where it is unnecessary to write a separate paper to record the size measurement of every virus?”14 But in Andrewes’s own assessment, each of Elford’s papers was significant because in addition to data about a new virus, it had something new to add about the ultrafiltration technique. In the mid-­1930s Elford also began to turn his attention to the emerging technique of ultracentrifugation as an independent means of verifying the size of different viruses. What the microbiologist Stuart Mudd had said of the “apparent simplicity” of filtration was equally true of the basic technique of centrifugation.15 It was even based on a similar principle of separation; namely, that of physical differences in the particles, although in this case it was density rather than size that was the differentiating factor. But centrifugation never faded into obscurity or came to be taken for granted by the scientists who invented and used them. For one, the 1926 Nobel Prize in Chemistry went to Theodor (The) Svedberg for his contributions toward understanding “disperse systems,” which was the basis for his invention of the ultracentrifuge just the year before.16 Centrifuges worked by spinning “mixtures of different substances at very high speeds. The heavier elements are pulled to the outer edge, and various measurements then allow the calculation of the weight of different molecules.” The machine constructed by Svedberg and his colleague allowed them to think about determining the size of particles that had eluded detection by the ultramicroscopic techniques—­another recent technical development, which just the previous year, in 1925, had garnered the Nobel Prize in Chemistry for its inventor, Richard Szigmondy.17 With his chemist’s acuity Elford very quickly devised certain techniques to eliminate errors and improve the utility of ultracentrifugation in the study of viruses, and within a few years greatly increased his already prodigious output on the sizes of different viruses. In all instances his results agreed well with data obtained from the earlier ultrafiltration experiments.18 Further corroboration would come later with newer, even more precise instruments such as the electron microscope. Size determination was simply the first of many contributions to virology from the ultracentrifuge, that “new whirligig of science,” as it was labeled by the New York Times.19 For that matter, Elford was not even the first researcher to attempt to deploy the instrument for this purpose, although he was certainly one of the most prolific. As Elford was quick to acknowledge in his first publication, the Hungarian chemWhat Viruses Became

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ist Martin Schlesinger had already successfully used the ultracentrifuge to estimate the size of bacteriophage particles based on differential sedimentation rates.20 In the meantime centrifugation also gained a lot of public attention as a key technique in virus research via the work of Wendell Stanley, a physical chemist assigned to help Rockefeller virologist Louis Kunkel isolate and characterize tobacco mosaic virus (TMV). Historian Angela Creager has shown how Stanley, after he had crystallized TMV, sought—­very successfully—­to “domesticate” the ultracentrifuge, primarily so that he would not have to depend on other laboratories to work out the finer details of virus structure. Later, in the 1940s, Stanley also turned his attention to the human influenza virus and subjected it to the same type of analyses as he had TMV; by then, his adoption of the centrifuges not only had helped establish it as a new analytical and preparatory tool to virology; it had also, reciprocally, led to improvements in the design of the apparatus for this very purpose.21 One beneficiary of the improvements to the design and operation of the ultracentrifuges with particular resonance for this book was chicken sarcoma research. The first forays in this arena were carried out in the mid-­1930s at the National Institute of Medical Research, by William Gye as well as by Andrewes and Elford.22 Gye’s contribution, in collaboration with J. C. G. Ledingham, the director of the Lister Institute, used high-­speed centrifugation to separate the infective “agent” from other components of avian tumor tissue. The publication of this report was quite a coup for Gye, because Ledingham had long been skeptical toward the viral identity not only of the tumor viruses but also the bacteriophages. For instance, at a 1933 talk on the current status of the bacteriophage by Burnet, whose doctoral dissertation he had helped supervise, Ledingham would confess to being agnostic on the matter.23 For that matter, agnosticism is also evident in the 1935 paper with Gye, manifested by the consistent use of the term agent (with quotation marks in most cases) rather than virus, despite Gye being an avowed champion of the virus theory. But Ledingham, unlike Murphy, appears to have eventually been swayed by the evidence; a couple of years after their joint paper, Gye would tell Rous, “Ledingham at a recent meeting in London nailed his colours to the mast. He is now a virus enthusiast. For years I have been arguing with him to the point of despair.”24 A second venue where the ultracentrifuge was deployed toward understanding the nature of the Rous sarcoma agent was the Rockefel-

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ler. The person to undertake this work was Albert Claude, as detailed in chapter 5. Given Murphy’s beliefs about the nature of the sarcoma agent, the first investigations Claude conducted had been chemical in nature, and these earliest studies culminated in a joint report in which Murphy’s theory of the agent as a “transmissible mutagen” was fully laid out in writing.25 But when Simon Flexner, in his capacity as director at the Rockefeller, urged the Murphy lab to use the techniques and instruments that had been successful in the virology lab in their quest for the sarcoma agent, Claude turned to centrifugation as the best tool for the job.26 Like Ledingham and Gye, he too appeared to use centrifugation primarily as a tool for isolating the virus rather than for size determination. In a series of papers published in 1937 and 1938, he reported the successful separation of active tumor agent from other components of tumor cells, using differential centrifugation.27 It was in the course of these studies that he obtained the raw material for the investigations that would eventually lead to his Nobel Prize for the founding of cell biology. Consequently, as claimed by Carol Moberg, in Claude’s hands the centrifuge became “the first critical instrument” for nascent cell biology.28 And yet, well before it was deployed for cell biology, the centrifuge had already proven critical in the quest for tumor viruses.

Crystalline if Not Completely Clear Visualizations In 1935, Stanley made national headlines with his announcement that he had successfully isolated a needle-­shaped crystalline protein with all the properties of an infective TMV. “Although it is difficult, if not impossible, to obtain conclusive positive proof of the purity of a protein, there is strong evidence that the crystalline protein herein described is either pure or is a solid solution of proteins,” he declared. Based on this evidence he concluded that the virus could be regarded as an “autocatalytic protein which, for the present, may be assumed to require the presence of living cells for multiplication.”29 Stanley’s results and conclusions seemed momentous because, as reported in a New York Times article published on the same day, they marked “the first scent on the trail of one of the ‘big game hunts’ of science” of whether a virus was a living or nonliving entity. Although proteins had been regarded as nonliving entities, because of their inability to grow and reproduce in the manner of living things, Stanley’s find appeared to indicate that a virus—­and by extension a protein—­was “an organism on the very border-­line between the living and non-­living.”30 Stanley’s announcement is of historical significance and bears reWhat Viruses Became

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visiting at this point, not so much for its contribution to the actual knowledge about viruses as for its immediate and profound impact on virus research.31 In terms of knowledge or content, as the historian Lily Kay pointed out in her detailed analysis of Stanley’s work and its consequences, the actual report on the crystallization of TMV had been “flawed by technical errors and misconceptions.”32 Few if any of his specific scientific claims, whether about the chemical composition of the virus crystals, their purity, or the autocatalytic nature of the proteins, were entirely accurate. These errors were in fact, suspected and corrected very soon by British biochemists Frederick Bawden and Norman Pirie, who, along with a pair of crystallographers, John D. Bernal and Isidor Fankuchen, soon identified the correct chemical composition of TMV.33 As Bawden recounted many years later: “Our reluctance to accept [Stanley’s claim] was not the prejudice of the many biologists who were unwilling to accept that a virus could crystallize, but because of his description of the isolated material. Not only was 20% [nitrogen] unusual for a globulin [protein], but some of the properties attributed to the material fitted ill with what was known about tobacco mosaic virus. . . . Our suspicions were soon confirmed for, instead of a crystalline globulin, we obtained a liquid crystalline nucleoprotein, containing 0.5% ribose nucleic acid.”34 Bawden and Pirie’s results were persuasive enough for Stanley himself to replace the word protein of his earlier descriptions of TMV with nucleoprotein, almost immediately.35 His transition was so seamless that in 1946 it was he, not the British biochemists, who was named as a recipient of the Nobel Prize in Chemistry, together with enzyme biochemists James Sumner, who received half the total prize for being the first to show that enzymes could be crystallized, and John Northrop, Stanley’s colleague at Rockefeller, who had further extended the work with the purification and crystallization of additional enzymes.36 As a matter of interest, the records show that Bawden and Pirie had also been nominated for Nobel Prizes both in chemistry and in physiology and medicine, both in 1939 for their work on TMV. Stanley, in contrast, was named in forty-­six nominations between 1938 and 1947. The nominator for the chemistry prize in 1939, the Swedish chemist The Svedberg, had in fact split his recommendation in half between Stanley and the British pair, but this was the sole instance of the forty-­seven nominations in which the nomination was shared.37 At least part of the reason for Stanley’s great success might be attributed, as Kay has argued, to the symbolic importance of his an-

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nouncement, especially for the proponents of the chemical view of life.38 The philosopher Alfred North Whitehead, who once famously proclaimed that it was more important for an idea to be fruitful than correct, would have no doubt approved. Although far more accurate, Bawden and Pirie’s data was a corrective and so lacked the startling power stemming from the novelty and originality of Stanley’s claims, erroneous or otherwise. What if the crystalline needles he had isolated were not pure? They were very nearly so: “We now call this the paracrystalline state,” the virologist Heinz Fraenkel-­Conrat would say, dismissing the problem.39 Similarly, although Stanley had missed the fact that viruses contained nucleic acid as well as protein, he was still within 0.5 percent of the correct composition, which was well within an acceptable margin of error given the precision of chemical purification of the times. His mistakes do not negate the fact that for the first time since the discovery—­or rather conjecture—­of the existence of viruses, scientists could begin to think about seeing or putting some sort of face or form to the name of virus. Bawden had remarked in a commentary on the impact of crystallography on the study of viruses, “Before 1936 it was tacitly assumed that all viruses were incompressible spheres.”40 The needle-­like crystals of TMV showed otherwise: here was one virus at least, that bore no resemblance to a sphere. But this finding was just the tip of the iceberg, a mere prelude to the deluge of revelations that the electron microscope would provide.

Through the Electron Microscope Centuries ago, in his landmark publication Micrographia (1665), the polymath scientist and first secretary of the Royal Society, Robert Hooke, had remarked, with specific reference to bacteria and microfungi, that the optical microscope had helped open up a whole “new visible World discovered to the understanding.”41 Deployed toward rendering viruses visible just a few years after Stanley crystallized TMV, the electron microscope similarly endowed researchers with a “newly found sight” that transformed the study of viruses from its status as a subfield of bacteriology to that of an independent scientific discipline.42 What electron microscopy revealed about the structure and composition of viruses as well as the diversity of the population—­which also had unexpected implications for their classification—­played a definitive role in shifting the definition of viruses from pathological to biological and molecular criteria.43 All microscopes work on the same basic principle, producing magWhat Viruses Became

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nified images of objects by reflecting and scattering incident radiation. The extent to which the images are magnified, namely resolution power of an instrument, depends on the wavelength of the source of radiation. Optical microscopes—­likely more familiar to more people—­rely on visible light and ultraviolet (UV) light rays, which can resolve distances between two points that are about 200 nanometers (nm), or 2,000 Ångstrom units (Å), and 100 nm, or 1,000 Å, respectively. Instead of light rays, electron microscopes use electron beams, which emit radiation of wavelengths several orders of magnitude smaller than that of visible or ultraviolet light. Consequently, the two pieces of equipment look and function rather differently. As explained by the pioneering American electron microscopist Thomas Anderson, Each element of the light microscope has a counterpart in the electron microscope. The lamp which serves as a source of light is replaced by an electron gun in which electrons emitted from a hot tungsten filament are accelerated by 60,000 volts to form a beam of electrons moving with a velocity almost equal to that of light; a magnetic condenser focuses this beam on the specimen; a magnetic objective lens forms an electron image of the specimen at a low magnification which is further increased by a projector lens corresponding to the eyepiece of the light microscope. The electrons which form this magnified image strike a fluorescent screen where their energy is converted into a light image which can be seen at a magnification which can be varied from 500 to 50,000 times.44

The first prototype of the electron microscope was built in 1931, an invention of Ernst Ruska, a German electrical engineer. While yet a student at the Technological University in Berlin, Ruska began his work on what would quickly go on to become “one of the most important scientific instruments of the 20th century.”45 But the significance of his invention was not immediately apparent, and it would be many years before he and the electron microscope garnered widespread recognition and accolades. In the context of this book, it is particularly interesting to note the parallel between Ruska and Rous: it took fifty-­five years (the longest gap in Nobel Prize history) for the Nobel committee to deem either man’s signature achievement worthy of the prize—­Ruska was the recipient of the physics prize in 1986. Like Rous, he too faced naysayers, in his case those who questioned the utility of the new instrument, especially in biology. Many physicists, for instance, were skeptical because

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they felt that the high heat produced by the electron rays when they bombarded the specimens would damage biological material such as cells or bacteria. The British physicist Dennis Gabor, for instance, predicted that everything exposed to the electron beam “would burn to a cinder.”46 Biologists were also hesitant at first to wholeheartedly embrace the electron microscope. To them, “the idea of using an electron beam in a microscope sounded just as absurd as letting lightning strike a person in order to photograph him.”47 This general attitude was brought vividly to life by Jules Bordet exclaiming at a 1934 meeting discussing the earliest results and the future of electron microscopy in biology: “Oh, no, no! please no electron microscope; we have already enough trouble to interpret the images obtained with the light microscope.”48 Such opinions notwithstanding, Ruska, unlike Rous, never had to face any conceptual opposition to his work, because he had developed a physical instrument, the existence of which could not be disputed. What was needed to harness its full potential were the techniques to prepare the specimens to be viewed, and also to accurately and efficiently record the images produced by the deflected electrons. Such improvements, which proved as important if not more so than improvements to the instrument itself, were developed almost hand in hand with the microscope and proponents of the technology very soon gained the upper hand over their detractors.49 Early in the history of the development of the electron microscope, Ernst Ruska’s younger brother, Helmut, training at the time as a medical doctor, had seen the potential for the new technology in his own field, especially in the visualization of “submicroscopic” agents of disease.50 Together with other minute things, such as cellular debris and subcellular components, viruses seemed to be good candidates for electron microscopy because “they automatically satisfied the requirements” for the techniques. By 1938, the Ruska brothers, together with Bodo von Borries, a physicist who helped Ernst improve the instrument design (and who was, incidentally, their brother-­in-­law), had published the first electron microscopic images of viruses.51 Other laboratories in Europe, the United Kingdom, and the United States soon followed suit—­simultaneously improving both the instrument and specimen preparation techniques. Within two decades, as the historian Nicolas Rasmussen noted in his comprehensive analyses of the taming of the electron microscope to the life sciences, “the electron micrograph of a virus [had become] its official, definitive portrait.”52 What Viruses Became

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Perhaps the biggest surprise electron microscopy had in store for the virus hunters was the fact that the viruses constituted a population as diverse in size range and morphological form as the bacteria. Especially astonishing were the revelations about bacteriophages, the tiniest known viruses, the earliest electron micrographs of which had seemed to confirm the impression that phages were relatively simple “kleines rundes Körperchen,” or small round bodies.53 In fact, ironically enough, Max Delbrück had initially chosen bacteriophages as his model system because of his assumption that all viruses were very simple biological objects. But the revelations from both sides of the Atlantic forced him and his group to rethink their theoretical assumptions, as further electron microscopic investigations by Anderson revealed that, in fact, bacteriophages “had amazingly complex structures.54 They had little heads, which were attached to what we then called a tail. . . . [N]ow we see, with improved resolution, that the tail itself is a very complex structure. It has a star-­shaped base to which very fine fibers are attached, and it seems that it’s these very fine fibers themselves that make the initial contact with the host cell. Then the phage particle appears to be drawn to the surface of the bacterium and the sheath on the tail contracts to expose a central core, and apparently injects that into the bacterium. So it acts like a tiny syringe needle through which the nucleic acid of the phage particle enters the bacterial cell.55

Anderson likened the Shope papilloma virus, then recently visualized by Robley Williams at Berkeley, to “mines that were used during the war; spherical particles that had a lot of protuberances on them.”56 Further studies of TMV showed that although it conformed largely to its tubular or “cigarette” shape, indicated by both crystallography and the earliest electron micrographs, it was not a simple needle-­shaped nucleoprotein as originally thought. Rather, as demonstrated through a series of experiments from various laboratories investigating this virus, the TMV rod was “a gigantic helix composed of protein subunits wrapped helically around an RNA core-­like structure.”57 As the virologist Leon Dmochowski would rightly emphasize later, the electron microscope showed that viruses were not mere molecules, as Stanley had posited, but had “a complicated structure like that of cells.”58 Simply the fact that it revealed a world far more abundantly populated and diversely formed than ever imagined might have been enough to place the electron microscope at the forefront of the new

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technologies that brought viruses “out of the fog” of uncertainty about their physical nature, and in some cases, their very existence.59 But, in fact, the electron microscope had much more to offer to virologists. It provided the basis for a nomenclature and classification of the viruses that was not dependent on their pathophysiological effects in the hosts but rather on the morphologies of the virus particles themselves.60 The development of ever more sophisticated methods for preparing and observing specimens also meant that researchers could think about investigating the nature of the interactions between viruses and their hosts. As Ladislaus Marton, very cleverly adapting a poem from Lewis Carroll’s Through the Looking-­Glass, aptly summarized, in rhyme, no less: “The time has come,” the Walrus said, “to talk of applications, “The uses of the microscope in our investigations. “To take a peek at things that were our former speculations.”61

An example of a speculation mentioned by Marton included the way in which viruses attached themselves to the surface of their host cells, and the stages of formation of new virus particles while within the cell.62 Anderson’s graphic description of the phage particle as a syringe quoted earlier gives not only details about the structure of the components but also the way in which the bacteriophage uses its tail to interact with the surface of its host bacterium before “injecting” its nucleic acids into the cell. Such precise detail—­first about the bacteriophages and later about animal viruses as well—­also demonstrated how viruses and bacteria were fundamentally different creatures that differed not only in their size, composition, and structure but also in more basic functions such as replication and multiplication. The mechanisms of bacterial multiplication by binary fission had been elucidated in the late nineteenth century, but save for Beijerinck’s conjectures about the obligate parasitism of the tobacco mosaic agent, virtually nothing was certain about the way in which they replicated their components or produced new virus particles. The electron microscope not only confirmed that bacteriophage particles did not—­could not—­undergo binary fission but also provided scientists with visual confirmation of what had earlier been indicated through microbiological and biochemical means, and thus helped reveal the nature of the replication cycle.63 Meanwhile, Albert Claude, who had put the ultracentrifuge to such good use in separating various cellular components in search of the chicken sarcoma virus, would, along with his colleagues Keith Porter, What Viruses Became

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Christian de Duve, and George Palade, go on to virtually invent the discipline of cell biology, springboarding from his electron microscopic investigations of the sarcoma agent. While at it he also supplied some structural details about the sarcoma virus itself.64 In light of these and many other achievements, one cannot help but feel that the inventors and users of the first electron microscope were more than vindicated in their 1939 claim “der Begriff des ultravisiblen Virus nicht mehr länger berechtigt ist” (The concept of the ultra-­visible is no longer justified).65

Advances in Virus Cultivation By rendering the viruses visible, the electron microscope effectively transformed two of the “negative” defining characteristics decried by Rivers into positive, describable attributes of morphology and structure: their invisibility under ordinary microscopes and inability to be retained by filters. But the third negative—­t he inability of viruses to be propagated in artificial media—­remained beyond the reach of the electron microscope. And surmounting that barrier was vital both for understanding the fundamental difference between viruses and other infectious entities, and even more, for progress to be made in working with them in the laboratory. This problem was recognized by workers in the field; in a 1931 assessment on the status of research on “filterable viruses,” Earl B. McKinley, for instance, noted that just as “the cultivation of the first bacterium on artificial medium gave to the science of bacteriology a fundamental working tool,” the cultivation of viruses was necessary for virology to similarly advance.66 And Sven Gard, a Swedish biologist and member of the Royal Karolinska Institute in Stockholm, acknowledged when awarding the 1954 Nobel Prize in Medicine to a trio of virologists—­John Enders, Thomas Weller, and Frederick Robbins—­for their successful cultivation of the polioviruses: “It is not difficult to find the reason why the virologists have failed where the bacteriologists were so successful. They have been severely handicapped by the difficulties connected with the cultivation of viruses.”67 At the root of the difficulties that viruses posed for cultivation in the laboratory lay the property that fundamentally sets them apart from all other living creatures—­t heir obligate parasitism in living hosts. But as detailed in chapter 3, there was very little consensus on the nature of viruses. Although Beijerinck had already identified obligate parasitism as a distinguishing characteristic of the tobacco mosaic agent in

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his very first report on the disease, he had been unable to expand on the notion at the time. Aside from asserting his view that “propagation results only when the virus is connected with the living and growing protoplasm of the host plant,” he had not been in a position to offer meaningful insights about the possible mechanisms of interaction between the virus and its host. “The question is naturally of special importance [and] new experiments in this direction are to be expected,” he wrote, but he himself did not pursue the matter any further because shortly thereafter, he ceased active laboratory work on tobacco mosaic virus altogether.68 In the absence of an active champion as well as the conceptual and experimental means to test them, Beijerinck’s ideas about the special nature of virus multiplication faded into obscurity for many years. His interpretation of data on viral multiplication was not widely accepted, nor were its implications appreciated by most bacteriologists. More scientists found it easier to believe Ivanovsky’s assertion that the “contagium of the mosaic disease is able to multiply in the artificial media.”69 Consequently, as Rivers recalled late in his lifetime, the notion that “viruses were small bacteria that were merely a bit difficult to grow on regular media” persisted for a very long time.70 When, in 1927, he wrote his comprehensive review of the state of knowledge on viruses, Rivers observed that, despite numerous reports of successful in vitro cultivation of viruses, there was in fact no persuasive evidence or independent verification for such claims. Rather, he stressed that “no worker has proved that any of the etiological agents of [viral] diseases is susceptible to cultivation in the absence of living cells. . . . Viruses appear to be obligate parasites in the sense that their reproduction is dependent upon living cells.”71 The one concession he allowed was that the location of the viral reproduction—­t hat is, whether it was intra-­ or extracellular—­was open to question. Although there was an increasing amount of evidence in support of the absolute requirement of living cells for the propagation of viruses, Rivers would recall later that he needed to defend the idea of their nature as obligate parasites “pretty vigorously” for many years: “For the life of me, I don’t see why it was so hard for me to convince people. The only reasonable explanation I can find at this late date is that sometimes science is held up because people of great renown hold on to their ideas and refuse to give them up.”72 Although Rivers did not specify the particular “people” who were difficult to convince, some of his comments earlier in the same interview referred to various bacterioloWhat Viruses Became

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gists whose ideas he disagreed with. This list included Arthur Kendall, George Eagles, John Ledingham, and most of all, his senior colleague the bacteriologist Hideyo Noguchi: “Noguchi had a tremendous influence on scientists throughout the world, and at one time most of them believed that you could cultivate just about anything in the Noguchi medium. All you needed was his long narrow test tube, a bit of rabbit testicle or kidney, a deep layer of broth and the virus or bacteria you were working with and you were in business.”73 Whereas the realization of the obligate parasitic nature of viruses was a largely conceptual barrier, in purely pragmatic terms—­regardless of whether they acknowledged or even recognized it—­researchers needed to contend with the property in order to investigate viral diseases properly. Nearly a decade after Rivers published his seminal review, for instance, Burnet, who was by then studying infectious viral diseases, would note that the ability to investigate viruses in the laboratory “depended entirely on their maintenance by constantly repeated passage from animal to animal.”74 The need for animals to grow viruses presented several drawbacks to the advance of animal virology, not the least among which was economic, as animal facilities were expensive to maintain. The host specificity of many viruses—­for many years the only way to propagate poliomyelitis viruses was in monkey hosts—­ meant that a facility needed to keep different pools of animals for different studies, which increased costs still further.75 Unsurprisingly perhaps, given that cultivation methods for bacteria has already been standardized in the late nineteenth century, the first viruses to be successfully and routinely cultured in the laboratory were the bacteriophages; indeed, their cultivation and discovery went hand in hand. By some strange twist of fate, it had been in the context of attempts to cultivate “ultramicroscopic viruses,” specifically the vaccinia viruses, that Frederick Twort stumbled upon the phenomenon of bacteriophagy—­or as he called it, glassy transformation—­in the first place.76 But although he made some suggestive remarks likening the transmissible material responsible for bacterial lysis to “an acute infectious disease” of bacteria, he did not pursue the investigation any further. When Felix d’Herelle encountered the same phenomenon in dysentery bacilli two years later, however, he was more explicit. Drawing clear analogies between the ability of the bacteriophages to form plaques on lawns of bacterial culture and the formation of bacterial colonies on artificial media, he designed a protocol to observe the growth of bacteriophages.77 Consequently, although the technique

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would be refined, first by Burnet in 1929 and later by Emory Ellis and Max Delbrück in 1939, the basic tools for growing bacteriophages were already in hand by the 1920s.78 The development of these techniques played an important role in advancing bacteriophage research at a much faster pace than research on other, especially animal, viruses. The first successful attempts to grow viruses of plant and animal hosts—­namely, multicellular eukaryotic organisms—­in an assayable manner emerged in the late 1920s and early 1930s. Living up to the label of “almost always the first” that Fraenkel-­Conrat would later bestow upon it, TMV was a frontrunner in these efforts.79 The plant pathologist Francis O. Holmes, working at the Boyce Thompson Institute for Plant Research in Yonkers, New York, had noted that upon injection into the leaves of susceptible tobacco plants, the infectious material produced small necrotic lesions that were proportional to the concentration of virus. Based on this finding he developed a technique for growing and quantifying TMV, which he claimed “gives as rapid and as accurate results as the determination of bacterial numbers by plating methods.”80 It is a testimony to the power of simplicity and convenience in scientific research that Holmes’s “local lesion assay,” like the plaque-­counting techniques for assaying bacteriophages, is still among the widely used tools in basic virus research.81 One of the earliest reports of a success in studying animal viruses removed from their known hosts came from the laboratory of none other than Peyton Rous at the Rockefeller. Very soon after Murphy had joined him, the two researchers had produced tumor-­like growths, or nodules, in chicken embryos by injecting cell-­free filtrates of tumor extracts, which could transmit sarcomas to unaffected hens into the chorioallantoic membrane of fertilized eggs. Their main aim in transplanting the tumors to the chicken embryos had been to compare the growth of neoplastic and embryonic cells in connection with some theories of tumor origins.82 The experiments with cell-­free filtrates were incidental, born of Rous’s discovery of the filterable transmissible agent earlier that year. But since only Murphy pursued chicken tumor research after 1915, and he did not believe that the sarcoma agent was a virus, any notions of developing virus assays were nipped in the bud. Some twenty years later, Rous and Murphy’s experiments resurfaced in the scientific literature pertaining to the problem of virus cultivation or propagation. At that time, finding various experiments to culture animal cells “inconclusive,” and the animal hosts themselves expensive and time-­consuming, Ernest Goodpasture, an infectious disWhat Viruses Became

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ease pathologist at Vanderbilt University, who was studying various viral diseases with the aim of developing vaccines against these diseases had begun to explore alternate means of growing and maintaining viruses. Looking for clues in past work he found that Rous and Murphy’s experiments had indicated that fertile eggs—­a source of live cells that were cheaper and more readily and consistently available in far larger quantities than experimental animals—­might serve as a breeding ground for animal viruses. On further exploration, Goodpasture and his colleague Alice Woodruff found that the “egg-­membrane was a very favourable site for the study of virus infections, and that a number of viruses—­fowl-­pox, vaccinia and herpes febrilis could be propagated thereon in indefinite series.”83 The egg membranes offered one further advantage over live animal hosts as a growth medium for the viruses; rather than merely increasing the quantity of viruses, they also developed visible and traceable signs of infection in the form of “pocks” of spots of damaged and dead cells on the membrane. Thus, not only were these lesions distinctive enough for a visual identification of different types of viruses but they also opened the doors to methods for assaying the viruses. Like bacterial colonies, bacteriophage plaques, and the local necrotic lesions on tobacco leaves, pock formation on the chorioallantoic membranes varied with the concentration of virus. Applying the lessons learned from extending d’Herelle’s plaque counting method, Burnet further developed Goodpasture’s basic approach into a broadly applicable experimental technique for growing a variety of viruses. Of particular significance was his success in developing the technique to cultivate and assay the influenza viruses, which he observed made the virus “almost as easy to handle in the laboratory as a bacterial virus.”84 That the significance of cultivation was not lost on contemporary virus researchers is indicated by the fact that the nomination archives of the Nobel Prizes show a steady stream of nominations for Goodpasture between 1937 and 1953, most of which specifically cite him for his work on virus cultivation in eggs. He shared two of these nominations with Burnet, who was in his turn nominated for the assay methods that he developed with the influenza virus.85 In a tribute to Goodpasture after his death, Burnet gave considerable time and attention to the topic of growing viruses in chick embryos. At a personal level, he noted, his own work on influenza virus stemmed directly from Goodpasture’s experiments and more broadly, he claimed that the latter’s experiments had opened “a new approach to virology [that] for another 10 to 12

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years was to represent the mainstream of virus research.” But then, he mourned, “but for a few specialized applications it rapidly disappeared. The chick embryo was displaced probably forever by the tissue culture techniques.”86 Burnet’s observation about the dominance of tissue culture over other methods for virus cultivation remains as true today as it did at the time of his lecture. In fact, growing viruses is just one of many applications of tissue—­or rather cell—­culture, the origins of which trace back to problems that were quite removed from virology; if anything, as scientists would learn, contamination with viruses would pose problems for growing and maintaining cells in vitro.87 The following summary by the British public health virologist Philip Mortimer, in an appreciation of the classic work of Enders, Weller, and Robbins, gives some idea of the different branches of biology and medicine that needed to come together for the successful cultivation of viruses: “Like many really useful medical techniques in vitro virus propagation is a hybrid derived from various strands. . . . It evolved out of practical advances in cell biology such as the use of trypsin to disperse cells from solid tissue and the creation of continuous and semi-­continuous cell lines; also out of the availability of antibiotics to maintain culture sterility. Virus isolation technique was then refined by the development of methods for recognising occult viral replication on monolayers, aided by skills acquired in passaging and maintaining the cultures long term.”88 The origins of techniques of growing cells in vitro; that is to say in the laboratory outside the context of their natural organismal environments, have a longer history quite independent of their use for growing viruses.89 As early as 1907, Ross Harrison, an anatomist at Yale, presented a short report about the growth of the tips of nerve fibers by isolating pieces of embryonic nerve cells of frogs and fixing them on glass slides in drops of lymph from the same animal.90 His main purpose in devising this method was to be able to observe the growing nerve while it was still alive, and he was able to keep the tissues alive for up to a week and in some cases for up to four weeks. His report stimulated such interest and research activity that by 1911 the American Association of Anatomists had devoted an entire symposium to the topic of tissue culture, proceedings of which were published the following year. Although none of the work presented at this symposium dealt with the applications of the technique toward the cultivation of viruses, the implications for the same were evidently realized by interested parties, because the earliest reports of attempts to grow viruses in such What Viruses Became

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tissue cultures began to appear soon thereafter, in 1913.91 But as evidenced by the remarks at the 1954 Nobel ceremonies, it would take a few decades for the routinization of tissue culture for growing viruses. One figure whose involvement in the early history of tissue culture who bears at least a passing mention here—­in part, to highlight the complex and often paradoxical nature of scientific progress and also, because of his work’s intersections with key players in the main narrative—­is Alexis Carrel, whose ideas about the possible similarities in the mechanisms of bacteriophagy and sarcoma causation I invoked at the beginning of this book. Probably best known for his pioneering work on vascular surgery and transplantation, for which he received the Nobel Prize in 1912, Carrel was also a pioneer of tissue culture, the person who gave the technique this label (which others have deemed a misnomer), and “its chief publicist.” But his role in the history of technique is complicated. The molecular biologist Jan Witkowski has made the case that although Carrel made a number of contributions to developing the technique and established it as a method of wide applicability, his writings about the difficulties of tissue culture deterred so many researchers that he may have had “an adverse effect” on its development.92 The cancer researcher Jacob Furth, who spent 1926–1928 training at the Rockefeller, credits Carrel with arousing a lifelong engagement with tissue culture, but also recalls that by then, Carrel was “isolated and unpopular.”93 This impression is also borne out in Thomas Rivers’s remarks regarding the surgeon’s contribution—­on the subject of the application of tissue cultures to virus research—­to his own magisterial edited volume Filterable Viruses.94 As he would confess to the historian Saul Benison, he chose Carrel as a contributor “for his name value and not because he had anything in particular to contribute.”95 Rivers’s remarks about Carrel may have been a trifle unjust to his former and by then long-­deceased colleague, and give credence to Witkowski’s observation that the justified criticisms against Carrel’s approach to tissue culture had made his legitimate contributions altogether too easy to overlook.96 Carrel’s brief chapter in Rivers’s edited volume provides a perfectly adequate—­if nonrevolutionary and somewhat generic—­picture of the status quo on the application of tissue culture techniques to virology, complete with a fairly representative bibliography of work done in different laboratories at the time. He rightly pointed out the usefulness of the technique, regardless of one’s thinking about the nature of the viruses themselves:

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A virus may be a very minute organism, or it may be a chemical substance manufactured by cells themselves. In either case, its multiplication depends upon the activity of a living tissue. Since [in tissue culture] pure strains of cells are caused to live in vitro under such simple conditions that the effects of viruses on cell morphology [and] the relation of the nature and metabolic condition of the cells to the viruses may be analyzed, new discoveries will certainly be made. It is obvious that the cultivation of tissues . . . reduces to very simple terms the problem of the relations of virus and cells.97

Carrel himself was especially interested in applying tissue culture toward the study of mechanisms of the genesis of cancer. Barely had Rous received the sarcomatous hens from the farmer and begun his own investigations into their etiology when Carrel published a report on the in vitro cultivation of sarcoma tissues.98 Later, soon after Gye had reawakened interest in the viral etiology in 1925, he also published a paper describing the conditions necessary for propagating the sarcoma agent in vitro, “Some Conditions of the Reproduction in Vitro of the Rous Virus.” Interestingly, although he used the term virus in the title, he was more circumspect within the paper and opened with the caveat: “Although the Rous virus, so called, of chicken tumor multiplies readily in vitro, we cannot consider that it has been positively shown to be an ultramicroscopic organism.”99 With Rous out of the picture from active tumor virus research and Murphy a firm disbeliever, however, Carrel’s ideas about tumorigenesis do not seem to have gained much traction either within the Rockefeller or in wider circles. The jump from Carrel to Enders might seem rather abrupt and indeed there does not appear to be any direct connections between their work. Serendipity and coincidence, rather than any concerted or programmatic endeavor, appear to have played a significant role in directing the three 1954 Nobel laureates toward their great success in cultivating the poliomyelitis virus in tissue culture. Indeed, they said as much in their Nobel lecture, recounting that in the course of working with culturing other viruses—­mumps and varicella—­and having a ready supply of a strain of poliomyelitis virus in storage, “it suddenly occurred to us that everything had been prepared almost without conscious effort on our part for a new attempt to cultivate the agent.”100 Of the three men, Enders, who was considerably senior to the other two, probably had the most meandering pathway to this achievement. He began his academic career with degrees in English and only switched to microbiWhat Viruses Became

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ology as a graduate student at Harvard. After receiving his doctorate in 1930, he remained at Harvard, where his first major contributions to research were in the area of bacteriology. As his fellow laureates recounted, it was only in 1937, when there was an outbreak of a virus disease in a stock of kittens in the experimental animal facility, that Enders was initiated into virology. From initial investigations into the growth of the herpes simplex virus, he developed “an enduring preoccupation with pathogens of this class and was followed by attempts to isolate the agent of measles. In these experiments the tissue culture method was employed with uncertain results. But the conviction was gained that it represented a basic tool for the study of viruses of which the possible applications were almost unlimited.”101 Weller and Robbins had a more direct route to virology than Enders, in that they both trained in medicine, and while in medical college at Harvard even shared living quarters for a time. Sometime in 1939 or 1940 Weller, then a fourth-­year medical student who had become interested in infectious diseases, applied for and was accepted to work on a project in Enders’s laboratory, where he was introduced to viruses and problems of growing them in tissue culture.102 Although this work was interrupted temporarily in 1941 due to the obligations of serving in the war, it was resumed in 1947 when Enders was asked to establish a virus research laboratory at the children’s hospital in Boston. Weller was among those involved in setting up this laboratory, and soon thereafter they were also joined by his old roommate Robbins, freshly returned from war duty in Italy, where he had gained recognition for his investigations into rickettsial diseases. Given Enders’s belief in the potential of tissue culture for virus research, they pursued this line of investigation, building on the promising approach devised by Johns Hopkins researcher George Gey for culturing cells in a large scale.103 Their first successes were obtained with the mumps virus, chosen because it had been shown to be easy to handle in the laboratory; there were, they later admitted, no particular plans to focus on the poliomyelitis virus, and it was just one of many: “In rapid succession mumps, the poliomyelitis viruses, Coxsackie viruses, and Echo viruses were grown in vitro.”104 Poliomyelitis became the focus of the Enders laboratory after 1948 and garnered these men their Nobel Prize, but the technique they had helped hone had a much broader reach, becoming integral to the routine study of viruses—­including the tumor viruses.

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Molecularizing Visions of Viruses The historian and philosopher Hans-­Jörg Rheinberger has suggested that it was the “conjuncture” of electron microscopy and cell culture that galvanized the “comeback of the tumor agent as a virus.”105 And while both these techniques played fundamental roles, I would add a third component to this conjuncture: the blossoming of molecular biology. It is virtually impossible to think about progress in any field of biology from the mid-­twentieth century onward without a consideration of this discipline, and virology is no exception. Indeed, I would add that virology has something of a special relationship with molecular biology, because more than any other discipline, it fed, as much as fed off, the newly burgeoning discipline. Today molecular biology is defined as the branch of biology that deals with the structure and function of molecules essential for life, but in fact, much like virus, the phrase molecular biology has its own history of variance, albeit with a more definite starting point. In 1938, Warren Weaver, then director of the Division of Natural Sciences at the Rockefeller Foundation, introduced the phrase molecular biology in his annual report to the board of trustees as “a relatively new field . . . in which delicate modern techniques are being used to investigate ever more minute details of certain life processes.” He went on to describe diverse projects that fell within the province of this new discipline, to which the foundation had awarded grants, among them investigations into nerve and muscle action, carbohydrate metabolism, X-­ray analysis of biological tissue, and cellular physiology.106 By 1950 the British X-­ray crystallographer William Astbury, in a Harvey lecture titled “Adventures in Molecular Biology,” expressed his pleasure at the fact that the term was in fairly common use. To him it was “an approach” to studying biology, which was concerned with “the forms of biological molecules, and with the evolution, exploitation, and ramification of those forms in the ascent to higher and higher levels of organization. Molecular biology is predominantly three-­dimensional and structural [but] must of necessity enquire at the same time into genesis and function.”107 Considering that viruses consist of little other than the basic molecules of life—­proteins and nucleic acids—­t hat are the focus of molecular biology, it not difficult to see why the histories of the two disciplines are so intricately intertwined. One major factor in solidifying the growing status of virus research

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as an independent discipline in the 1940s and 1950s was the fact that “many biologists viewed viruses as good experimental tools for investigating the physical nature of heredity.”108 It was precisely with this objective in mind, for instance, that Max Delbrück had worked with Emory Ellis to develop the single-­burst assay for the bacteriophages, which were consequently given pride of place in the title of the book often touted as having “effectively launched the history of molecular biology.”109 Prepared as a festschrift to honor Delbrück on his sixtieth birthday, Phage and the Origins of Molecular Biology is a compilation of essays by his former colleagues and students—­t he American Phage Group—­ who had been asked by the editors to “describe some significant contribution that they had made to molecular biology.”110 The resulting collection of some thirty essays was criticized by one of its first reviewers, the British crystallographer John Kendrew, for presenting the molecular biology as if the discipline were exclusively about “biological information.” Kendrew himself was an exponent of what he called the “British school of molecular biology,” which privileged structure over information, focusing on techniques such as crystallography to attempt to understanding the workings of different molecules through their three-­dimensional structures.111 For all his criticism of Phage and Origins and its view of molecular biology, he did not, however, not take issue with the prominence given to the bacteriophages in the title. Bacteriophages and for that matter other viruses showed no special propensity for either side of the divide, and featured prominently in both the informational and the structural camps of molecular biology. On the structural side, viruses had been involved in the “symbolic beginning” of the discipline, marked by the crystallization of the tobacco mosaic virus—­over that of several enzymes that had already been crystallized—­ in 1935, long before the festschrift was published.112 Where viruses provided the experimental material and tools to shape and advance molecular biology, molecular biology, reciprocally, provided both technical and conceptual tools for furthering the understanding of what viruses were made of and how they functioned. As elaborated in the next chapter, the revelations from molecular biology—­ especially, though by no means exclusively, about the functions and structure of DNA—­would enable virologists to achieve a “genetic visualization” of both the bacteriophage lysogeny and tumor causation by viruses.113 Armed with the knowledge that the genes of an organism were housed in its DNA, they could conceive of the integration of the viral DNA with that of the host cells.

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The modern concept of viruses is often presented as the culmination of research in molecular biology—­in its fullest sense—­t hat made it possible to better understand the structure and functioning, and in particular, the replication of viruses.114 The most significant breakthrough in this regard was doubtless the resolution of the phenomenon of lysogeny about which Burnet and Eugène and Élisabeth Wollman had shared their insights in the 1920s and 1930s. Soon thereafter, as discussed in chapter 4, the topic had entered a “long sleep” in the wake of the Wollmans’ tragic deportation and execution as well as Burnet’s shift to other topics. It was the blossoming science of molecular biology that served as the morning call to reawaken lysogeny, which then proved to be the linchpin that would bring the study of diverse viruses under one common disciplinary umbrella.

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7KNITTING DONE

LYSOGENY AS LINCHPIN

Lysogeny, once a concept out of favor, is the basis of our understanding of relations between cell and virus. —André Lwoff, 1966 The virus becomes equivalent to a cellular gene controlling cell morphology. —Howard Temin, 1960

Of the many twists and paradoxes that have peppered this tale of two viruses, there is perhaps none bigger than the role played by lysogeny. Considering that it was discovered by one of Felix d’Herelle’s earliest and most implacable opponents, Jules Bordet, and wielded for many years as the most formidable argument against d’Herelle’s claims about the viral nature of bacteriophages, it is extraordinarily ironic that it later became the linchpin that “led to the definition of . . . the very concept of virus,” and also enabled an understanding of cell-­virus interactions, according to the scientist most directly instrumental in effecting these changes in vision.1 The resolution of the problem of lysogeny brought together many ideas and virus-­related phenomena earlier considered as unrelated or even, on occasion, contradictory to one another. Specifically in the parallel histories of bacteriophages and tumor viruses, it proved to be the focusing lens that brought about the convergence of the thus far largely separate trajectories of research on the bacteriophages and cancer viruses. Before lysogeny could play this pivotal role, however, it needed to be reawakened from the “long sleep” that it had gone into in the 1930s

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following the work of such scientists as Eugène and Élisabeth Wollman and Frank Macfarlane Burnet. Their hypotheses—­for example, that a “virus” somehow became a “gene” (Burnet) or that bacteriophages alternated between an infectious and noninfectious or lysogenic phase (Wollmans)—­were simply not testable given the state of scientific knowledge about the nature and makeup of viruses or genes at the time.2 As the French microbiologist André Lwoff, whose “Prince Charming kiss” awoke lysogeny from its big sleep (to paraphrase the analogy used by molecular biologist Melvin Cohn), later observed, these theories had been advanced in an era “when genetic material had not been identified, [and] a few bacteriologists had understood the strangeness of lysogenic bacteria, but knowledge concerning viruses and their reproduction was too cloudy.”3 The discovery that nucleic acids were the key substances that carried and transmitted genetic information was thus an important stepping-­stone to resolving the problem of lysogeny. But it was not until 1944 that Oswald Avery and his group at the Rockefeller demonstrated that the bacterial “transforming principle” was composed of DNA.4 Even this discovery was not in and of itself enough, for due to a whole slew of reasons discussed in depth by other historians, the findings of Avery’s group were not immediately well received by the scientific community at large. But by the late 1940s, when Lwoff began his bacteriophage work in earnest, the idea had gained considerable traction. But there was also another reason that Lwoff, in particular, identified for the long lapse between the discovery of lysogeny and its definition, one that goes back to Bordet’s ideas about the phenomenon and its implications, specifically his theory that lysogenic bacteria “secreted” bacteriophages, which in his estimation were emphatically not viruses. According to Lwoff, the secretion theory itself was “probably unconsciously reached by the following reasoning: lysogenic bacteria live and multiply, lysogeny is not lethal; lysogeny being phage production, phage production is not lethal . . . lysogenic bacteria adsorb homologous phage without being killed; phage is not lethal. Thus, in lysogenic bacteria, prophage is not lethal, perpetuation of prophage is not lethal, phage is not lethal. How should a normally innocuous particle be lethal only when producing another similar innocuous particle? No example of this type of phenomenon was known. Phage must be secreted. Phage is secreted. Phage was secreted.” When, he went on to argue, “a scientifically minded bacteriophagist” was unable to verify the secretion theory experimentally, the conclusion was that phage-­ Knitting Done

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secreting bacteria and consequently, lysogeny itself, did not exist: “A wrong definition of lysogeny had led to the condemnation, not of the definition, but of lysogeny itself.”5 Although Lwoff did not explicitly say so in this instance, his line of reasoning would account for why members of the American Phage Group—­who played the most prominent role in shepherding bacteriophage research during the years lysogeny lay fallow—­could ignore the existence of the phenomenon for so many years.6 But, adding still another layer of irony to this unfolding history, the same group’s contributions to knowledge about bacteriophages and thus molecular biology would go a long way toward ultimately facilitating the resolution of lysogeny. Lwoff pointedly drew attention to this paradox in the short section that he allocated to lysogeny in a reprise of his research life: “Max Delbrück had paradoxically played a role in the development of lysogeny. I say paradoxically because the founder of the ‘phage church’ did not believe in the existence of lysogeny. Falling from the lips of Max Delbrück, the death sentence, ‘I do not believe’ had been often heard by many of us. It was an excellent catalyst.”7 Lwoff himself played a role in virtually all facets of the resolution of lysogeny; directly in some instances and indirectly in others. He not only provided an explanation of the phenomenon in molecular terms but also applied the lessons learned toward providing what is arguably the best general definition of viruses to this present day—­in a nutshell, as obligate intracellular parasites comprising a protein coat and a single type of nucleic acid only.8 Later it was the mechanism that he described for bacteriophage lysogeny, that became—­first by analogy and later with experimental evidence—­t he basis for understanding how viruses could induce tumors in their hosts. Therefore it is with him that I begin this final chapter, before going on to knit the various narrative threads left hanging earlier into a coherent conclusion.

Lysogeny and Dr. Lwoff Highlighting the aforementioned irony of the role of lysogeny as linchpin in this book to an even greater degree is an earlier essay by Lwoff, his contribution to Delbrück’s festschrift, Phage and the Origins of Molecular Biology. The essay was entirely about lysogeny, the very topic that Delbrück and the rest of the Phage Group (with the exception of Elie Wollman, the son of Eugène and Élisabeth Wollman) ignored. But Lwoff nevertheless paid the group what he considered due tribute: “In reality, lysogeny is one of the manifold aspects of the biology of bacte-

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riophage and [it is the latter that] paved the way for the development of virology and played such an important role in the blooming of molecular biology. That is why I, like so many others, feel indebted to Max Delbrück.”9 Justified though this feeling of gratitude undoubtedly was, there are at least two other people to whom Lwoff owed prior, and arguably larger, intellectual debts for his success in resolving lysogeny: first, the protozoologist Édouard Chatton, under whose guidance he, as a medical student, conducted his earliest research work, and second, Eugène Wollman, who introduced him to the phenomena of bacteriophagy and lysogeny. As Chatton’s assistant Lwoff studied particles called kinetosomes, visible organelles in protozoan cells, which are responsible for the formation of cilia, the hairlike extensions of the cell that endow these organisms with their powers of locomotion. Tracking the behavior of the kinetosomes over multiple generations of different species, Chatton and Lwoff observed that the patterns in which they were organized inside the cell changed over the course of the life cycle of the organism, but were constant for any given stage of the life cycle. The fact that these changes were not random was evident from the observation that for a given species, the kinetosomes occurred in the same numbers and organizational patterns at each particular step of the cell life cycle generation after generation. This seemingly hereditary pattern led them to the rather controversial claim that kinetosomes derived from other kinetosomes—­just as cells themselves derived from other cells—­rather than being synthesized de novo at the time of cell division like other cytoplasmic contents.10 In other words, Chatton and Lwoff saw the kinetosomes as particles endowed with an autonomous “genetic continuity” within the protozoan cells, in whose complexity they nevertheless remained embedded.11 It was while he was deeply immersed in this work that Lwoff first met the Wollmans. “In 1921 I entered in the Pasteur Institute. I rapidly became acquainted with Eugène Wollman, [who] was working with bacteriophage and lysogeny, [and who] liked to show his experiments and was eager to discuss his ideas. Thus, especially between 1930–1939, I was introduced to Bacillus megatherium.  .  .  . I should have been impressed by the bacteriophage. Shall I confess that I was not? At least not deeply enough.”12 This description by Lwoff, written much later in his life, should not be taken at face value but with a recognition of the irony in his self-­deprecation. For although it is true that he did not work on bacteriophages until nearly three decades earlier, it is evident from Knitting Done

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both his work and his writings that the concurrence of his research on the ciliates and conversations with Wollman had a profound influence. In his own later assessment, “the kinetosomes, their movements, the factors that govern their specific activity, served me as a model and a guide for attacking the problem of lysogenic bacteria.”13 Specifically, his findings about the genetic continuity of the kinetosomes predisposed him toward accepting Wollman’s idea—­still in development during these years—­t hat the bacteriophages had a biphasic life cycle alternating between its infectious (mature) and noninfectious (latent or lysogenic) forms. Where the direct production of cilia offered Lwoff a model for understanding the autonomy of the free bacteriophage particle, the reproduction of kinetosomes as well as their patterns of organization through multiple generations served as a guide to explain the latent—­genetic continuity-­providing—­bacteriophage stage.14 But these ideas would not flower fully in Lwoff’s brain for decades to come. In the meantime, he, still “in love with other creatures”—­not just the ciliates but also marine creatures called copepods and a bacterial species named Moraxella—­produced a formidable body of work on such diverse research topics as nutrition, growth factors, morphology, morphogenesis, and microbial evolution through loss of function, and established a solid reputation.15 It was not until 1949, following discussions about adaptive enzymes, bacteria, and bacteriophages with his colleague (and later corecipient of the 1965 Nobel Prize) Jacques Monod, that he finally turned his attention to the bacteriophages and problem of lysogeny. Well acquainted by then with Delbrück and the American Phage Group, and their skepticism toward lysogeny, Lwoff “took it as a personal challenge to demonstrate the importance of observations made by the Wollmans.”16 In his own words: “because of Eugène Wollman,” he took up the problem of investigating the lysogeny phenomenon and following his example, used B. megatherium as his model host organism.17 Lwoff’s model system proved to be a lucky choice, because the relatively large size of B. megatherium enabled him to work with single organisms at a time, which he claimed was not only enough but necessary in order to “know what was really happening, to know how the lysogenic bacteria released the bacteriophages they produced.”18 As he explained later, experimental results—­for example, bacteriophage-­to-­ bacteria ratios—­working with a population of lysogenic bacteria, would have been identical, regardless of whether the phages were produced one per bacterial cell or many by a single organism. 19 The power to

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Fig. 7.1 André Lwoff working with a micromanipulator, circa 1954. Photograph courtesy of the Caltech Archives, California Institute of Technology.

observe the phenomenon at the level of single bacteria obviated indirect means to getting at the truth. Using specialized equipment—­a micro-­manipulator with the help of which one could work with extremely small volumes (drops) of medium—­fitted with a microscope for observation, Lwoff was able to “play with individual bacteria,” and begin to learn something about the events unfolding within the bacterial cells.20 By inoculating individual lysogens into individual micro-­drops of medium, and observing their growth, division, and behavior in the presence of bacterial strains sensitive to the bacteriophages produced by the lysogenic strain of B. megatherium, for instance, he confirmed that these bacteria grew, divided, and multiplied normally without releasing bacteriophages. The occasional and sudden lysis of a small percentage of these bacteria followed by the release of several bacteriophages—­he calculated an average of seventy-­two phage particles per bacterial cell—­indicated that the ability to produce bacteriophages was retained over successive generations of these bacteria.21 It is worth mentioning that these earliest experiments by Lwoff and his collaborators did not contribute anything radically new or difKnitting Done

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ferent; rather, they seem to reaffirm the decades-­old findings and claims of the Wollmans and to some extent Burnet, about the phenomenon of lysogeny. Perhaps the biggest advantage that these new experiments offered, however, was that the nature of the experimental system that Lwoff used in producing the results made those same claims about the reality of the phenomenon and the production of bacteriophages much more difficult to argue with or refute. Furthermore, they began to narrow down the plausibility of various theories—­eliminating, for instance, the secretion theory as indicated earlier. Even so, conflicting ideas and explanations about the nature of lysogeny continued to coexist. For example, as late as 1948, bacteriophage researchers Alfred Hershey and Jacques Bronfenbrenner would claim that “lysogenesis, frequently cited as evidence for the spontaneous intracellular origin of viruses, can best be explained as one type or another of association between exogenous virus and incompletely susceptible bacteria.”22 According to the molecular virologist Wolfgang Joklik, it was through a set of experiments using ultraviolet (UV) irradiation of lysogenic bacteria, conducted in collaboration with Lou Siminovitch and Niels Kjeldgaard, that Lwoff most dramatically revealed “the true nature of lysogeny.”23 Subjecting lysogenic bacteria to UV radiation—­by then known to be mutagenic24—­t hey found that whereas only a small percentage of such bacteria growing normally in a complex medium would produce bacteriophages, the irradiation of such bacteria with ultraviolet light induced the termination of the lysogenic state and cause all the bacteria to replicate, be lysed, and release bacteriophages. The fact that nonlysogenic bacteria under identical conditions—­ namely, growing exponentially in complex media—­did not undergo lysis or release bacteriophages when treated with UV radiation led them to the conclusion that the lysis of the lysogenic strains was “not a direct effect of the UV irradiation.”25 Rather, the ultraviolet radiation seemed to act as a trigger to unlock or stimulate the lysogenic bacteria to start producing bacteriophages. In other words, these experiments suggested that the bacterial gene possessed a potential or capacity for producing bacteriophage, which was somehow either blocked or repressed in the normally growing lysogens and released when induced by ultraviolet radiation. In a followup paper published later that same year, Lwoff described this capacity as a “non-­virulent, latent ‘intracellular bacteriophage’ as it exists inside lysogenic bacteria,” and label it a “probacteriophage.”26 He also introduced the term prophage as a synonym for the probacterio-

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phage, which, although appearing just once in this paper, is the version of the label that stuck. In his landmark publication of lysogeny a few years later, it was probacteriophage that was relegated to the single mention, and by the time he wrote his essay for Delbrück festschrift, “The Prophage and I,” there was nary a mention of the longer label. Lwoff’s experiment would, at first glance, appear to have been corroborating the by then decades-­old idea that the lysogenic bacteria contained an anlage of the bacteriophage in their hereditary constitution. He would even later concede that Burnet and Margot McKie had rightly concluded that the lysogenic bacteria were perpetuating something specific from parent to progeny. That something, he elaborated, was a “specific noninfectious particle endowed with genetic continuity.”27 Worth stressing here is that the term genetic in this context was not used in the more common sense pertaining to science of heredity per se but rather “used in the original sense of the word, which refers to the ‘genesis’ or ‘development’ of something.”28 Thus, the continuity implied in Lwoff’s description reiterated his notion of lysogeny as the potential or capacity to produce bacteriophage rather than the physical bacteriophage itself. Whereas the earlier researchers had not offered any details about the physicochemical nature of this particle, Lwoff’s conclusions implicitly convey the sense that prophage was made of DNA; for example, in his description of lysogeny as a process in which “the genetic material of the bacteriophage is ‘reduced’ into prophage.”29 By this time it should be noted that although it was by no means considered a given, the identity of DNA as the material basis of heredity as suggested earlier by Avery was certainly one that scientists were aware of. Consequently it was possible to visualize the reduction of the genetic material of bacteriophage—­its DNA—­into a prophage inside a bacterial cell, which had not been the case with Burnet and the Wollmans’ theories. Is there any more substantial difference between Burnet’s anlage and Lwoff’s prophage other than the fact that, whereas the chemical basis for the former was completely unknown, the latter could have been DNA? Answers vary depending on context: both on who is asking and who is answering. Burnet certainly believed they were the same; in his memoirs, he would recollect getting “a twisted pleasure in watching Americans or Frenchmen discover in the forties what we had clearly described between 1927 and 1935.”30 Lwoff, on the other hand, was insistent that the anlage and prophage were distinctly different conceptions. Burnet’s anlage was “as an intracellular phage multiplying by bipartition,” he argued, whereas his own prophage was by definition, just Knitting Done

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Fig. 7.2 Bacteriophage as Madame Defarge, earmarking victims for execution by knitting itself into the hereditary constitution of the bacterial host. Cartoon by Sanja Saftic.

the “hereditary power to produce bacteriophage” and not itself an intracellular bacteriophage.31 Referring to his 1950 publication with Siminovitch and Kjeldgaard, Lwoff would emphasize the point that “lysogenic bacteria do not, cannot, perpetuate a latent or masked phage particle; the production of phage from prophage thus appears, not as an unmasking, but as a development.” Lwoff’s reference to masking recalls the phenomenon described in the 1930s by Richard Shope and Rous, whereby certain animal viruses, not directly detectable in their host cells by direct methods, could be detected by such indirect means as the presence of antibodies.32 Shope had, in fact, explicitly compared masking to bacteriophagy but as is evident from his explanation, considered their mechanisms to be different: “If in the case of the bacteriophage, the time gap between the disappearance of the initial virus and appearance of the mature virus in an infected cell were a matter of days instead of minutes, the phage during this period might possibly be thought of as ‘masked’ in the sense in which that term can be used in the animal virus field.”33 That lysogeny was not a masking of the sort described by Shope and Rous was true enough—­in fact, in his analogy Shope was speaking of lytic, not ly-

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sogenic, bacteriophages—­but Lwoff’s comparison of the anlage to the masked viruses seems misplaced as well as anachronistic. Save for the fact that both investigators conceded that the mechanisms for their proposals of virus (or bacteriophage) behavior were unknown, Burnet’s 1929 claim that the lysogenized bacteriophages had become part of the bacterial gene was not analogous to the 1937 descriptions of the masking of virus particles in the host cells. His interpretation of Burnet’s work notwithstanding, Lwoff’s insistence on the fundamental difference between the prophage and the bacteriophage was justified on the basis of the molecular knowledge at the time he came up with the model. Knit into the genome of its host, the prophage provided the host with the power or potential to produce all the necessary components for the making of the lethal bacteriophage that would lyse it. Like Dickens’s Madame Defarge, who attended every guillotine execution and knit the names of the victims into her list without killing them herself, the prophage earmarks its bacterial victims for lysis by knitting itself into their genomes. But it is not in itself a bacteriophage any more than any other gene is a gene product.

From Prophages to Viruses The impact of Lwoff’s prophage hypothesis was both immediate and profound. This effect was first and perhaps most compellingly manifested in the program of the Cold Spring Harbor symposium that same year. For the first time since its founding some twenty years earlier, the 1953 symposium was devoted entirely to the topic of virus research and is singled out in the archives both as heralding the “golden age” of the symposium series and for having taken on a “mythic quality” in the history of molecular biology. Underwritten by the National Foundation for Infantile Paralysis, the main purpose of the symposium that year, according to the organizer and director of Cold Spring Harbor Laboratory, Milislav Demerec, was “to bring together research workers in [the field] and to provide an opportunity for those specializing in bacterial, animal, and plant viruses to correlate their findings.”34 Although Lwoff was not a speaker at the meeting, his concept of the prophage—­appearing in its more generalized form of “provirus”—­was a strong, if somewhat mysterious, thematic presence. As Delbrück explained in his introductory remarks at the symposium: “In formulating the program, we start out with the doctrine of the trinity of virus: infective (or mature) virus, vegetative virus, and provirus.”35 The last, which he described as a “shadowy character” with whom everybody wished to Knitting Done

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get better acquainted, was the sole focus of the second day of the symposium. Barely four years after the publication of his review article, Lwoff was invited to deliver a lecture to the Society for General Microbiology. Titled “The Concept of Virus,” this lecture holds an indisputable and well-­deserved place in the history of biology as containing the first formulation of the “modern” definition of virus. But it is fairly clear from its content and tone that the lecture was a synthesis rather than a presentation of new data or ideas. Drawing on history—­of work on bacteriophages and experimental evidence from bacteriology (both his own and that of others)—­as well as philosophical and scientific discussions on the nature and origins of life, Lwoff detailed his “ambition to show that the word virus has a meaning, [and] to defend a paradoxical viewpoint, namely that viruses are viruses.”36 This statement, characterized by Lwoff as “prosy, coarse and vulgar,” which he reiterated at the conclusion of his lecture, is famous among virologists and historians of biology alike for its seemingly uninformative stance. But it is not, in fact, as opaque as it might seem at first. As suggested by the historian William Summers, the statement tacitly assumes that a virus is a “natural kind in the Aristotelian sense.”37 Such an assumption automatically sets it apart as a separate category from other natural objects, such as bacteria, along with which it was earlier classified. In fact, Lwoff directly brought up this point in his talk, pointing out that the definition of anything “must not only include some objects, but exclude other objects.” With regard to viruses, he specified, “We speak of viruses as different from bacteria, protozoa, fungi and algae. This implies the existence of a category of infectious agents, viruses, which are different from the other infectious agents.” One of the principal ways in which he stressed that these infectious agents were different was the physical nature of the entity that actually infected its host, what he called the “infectum.” Whereas in the cases of bacteria and protozoa, for instance, the infectum was the whole organism, “when dealing with the bacteriophage, the infectum is its genetic material.”38 Lest readers be thrown by his use of the word bacteriophage rather than virus more generally, it should be noted that Lwoff explicitly pointed out that he was using the bacteriophage, with which he worked with in the lab, “as a model of virus.” Already in his earlier paper on lysogeny, there are clear harbingers of the generalization from bacteriophages to viruses. For instance, one of his stated aims in the

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earlier article had been to direct attention “to possible analogies between the viral diseases of bacteria on the one hand, and of animals and plants on the other.” To this end, he posed the question of “whether viruses of plants, insects or animals may or not be perpetuated in the form of a provirus,” introducing the more expansive, hypothetical entity, which, as seen, was Delbrück’s choice of terminology at the Cold Spring Harbor symposium. Conceding that the question was far from resolved, Lwoff did, however, go on to enumerate several similarities between the bacteriophages and the different types of viruses, notably in their constitution; in their life cycles: there was no evidence of binary fission in either case; and the fact that many viruses, like lysogenic bacteriophages, lost their infectivity upon entering their host cells.39 By 1957 he had clearly integrated these ideas to provide the general and modern definition of the viruses, inclusive of bacteriophages, as: “strictly intracellular and potentially pathogenic entities with an infectious phase, and (1) possessing only one type of nucleic acid, (2) multiplying in the form of their genetic material, (3) unable to grow and to undergo binary fission, (4) devoid of a Lipmann system.”40

The Bridge from Bacteriophage to Tumor Etiology Whereas the virus posed, in Lwoff’s estimation at least, “a pons asinorum of microbiologists,” (in Latin, an asses’ bridge) his elucidation of lysogeny would prove to be a bridge of a very different sort.41 Rather than pose difficulties, it served as a bridge to understanding, a pontem intellectum (to borrow from Latin as Lwoff did), not only of the nature of viruses but also, most pertinently here, of their role in tumor causation. Of course there had been a constant exchange of models and hypotheses between genetics, cancer etiology, and the developing field of virology throughout the early twentieth century, but as historians of molecular biology Nadine Peyrieras and Michel Morange have contended, the resolution of lysogeny added something more to the picture; namely, “the possibility of an alternation between inactive and active phases that characterized both certain viral diseases and the evolution of cancers.”42 The same idea evidently occurred early on to Lwoff, who in 1953 had already discussed the analogies between the two phenomena: The neoplastic potentiality of a cell could be visualized as perpetuated in the form of the genetic material of the neoplastic particle. But, whereas the development of phage in a lysogenic bacterium is lethal, the neoplastic agent is not pathogenic for the neoplastic cell.

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It is the neoplastic cell which is pathogenic for the organism. This difference being duly taken into account, it is possible that, in potentially malignant cells, the initiation of malignancy, i.e., the initiation of the development of a neoplastic agent, may have something in common with the initiation of phage formation from prophage in a bacterium potentially able to produce bacteriophage.43

It is clear from his language that Lwoff accepted the notion that viruses could be involved in causing tumors, and moreover did not feel the need to justify his reasons for doing so. His stance presents a striking contrast to that of Christopher Andrewes, who just a couple of decades earlier had felt the need for a private forum to let himself go on the matter, and even when he gave a public lecture in 1939, offered his ideas only tentatively.44 The difference in the intellectual climate in which the two men forwarded their ideas may be attributed in equal measure to the broader influence of various technological and conceptual advances described in the previous chapter, as well as the influx of data from various quarters during the intervening period.45 As cancer researcher Howard Temin would fittingly remark many years later, the final push toward understanding Rous sarcoma virus in particular depended equally on conceptual advances in science—­specifically naming the discoveries of the Avery lab, James Watson and Francis Crick, and Lwoff—­as well as the development of such technical tools as “those of quantitative virology and of the study of animal viruses in cell culture,” developed by Delbrück, John Enders, Thomas Weller, and Frederick Robbins, and Renato Dulbecco.46 Save for the last, all of the discoveries in the list and the implications thereof have already been mentioned or discussed at length, and at this chronologically and narratively apt moment, I turn to a discussion of that exception, Dulbecco, among the earliest immigrants, so to speak, from bacteriophage to tumor research. A medical student at the University of Turin in his native Italy during the mid-­1930s, Dulbecco, who was interested in medical research, was selected as an intern in the laboratory of his professor of anatomy, Guiseppe Levi. In his estimation, “Levi [was] interested in tissue culture. And that was the thing that was important for me, because I immediately felt it was a really exciting idea. . . . So I learned about tissue cultures.”47 Also during this period, Dulbecco shared laboratory space and struck up an enormously influential friendship with

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the future Nobel Prize–winning neurobiologist Rita Levi-­Montalcini, then training as an experimental embryologist. Nearly a decade after graduation, having completed his mandatory military service and served as a physician in World War II, Dulbecco returned to Torino and reconnected with Levi-­Montalcini (fortunately she had escaped the tragic fate of many other Italian Jews). This encounter, he later said, was instrumental in shaping his future: Rita is a very wise person.  .  .  . We talked continually about the future—­what to do, what will come next, what science. And I remember, on the question of genes, we had a vague notion that there was such a thing[, but] at school, no one had taught us genetics. . . . So there was nothing that could direct us toward genes, but we knew something. . . . And then she came with a proposal to try to visualize new methods for studying genes. And she said, “Since you’re good at mathematics, why don’t you enroll in physics, because that may be useful in this way of thinking.” So I did that.

According to Dulbecco, Levi-­Montalcini was also instrumental in his decision to move to the bacteriophage laboratory of Salvador Luria in Bloomington, Indiana. A former senior colleague at Turin, Luria had left Italy for the United States at the beginning of the war and by the mid-­ 1940s was well established at the University of Indiana in Bloomington. While on a visit to Italy after the war, Luria had visited Levi-­Montalcini, who suggested a meeting with Dulbecco due to their mutual interests. “So we talked, and Luria asked what I was doing. And I explained this idea to try to study viruses, for their genes, with physical methods, especially radiation. And I was studying physics because of that. And he said, ‘By gosh, that’s what I’m doing! . . . Why don’t you come and work with me?’ Oh, immediately I said, ‘Sure I will!’” In 1947, then, Dulbecco left for the United States on what was supposed to be a one-­year visit but turned out to be a long-­term move. He proved his worth as a bacteriophage worker in short order in Luria’s laboratory, neatly discovering that the mechanism through which bacteriophages inactivated by ultraviolet radiation could be “reactivated” to multiply again.48 His work attracted the attention of Delbrück, who invited him to join the faculty at Caltech: “I remember receiving this letter from Max, and Jim Watson was sitting there. And I said, ‘What do you think, Jim? Do you think I should go there?’ And he said, ‘Oh, absolutely. Caltech is the best place in the United States, so you should go there.’” So two years after moving to the United States, Dulbecco packed his bags again and made Knitting Done

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another long haul to Pasadena, where in a short time he shifted his focus to animal viruses. The impetus for Dulbecco’s switch in direction was, like so many instances in scientific research, driven by financial considerations. Readers may recall from chapter 2 that James Murphy had been on the verge of quitting cancer research around the same time Rous abandoned it in 1915 and had only become the head of the laboratory due to an endowment. Sometime in the late 1940s a well-­to-­do patient in Los Angeles, afflicted with shingles (herpes zoster), learned from his firsthand discomfort about the relative paucity of knowledge about the viruses that affected humans. This patient was persuaded to make an endowment to Caltech to promote advancement in that field by harnessing the knowledge already gleaned from work on bacteriophages toward understanding this considerably more intractable group of viruses. As Dulbecco recounted later, “Since Delbrück knew very little about animal viruses, he thought it wise to arrange a meeting of virologists of all kinds to provide inspiration.” But when this meeting, which was held in 1950, failed to produce any immediate tangible results of the sort that Delbrück was hoping for, he turned to what Dulbecco described as “a more pragmatic approach. One day Seymour Benzer and I were called to his office: Delbrück pointed out that animal virology appeared to be ready for major advances. Would either of us be interested in trying his hand at it? To me it sounded wonderful . . . so I immediately expressed my interest, before Benzer could say anything. At that moment I became an animal virologist.”49 He explained this decision more fully in an oral history interview: At the time, phage work didn’t appeal to me so much. I needed something new, so I said yes. Also, because with my background, I had an MD and viruses are a medical problem. Then, the other thing that I immediately thought was that to do anything in the field, you needed tissue cultures. And that I knew; I already had a background in that. So I said yes. Then we discussed what to do. I said that I did not know anything about animal viruses, and if I was to do anything sensible, I should go and visit laboratories where they work with these viruses, in order to see what people do now—­a base to start from. And they all agreed. And so I started on an almost three-­month trip throughout the country.50

His experiences in the Luria and Delbrück labs had given Dulbecco a keen appreciation for the one-­step growth assays for the phages based

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on counting plaques, so much so that he believed that one of the main reasons why animal virology had not kept pace with phage biology was the lack of similar assays.51 The chick and other animal embryos that various labs used to cultivate these viruses were simply not enough for obtaining reliable and consistent results because of the natural variation among the host animals. Consequently, even before embarking on the trip, Dulbecco had set his first goal: “to develop a plaque system like that of phage, because that is the way you can get a good assay. And for that, you need a uniform layer of cells. And there was no way to do that at the time.”52 The trip was a worthwhile investment of both time and money. One of the researchers that Dulbecco met during his trip was Wilton Earle, a senior cytologist at the National Cancer Institute, who was also trying to grow cells. Soon after Dulbecco’s return to Caltech, “Earle sent me a photograph of something he called, in the accompanying letter, a ‘steak’: a wonderful layer of cells growing at the bottom of a flask. He knew of my attempts to grow cells in a layer [and] since he had succeeded in obtaining that result he sent me the recipe.”53 Dulbecco adapted the techniques for his own needs and was successful enough in growing monolayers of cells in vitro to begin experiments, growing different animal viruses on them. As he recounted: Maybe for a week or two, nothing seemed to happen. And then one day I took one of these cultures to see whether there were any of these plaques. And for some reason, I put the culture in a tangent light, and I saw that it was full of plaques. They were not visible in transmitted light, . . . because obviously when the cells are killed, they make very fine granules, which you [only] see by the scattered light. So then we discussed it with this old man that was there to do something, because the plaques were very difficult to see. He suggested we use a lighter stain—­t hat stains living cells but not dead cells. And so we added this to the medium, and we could clearly see the cells as . . . a red background, and these holes.54

Within a year of his return, Dulbecco had published these results and soon thereafter developed the now widely used plaque-­counting assay for the animal viruses.55 But this “attack mode of the Phage School” toward studying the animal viruses was not immediately accepted unanimously or without argument.56 For instance, Dulbecco recalled that when he presented his results and ideas at the 1953 Cold Spring Harbor Symposium on viruses, it was not easy to persuade everyone that the Knitting Done

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proportionality between virus concentration and the number of plaques meant that a single plaque was formed in response to infection by a single virus particle. The irony of his position—­t hat he faced the same type of objections that d’Herelle had when he, in turn, had tried to persuade his contemporaries about the bacteriophages—­was not lost on Dulbecco: “I relived the predicament of d’Herelle and I resorted to his ultimate weapon, namely citing Einstein on my behalf.”57 Rather more quickly than d’Herelle, Dulbecco was proven to be right. By 1966 he could remark with justifiable pride on how “a single development, such as that of a plaque assay, could have great consequences” in so many different areas of basic and medical virus research. In the years immediately following the development of the plaque assay, Dulbecco, working in collaboration with a number of different colleagues and students, added further foundations for making animal virology quantitative. And it was this quantitative turn that would prove crucial for further progress in understanding the role of viruses in tumor etiology. In the meantime, although there was a revival of widespread interest in problems around tumor virology due to the discovery of a bona fide cancer-­inducing virus in mammals, there was little increase in “knowledge of how viruses cause cancer until quantitative methods were introduced for studying virus-­cell interactions,” according to veterinary virologist Harry Rubin, who would soon become an active player in this field.58 Unlike other animal viruses, tumor viruses did not kill their host cells, which meant that they would not form plaques in cell cultures. The obvious alternative to plaque production was to “attempt to induce and recognize the transformation of normal cells into malignant cells in tissue culture.” It was precisely this task that Rubin set out to accomplish upon his arrival as a postdoctoral fellow in Dulbecco’s laboratory in 1953, choosing the Rous sarcoma virus, the “canonical ‘cancer virus.’” as his experimental system.59 At first, Rubin later recalled, “I had no idea of how the malignant transformation, were it to occur in tissue culture, would manifest itself in the appearance of the tumor cells, [and] with a lively sense of fantasy, it was easy to imagine seeing the hoped-­for cell transformation quite frequently, but it could be neither quantitated nor reproduced.”60 For a time, he fell back on the methods for cultivating viruses on chick-­ embryonic membranes developed by Goodpasture and Burnet, and modified specifically for growing and assaying RSV by the Australian virologist E. V. Keogh. This method was limited in its ability to produce consistently reproducible results, but luckily others besides Rubin were

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on similar quests.61 In 1956 a pair of investigators clear across the country at Rutgers University in New Jersey reported that the infection of cultured chicken embryo cell with RSV produced discrete foci of altered cells—­t he “cell culture analogs to tumors in chicken”—­proportional in number to the concentration of virus.62 The arrival in Dulbecco’s laboratory, around that same time, of Howard Temin, a graduate student looking to change the direction of his studies, proved fortuitous. Rubin tasked the newcomer with converting the observations of the Manaker group into a quantitative assay, which was achieved “after a considerable period of trial, error, and frustration.”63 Instead of the plaques of dead cells produced by bacteriophages and infectious animal viruses, this technique relied on the formation of foci that were “easily recognized because the cells round up, become refractile and tend to heap up in layers.” In short order, Rubin and Temin applied the technique toward studying the dynamics of RSV infection and determined that the virus had a relatively long latent period between the initiation of infection and the appearance of progeny viruses.64 The technique also led to the demonstration that transformed cells could produce RSV particles, a holy grail that had been eluding scientists ever since Rous had first suggested that chicken sarcomas were caused by a virus.65 Thus, the successful development of “plaquing” of tumor viruses was instrumental in building in what Angela Creager and Jean-­Paul Gaudillière characterized as “the genetic vision of the ‘enemy within’” that is cancer.66 Meanwhile, although the RSV work Rubin and Temin conducted under his aegis had piqued his interest in tumor virology, Dulbecco himself did not immediately divert his energies to the cancer problem until 1958. An important part of the reason was that the nucleic acid component of RSV had recently been shown to be RNA rather than DNA.67 This fact raised some problems, because although the existence of RNA viruses was well known, the idea that the genome of such viruses could integrate into the DNA-­based genomes of their host cells ran contradictory to the so-­called central dogma of information flow in biology, which was taking hold around that time. Originally formulated by Crick as a guiding principle to think about the nature of protein synthesis, the underlying idea behind the dogma was that the transfer of biological information—­which he defined specifically as “the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein”—­was likely possible between nucleic acids or from nucleic acid to protein but impossible between proteins Knitting Done

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or from proteins to nucleic acids.68 Although Crick’s original idea did not differentiate between the nucleic acids, others thought that the information flow was more restricted in its directionality. According to Watson, for instance, “Virtually all the evidence then available made me believe that DNA was the template upon which RNA chains were made. In turn, RNA chains were the likely candidates for the templates for protein synthesis.”69 Perhaps because of his long association with Watson, beginning from their days together in the Luria lab in Indiana, Dulbecco’s understanding was colored by his view, for he later explained, it was difficult for him to imagine how the RSV genome could become a provirus: “How [was] it possible that [viral] RNA persists in the cells? I didn’t know any mechanism. So I thought, well, if I want to look at the problem, I should look at the tumor virus, which has DNA rather than RNA.”70 Opportunity knocked in 1958 when he learned of the discovery and isolation of a new DNA virus linked to cancer of the salivary glands of mice—­t he polyoma virus.71 This new discovery checked all the boxes that Dulbecco needed to turn his complete attention to tumor virology, for unlike RSV, in addition to having the right chemical makeup, the polyoma virus was also a mammalian tumor virus, that elusive long-­ sought-­after analog for human cancer. Primed by the recent success of Rubin and Temin’s focus assay, Dulbecco modified the technique to work for this new group of viruses, taking the decisive first step toward the topic that would remain an enduring interest for the rest of his career.72

The Linchpin in Action In 1959, the American Cancer Society, which had, a few years earlier, begun to sponsor a symposium series, chose as the theme of its third symposium “the possible role of viruses in cancer.” One of the main reasons for this theme, according to Harry Weaver, who had become the society’s research director just the year before, was the recognition of the “obvious need for a careful appraisal at this time of progress in this field.”73 Its theme alone would have merited a discussion of this symposium in this book, but adding to its resonance with the history here is the fact that one of the key points discussed at the meeting (if not in quite the same language that I have used) was application of the lessons learned from lysogeny toward understanding cancer etiology. In order to promote meaningful, in-­depth interactions among all

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attendees, the conference was limited to about thirty-­five participants (inclusive of the organizers), hailing from different areas of virus research. With the American Cancer Society headquartered at the time in New York City, it is not altogether surprising that Rockefeller University had been well represented in the organizing committee; in fact, three out of the five members hailed from its faculty. Interestingly, although Richard Shope chaired the committee, it was Rous who delivered the opening remarks and final closing commentary, likely (in part at least) out of deference to his seniority. By this time he was past eighty years old but still very much a prominent figure in the field, due to his work on the Shope papilloma virus. While it would be impractical to list the entire roster of attendees here, it should be mentioned that several key figures mentioned in earlier parts of this book and representing different areas of virology were in attendance, among them pioneers in viral research techniques such as Wendell Stanley (crystallography) and Enders (cultivation) and basic bacteriophage researchers including Luria and Lwoff. Among those representing the perspectives from work on the tumor viruses were the newly initiated Dulbecco, Rubin, and Rous’s long-­term correspondent and closest ally on the matter of tumor virus identity, Andrewes, self-­proclaimed as a “general-­purpose virologist.”74 In his customary spirited and provocative manner, Rous kicked off the meeting by turning the symposium theme on its head, focusing on “tumors in relation to viruses” rather than vice versa. Tumors, or neoplastic phenomena, as he called them, “stare us in the face and demand to be understood. Indeed, they have the final word, not only as concerns our thought on tumor causation and the relation of viruses thereto but as to whether our findings have any real significance for the cancer problem.”75 But, as he hastened to add, what he and fellow committee members “had not known and greatly needed to know were the various roles of nucleic acids” in viruses and in neoplastic phenomena.76 To him such revelations vindicated the choice to give “genetical virologists”—­t he bacteriophage researchers—­t he most prominent spots on the program. It was no accident, Rous added, that the committee had designed to program so that both the opening and closing talks were delivered by leading bacteriophage researchers.77 In the opening lecture, Luria seemed sympathetic to the cancer biologists’ worry that the idea of a viral etiology of cancer would raise the specter of cancer as an infectious disease. He was also mostly in agreement with the thinking that most cancers stemmed from genetic Knitting Done

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changes within the affected cells rather than from an extrinsic virus, a view that oncologists had adhered to most strongly. Nevertheless, Luria believed, the most rapid progress in cancer research would emerge from the study of virus-­induced tumors because the latter subset provided the “opportunity to analyze the role of individual genetic elements in the transformation of normal cells into tumor cells.” From his point of view, the viruses were “genetic elements, specialized for transfer because they possess certain specific genetic functions,” and a virus infection interpreted as “a kind of infective heredity.”78 In his commentary following Luria’s presentation Andrewes voiced many of same objections against the newer expression that he had forwarded against Murphy’s notion of the transmissible mutagen years earlier. He began his response politely enough, by lauding Luria for laying out facts about genetics relevant to the cancer problem, many of which he professed ignorance about. But then, pointing out that Luria was “trailing his coat in the hope of having some sharp arguments,” he took it upon himself to rise to the occasion, homing in on the latter’s representation of viruses as a form of infective heredity: This idea may or may not be a good one, but if [Luria] really thinks it is generally accepted it just convinces me that these geneticists never come out of their ivory towers. . . . If he went to a gathering of ordinary virologists he not only would not find it was generally accepted but that they would have great difficulty in understanding what he was talking about. When he says that “virus infection is a cellular mutation,” I am personally at a total loss. I don’t think it even occurred to me during the 1957 epidemic of Asian influenza that we were suffering in our hundreds of thousands from cellular mutations!79

Andrewes then dwelled a bit on his own idea about what viruses were and how they affected the host, recalling the ideas he had first put forth in his fairy story some two decades prior. Furthermore, he went on to suggest that Luria himself would have no quarrel with this idea, calling on for support a 1953 article in which Luria had speculated that “a virus may be both a regressed parasite and a cell-­component that has become infectious, depending simply on which phase of the evolutionary history of its genetic material we are observing.”80 How, or indeed whether, Luria responded directly to Andrewes’s critique is not recorded in the meeting’s proceedings, but the comment is just one example of the different points of view on a diverse range of

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issues at the meeting. This range is unsurprising given the number of prominent and opinionated scientists present there. But even the contradictory opinions, Rous observed, “were in the main brought forward for their intrinsic worth and for the new ideas to which they gave rise. [Much time] was given to new facts necessitating readjustments of thought.”81 Certainly, a perusal of the talks and discussions shows that disagreements notwithstanding, most attendees were converging on the idea that the genetic concept of virus infection provided a “workable prototype” for cancer etiology. Even the contrarian Andrewes would grant that Lwoff’s prophage concept was a very useful one, and that even though no one had provided any conclusive evidence that any other (animal) virus could go into an analogous provirus state, “If it [did] I think it would make sense.”82 Without a doubt, the most explicit discussion of concrete analogies between lysogeny and the tumor viruses at the symposium was provided by Dulbecco in his talk on the relationships between tumor viruses and their hosts—­t he neoplastic cells.83 In a manner reminiscent of the distinction drawn by the Wollmans between the types of bacteriophage interactions with the bacteria, he identified two distinct types of interactions of the tumor viruses with their hosts: an “integrative” one akin to that of the lysogenic phages, in which the infected cells were transformed to continue to grow and divide without producing infectious virus particles, and a “nonintegrative” type of relationship in which many progeny viruses were synthesized, ultimately resulting in the death of the host cells within a couple of generations. He reported that either type of interaction was possible for both polyoma and papilloma viruses, “depending on the type and on the state of the host cells, and that the two types of interaction can occur in the same virus-­cell system, but with different probabilities.” He also described the as yet unpublished experiments of his then student Howard Temin on the chicken sarcoma viruses, which displayed different transformative abilities in cell culture, once again underscoring their similarity to lysogeny in terms that echoed Burnet’s description from decades ago: “The transformation is hereditary, in that the descendants of the transformed cell maintain the acquired morphological type. . . . This is a clear example of conversion of the cells by the virus genome, analogous to conversion caused by bacteriophage.”84 Before going on to detail the further and considerable contributions of Dulbecco and then Temin toward unraveling the mysteries of the viral causation of cancer, I return briefly to Lwoff, whose concludKnitting Done

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ing address at the meeting was perhaps his last substantive offering in the arena of applying the lessons of lysogeny to cancer research.85 This address was not short by any means; neither his style nor the range and depth of the presentations and discussions at the meeting would have allowed for a pithy summary. For all his protestations that he was an “outsider” to the cancer research field, Lwoff had plenty to offer. Not only did he talk about the specific analogies between lysogeny and the possible mechanisms through which tumor viruses induced cancer but he also drew connections between these phenomena and the broader context of his bacteriophage work; namely, the genetic regulation of the cell’s activities. Rather than risk robbing anyone of the credit for their achievements, he chose not to cite anyone—­whether present or absent—­by name. Rous was the single exception to this policy, a decision for which Lwoff proclaimed, no explanation was needed. Interestingly, however, it was not the discovery of the sarcoma virus to which he drew attention but rather to a rather more recent publication in which Rous had remarked, “The individual cell is really the host of the tumor virus, not the organism.”86 Recalling this observation, Lwoff went on to remind his audience of another host-­parasite relationship that lay at the foundation of all considerations of cancer and tumor: “[I]f the host of the tumor virus is the cell, the host of the cell-­virus system—­t he malignant cell—­is the organism.”87 By setting up his talk in this manner, Lwoff deftly trained his audience’s focus on how essential it was for virologists to study the interrelations between viruses and cells. Less pointedly, he also set the stage for the various analogies to be drawn to his proposed mechanism for lysogeny—­already mentioned by Dulbecco as well—­which was, after all, an explication of the nature of the relationship between the bacteriophage (virus) and its host, the bacterial cell. With specific reference to the way in which a cell’s physiological functions were controlled by such molecular actors as enzymes and genes, Lwoff pointed out: “When one considers bacteria, and especially lysogenic bacteria, it becomes clear that viruses can play an important role in the physiological balance of the organism, and this is also true for oncogenic viruses.” Recapitulating the key features of “true” classical viruses, for which he had recently provided the definitive version, Lwoff went on to describe a viral infection as “the introduction into a cell of the genetic material of a virus.” When the infecting virus was oncogenic, he explained, the result was the transformation of the cell to a malignant form, provided the infection was of the integrative type (described by Dulbecco in his

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lecture) akin to lysogeny. This integration of the viral genome introduced new genetic information “into the balanced dynamic system” of both the individual host cell that was infected and the whole organism of which the cancer cell was part. Lwoff’s summation was not designed to lull symposium attendees into thinking that there were easy answers to the questions they sought, as indeed it did not. “A number of us still wonder—­in the stilly watches of the night, when perhaps we are feeling morose—­whether all neoplastic viruses are, in Dr. Lwoff’s phrase, genetic in character,” Rous said at the end. But, he added, the mood would pass, and on the whole, the entrance of the microbial geneticists had brought the gift of “important and astonishing facts” to the problem of viruses and cancer.88 Not only was he right in this respect but, in fact, the most important gifts were yet to come. Following hard on the heels of this symposium, Dulbecco’s group published a series of reports detailing their recently begun investigations on the polyoma viruses. Their studies confirmed that these viruses contained “DNA as the essential nucleic acid” and that cell cultures transformed by the virus revealed properties very similar to those of lysogenic bacterial cultures.89 A few years later, improving on the state-­ of-­t he-­art nucleic acid hybridization techniques invented by Sol Spiegelman’s group at Urbana-­Champaign, they found that cell cultures transformed by different polyoma viruses—­by then they were also working with another model, the simian virus 40 (SV40), known to cause brain and bone cancers in monkeys—­seemed to possess few or no free virus particles, but contained viral DNA integrated in the DNA in their nuclei.90 Here at last, was concrete evidence that animal viruses, or at the very least the polyoma viruses, were able to become proviruses.

The Teminal Heterodoxy What of RSV in the meantime? As Dulbecco had imagined, being an RNA virus, it would prove to be a harder nut to crack, a fact that Temin was in the process of discovering firsthand. The history of the introduction and reception of Temin’s DNA provirus hypothesis is a well-­documented story, with as many interpretations of why events unfolded the way they did as there are accounts. The details do not bear rehashing here; readers are referred to the numerous tellings by participating scientists and historians, as well as a number of obituaries of Temin, who died prematurely in 1994 (ironically, of lung cancer, albeit Knitting Done

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not a virus-­induced type), before he had even turned sixty. Instead I will limit myself in this section to sketching out those events most relevant to the way in which Temin’s hypothesis and pathway to affirming it exemplify the role of lysogeny as linchpin in the history of virology. Joining Caltech in 1955 right after obtaining his bachelor’s degree in biology from Swarthmore College, Temin had begun his graduate studies in the laboratory of the developmental biologist Albert Tyler. According to the molecular biologist John Drake, who arrived there at the same time with similar interests, both of them were quickly frustrated by their choice, seeing “no way to advance the field, the probes of the time often being no better understood than their target tissues.” Luckily for them, Watson, who was visiting Caltech around that time, recognized their quandary and suggested working with Dulbecco, who agreed and “introduced us to the members of his group who were to become our daily mentors [Marguerite Vogt and Harry Rubin]. Probably because of perceived parallels between developmental processes and the cellular modifications wrought by tumor viruses, Howard took up Rous Sarcoma Virus [with Rubin].”91 His instincts evidently served him well, because as indicated earlier, Temin achieved the first task Rubin set upon him in quite short order. Right from the start, Dulbecco later reflected, Temin “was interested more in the biology—­how does a virus cause cancer? . . . I remember we talked a lot about that.” His time at Swarthmore and the Tyler lab had not given Temin much depth of knowledge in bacteriophage research until that time, and it is possible that he was therefore likely more receptive to ideas about lysogeny and its application to the cancer virus question, compared to more established members of the Phage Group. Dulbecco especially attributed Temin’s interactions with the Swiss phage researcher Jean Weigle as a significant influence on the development of the younger man’s thinking about the viral etiology of cancer: [Weigle] was a physicist, actually, but like Max he had become a biologist. He was working with . . . a temperate phage—­a phage that establishes a relationship with a cell without killing it. . . . So there was, therefore, something which told us that it is possible to have a permanent infection within the walls of the cell without the cell suffering from it. . . . And in talking to our people, Howard [Temin] would say, “Yes, there’s no doubt. There must be something like this.” And he tried to do an experiment to show that the Rous sar-

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coma virus persisted in the cancer cells, but [had] nothing directly to show it.92

By 1960 Temin had written and defended his dissertation—­under the joint supervision of Dulbecco and Rubin—­focusing on the in vitro interactions between RSV and its host cells. And although he acknowledged the lack of direct evidence, he could not resist including some remarks about the possible existence of a provirus form of RSV. “The parallelism of the radiobiological results for RSV and temperate phage strongly suggests that RSV also interacts with the genome of the host cell,” he hypothesized, referring to data reported in a concurrent publication by Rubin and himself.93 In a section of his thesis discussing the relevance of his work in understanding the role of viruses in cancer, Temin went on to make a somewhat stronger claim: “The present work establishes some kind of a close relationship with the genome of the infected cell. It is reasonable to assume that this relationship causes the cell to become malignant. . . . The conversion of the same type of cell to different types of Rous sarcoma cell by mutants of the virus indicates that the virus can contribute information which determines the character of the Rous sarcoma cell.” It was not to be expected that such claims would pass uncontested, and indeed they did not. Delbrück, who was on Temin’s dissertation committee, was a major detractor, and at the defense, apparently told Temin: “You have no evidence, therefore you cannot say that,” in response to Temin’s hypothesis about the similarities between the effects of bacteriophage in lysogeny and of RSV in malignant transformation.94 In a short reprise of the events of the defense in his former student’s obituary, Dulbecco said that Delbrück’s reaction surprised him, at least in part because he believed that it was “essential” that a doctoral dissertation should go beyond the immediate validity of the experiments and look ahead to future avenues for investigation, which Temin was certainly doing.95 Moreover, as described earlier, Dulbecco himself regarded the analogy to lysogeny as “the main model” for virus-­host interactions by tumor cells, though he prudently chose to work with DNA viruses. In any event, Delbrück’s disagreement did not deter Temin significantly, for soon after his defense he went on to publish data on the interactions between RSV and the cells it infected and transformed, where he made the claim—­bearing an uncanny resemblance to the more than two-­decades-­old claims about lysogeny by Burnet—­ that the virus became “equivalent to a cellular gene controlling cell Knitting Done

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LYSOGENY

PROVIRUS Bacterial Cell

Cellular DNA Normal Animal Cell

Bacterial DNA

RSV Particle

Bacteriophage DNA

Viral RNA

Lysogenization

Reverse Transcription

Prophage

Transformed Animal cell Lysogenic Bacterium

DNA Provirus

Bacterial Cell Division

Bacterial Progeny

Cell Division

Tumor Cells

Fig. 7.3 Parallels between lysogeny and provirus formation in tumor cells. Illustrated by Annapoorna Mahesh, after flowcharts published in Harold Varmus, “The Pastorian: A Legacy of Louis Pasteur,” in Advances in Cancer Research, ed. George F. Vande Wude and George Klein (Academic Press, 1996), 12, fig. 14; 14 Left: Schematic diagram showing the main steps in establishing bacterial lysogeny described by André Lwoff,“Lysogeny.” Bacteriological Reviews 17, no. 4 (1953). Right: Schematic diagram showing the main steps in the formation of a DNA provirus. The parallels with lysogeny shown opposite could not be established until the discovery of reverse transcriptase. Howard Temin, “The DNA Provirus Hypothesis,” Science 192, no. 4244 (1976).

morphology.”96 In many ways, Delbrück’s objections to Temin should not be that surprising. For one, it seems entirely in keeping with his reputation for requiring hard evidence for any theory, and Temin decidedly lacked such evidence for many years. Also, it must be remembered that Delbrück in particular, and the Phage Group in general, had an uneven history with bacteriophage lysogeny. Far more astonishing from a historical perspective is the opposition Temin would face from Harry Rubin, his second supervisor on the dissertation. The challenge from these quarters was not as immediate as Delbrück’s; certainly there are

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no records of Rubin expressing his disagreement with Temin at the defense. If anything, publications from that period show Rubin quite aligned with Temin’s main idea: indeed, it was he who had introduced the possibility of the similarity between RSV infection of its host cells and lysogeny by temperate phages in a single-­authored paper published in 1955 even before Temin had joined the Dulbecco labs.97 Furthermore, their joint paper on radiological studies on the host-­virus interactions, which Temin also cited in his dissertation—­concluded with this fairly definitive statement: “It would appear that the genome of RSV, like that of temperate phage, is integrated with that of the host cell, but the mechanism of integration is somewhat different. This seems a logical consequence of the differences in nucleic acid type between RSV and phage, and in the degree of organization of their host cells.”98 But despite such initially favorable statements, Rubin would rather emphatically disagree with Temin’s notion that RSV operated in a manner analogous to bacteriophage lysogeny. Exactly what caused Rubin to change his mind is a matter of some ambiguity in the extant records. The easy, most obvious, answer is the one favored by Temin, for example, and explicated by Harrison Echols, a colleague first of Temin’s at the University of Wisconsin and later of Rubin’s at Berkeley. In their view, Lionel Crawford’s discovery of the RNA makeup of the RSV genome “ended Rubin’s—­and almost everyone else’s—­entrancement with the idea of an integrated provirus; clearly an RNA virus could not integrate its nucleic acid into host chromosomal DNA.”99 Certainly this explanation fits in with most later narratives of Temin’s work and its reception, but as certain discerning historians have remarked, the explanation is not quite as neat. In fact, when Temin first put forward his provirus concept, it was, as both he and Rubin later emphasized, a purely “genetic hypothesis [that] contained no implication about the molecular nature of the provirus.”100 There is even a letter from Rous to Rubin after his move to Berkeley, regarding a unpublished paper by the latter about which Rous commented: “Needless to say, your paper has interested me much, especially your suggestions about a possible integration of the genome of the virus with that of the cell.”101 It is interesting that this letter was addressed to Rubin rather than Temin. And finally, although he acknowledged the barriers posed by the RNA makeup of RSV, Rubin did not attribute his firm opposition to Temin’s idea to his disagreement over the issue of the chemical makeup of the potential provirus; indeed, a joint publication from 1958 shows both scientists to be well aware of Knitting Done

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the potential problems posed by an RNA genome, as well as other differences between the temperate (lysogenic) phages and tumor viruses: “Although there are striking similarities between temperate phages and tumor viruses, there are also important differences. For one thing, a Rous sarcoma cell can make mature virus and continue to divide, while a lysogenic bacterium cannot. A second important distinction is that bacteriophages contain DNA only, while it seems likely that RSV contains only RNA. Thus  .  .  . we must expect to encounter even more unique situations in this [latter] system and be prepared to deal with them in a unique and original way.”102 According to Rubin’s own account, the tipping point for him came in the guise of the patterns of the transmission of congenital infections of avian leukosis, caused by a virus closely related to RSV. Whereas the infection was passed along quite easily from parents to offspring from infected female birds, infected roosters did not transmit their infection to offspring. Rubin remarked, “The failure of male transmission is the first suggestion that the viral genome may be restricted to the cytoplasm. This evidence is complemented by the presence of high concentrations of virus in the unfertilized female gamete, a single cell with large amounts of cytoplasm, and the uniform success of congenital transmission by viremic females. [Such] experiments tend to rule out the type of intimate association between the viral genome and host-­cell chromosomes which occurs in lysogenic bacteria.”103 Such negative evidence was not enough to deter Temin, however. When, after completing his PhD and a one-­year fellowship at Caltech, he moved to the University of Wisconsin–Madison to take up his first academic position as an assistant professor of cancer research, he took the RSV problem and his ideas about it with him. Although the working conditions in his new home might be described as sparse—­in a later biography he recalled that his “first laboratory was in the basement, with a sump in my tissue culture lab and with steam pipes for the entire building in my biochemistry lab [and] I worked with only two technicians”—­he found the intellectual atmosphere congenial and remained at Wisconsin for the duration of his career.104 What made Temin, then a young researcher at the very beginning of his career and in impecunious circumstances, so tenacious in his adherence to the provirus hypothesis? By all accounts he was not a fanciful theoretical scientist. Rather, as his contemporary David Baltimore remembered, he was “first and foremost . . . an experimentalist.”105 So the experimental evidence should have led him, as it did Rubin, in the

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opposite direction to the notion that RSV was attaining something akin to a lysogenic state. But, according to Baltimore’s perspective as an outside observer of the events, it was Temin’s experimental focus—­on a different set of experiments—­t hat fed his conviction of the rightness of his idea: It was the transformed cells in the dish that were talking to Howard and he was listening. What they shouted at him was stability. The transformed state was a permanent one. Every transformed cell gave rise to more transformed cells. Most persuasively, he found that strains of Rous Virus that gave altered morphologies to cells did so stably—­t his was a key argument for the control of the transformed cell by the viral genome. It was also important that cancer was an irreversible process of cellular change. To Howard this meant that there had to be a change in the cell’s DNA.

Baltimore’s explanation rings true and is borne out to a certain extent by some of Temin’s own statements in early publications. In the same publication in which he proposed that RSV became the equivalent of a cellular gene, for instance, he also suggested two possible ways in which the virus could operate to effect the transformation of a normal cell into a tumorous one in vivo, either by contributing genetic information to the cell that enabled it to become tumorous or by activating a tumorous state. He also indicated his belief that the data appeared to favor the former possibility: “The present findings indicate that some genetic information responsible for the character of the Rous sarcoma cell is introduced by the virus,” he wrote, but conceded that there was no evidence to indicate that the differentiating and carcinogenic actions of the virus were due to the same viral action.106 As he explained in an article on viruses and cancer for a general audience around this time, “Once we know that the virus acts to cause a genetic change in the infected cell, we can ask how this genetic change is related to the production of a tumor.”107 At this point in time, it should be noted, Temin had not yet explicitly proposed the notion of DNA provirus—­t he specific idea that, in retrospect, has been deemed heretical by many scientific actors and narrators of this history. This idea would not be fully realized for a few more years, during which interim, as the publication output from Temin’s new laboratory in Wisconsin reveals, he pursued two distinct lines of investigation in order to parse out the details of his still-­nascent ideas. On one hand, there was the issue of carcinogenesis, namely how Knitting Done

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the viruses could cause cancer, and on the other, biochemical investigations to try to determine how an RNA virus could integrate its genetic information into the host’s DNA genome. The historian Susie Fisher has made a persuasive case that it was this dual “Janus-­faced mission” that set Temin apart from others tumor virologists. Rubin, for instance, went on to look for nongenetic explanations for transformation by RSV, while others pursued the matter of RNA virus replication as an independent problem. The fact that “for Temin solving either issue singly would have been quite unacceptable” likely made his working life more difficult at the time but his eventual success that much more meaningful.108 On the carcinogenesis front, he confirmed his idea that RSV’s ability to induce tumor formation by inducing morphological changes or conversions of the host cell was separate from its ability to induce the production of virus particles by the infected cells. Together with corroborating evidence from Dulbecco’s group, in the case of polyoma viruses, these findings buttressed Temin’s view that there was a common mechanism at work in tumor formation, despite their biochemical differences, which was “the presence of the viral genome at a specific site in the cell causing conversion and carcinogenesis.”109 On the biochemical front, meanwhile, he pursued the issue of the chemical nature of the provirus. He pursued the problem through two rather different “exploratory experimental sets”: both indirectly by examining the biochemistry of RSV replication in its host cell and directly by hunting for traces of the provirus in the genomes of transformed cells.110 In order to decipher the biochemistry of RSV replication, Temin built on the then recent findings of a group at Rockefeller headed by the renowned biochemical geneticist Edward Tatum that antibiotics such as actinomycin D had differential effects on the biosynthesis of different nucleic acids—­DNA and RNA—­depending on whether these nucleic acids were of cellular or viral origin. Whereas the antibiotic “selectively and irreversibly suppressed” the biosynthesis of mammalian cellular RNA, it did not appear to affect the synthesis (replication) of DNA in these cells. Its effect on the synthesis of viral nucleic acids growing in host cells was quite the opposite, however. The data showed an inhibition of the replication of DNA viruses such as the pox virus, whereas the replication of the mengovirus, a small RNA-­containing virus, was unaffected. These data had led these researchers to conclude that actinomycin D acted by interfering with that DNA-­directed RNA synthesis, regardless of the source of the DNA.111 Interestingly, howev-

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er, when Temin subjected RSV-­infected cell lines to actinomycin D, the virus did not appear to respond in the same way as the RNA-­containing mengoviruses. Rather, its replication was suppressed, albeit not irreversibly, which led Temin to surmise that the effect of the antibiotic on RSV production was probably mediated through DNA and that actinomycin D treatment prevented formation of viral RNA. In his control experiments, gauging the effects of the antibiotic on cell cultures in the absence of virus infection, his results agreed with those of Tatum’s group, that actinomycin D inhibited the synthesis of cellular RNA but not DNA. Therefore, he concluded, “it is suggested that the template responsible for synthesis of viral nucleic acid either is DNA or is located on DNA.”112 Temin also set about looking for direct evidence of the presence of RSV provirus—­namely, RSV-­specific DNA sequences—­in the genome of transformed cell cultures. The rationale for his approach was to find out whether RSV-­infected cells contain a new DNA that is homologous to the viral RNA. Using radio-­labeled viral RNA as probes, he tested both RSV-­transformed cells and uninfected cells of the same culture for the presence of complementary DNA sequences that could specifically anneal or hybridize to these probes. In his judgment, the results showed “a consistent difference between hybridization of viral RNA with DNA from RSV-­infected cells and with DNA from parallel uninfected cells . . . most simply explained by the hypothesis that the DNA of RSV-­infected cells has a region of new DNA, formed at infection, homologous to viral RNA.”113 Armed with what he believed was persuasive evidence from two different experimental sets on two separate but related problems, Temin publicly presented his DNA provirus hypothesis at an international conference on avian tumor viruses held at Duke University in 1964.114 That his hypothesis was not received favorably by most attendees, not only on this occasion but for some years afterward, is undisputed, but the various accounts of the reasons for poor reception have varied considerably. As mentioned earlier, Temin himself and many of his scientific friends and colleagues attributed the poor reception to the purported heretical nature of the DNA provirus. An examination of the scientific literature shows that while the directional information transfer implied by the central dogma as it was then understood was certainly influential, it was by no means an idea set in stone.115 Indeed, in a 1964 lecture on the biochemical aspects of cancer etiology to the American Association of Cancer Research in Chicago, the biochemist Van Knitting Done

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Rensselaer Potter, Temin’s chair at Wisconsin, explicitly drew attention to Temin’s hypothesis and his actinomycin D work to predict the possibility of an enzyme that could make DNA from RNA templates. Claiming that Temin’s data suggested that the provirus contained DNA, he said, “It presumably would have to be synthesized by a special RNA-­ directed DNA polymerase so far undiscovered. There is thus pending the distinct possibility that both RNA and DNA tumor-­producing viruses may become integrated into the genome of normal cells in order to convert them to tumor cells.”116 Such support notwithstanding, it is true that the majority of the scientific community did not accept Temin’s ideas, not so much because they were heretical—­a word that was not really used at the time of the debates but brought up in later descriptions of the work—­but because many believed that Temin may have overzealously interpreted the results of his antibiotic experiments in order to fit the data to his theory. In his reprise of the events at the time, Rubin, who was generous in according Temin credit for his tenacity and vision, did point out that when he first reported his actinomycin D experiments, Temin did not adequately account for the fact that the antibiotic did not act as expected on the multiplication of influenza viruses, another group of RNA viruses known not to integrate into the host genome.117 As for the hybridization experiments, Fisher has noted that they were a “one-­t ime operation” given the paucity of funds, equipment, and manpower during the early years of Temin’s appointment at Wisconsin, and that most contemporaries found the “numbers” unconvincing.118 In fact, that the paper was published at all was due to the process through which Proceedings of the National Academy of Sciences to this day accepts papers for publication. Rather than following the typical peer review process of many scientific journals, the National Academy of Sciences accepts contributions from its members via a special track. Temin’s paper was submitted by the famed geneticist James Crow, his senior colleague at the University of Wisconsin who was a member, and with whom he had built relationship of mutual intellectual trust in the four years since his arrival in Madison.119 Describing himself as “innocent of any detailed technical knowledge, but greatly respect[ing] Howard’s judgement,” Crow also painted what is probably one of the most complete pictures of Temin’s tenacity and vision, underscoring the importance of his Janus-­faced mission: “Howard’s critics found each experiment wanting in some important respect, yet the several separate lines of evidence all pointed in the same direction.” Later, Dulbecco would make a simi-

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lar observation: “All his critics were looking at the results of the experiment, not at the whole picture, as Howard did.”120 Over the next years, Temin determinedly persevered in his goal of gathering evidence to defend his hypothesis, but it proved something of an uphill battle. Rubin would later recall how even independent confirmation from a group of researchers at the National Institutes of Health—­t hat DNA synthesis was an absolute requirement for the synthesis of RSV particles in transformed cells—­failed to persuade the skeptics.121 It was not until the 1970 discovery of the enzyme reverse transcriptase, which is capable of using RNA templates to synthesize DNA, that the scientific community came around to accepting the entirety of Temin’s vision.122 The discovery of the enzyme was an instant success in the scientific community, and virtually overnight caused Temin to go “from rebel to establishment” in the scientific community.123 Within a mere five years of their discoveries, Temin and Baltimore received the 1975 Nobel Prize in Physiology (shared with Dulbecco), a striking contrast to the fifty-­five-­year gap between Rous’s discovery and his prize. Certainly reverse transcriptase helped clinch matters because it provided both physical evidence and the theoretical mechanism for the possibility of the conversion of the RSV genome into the DNA provirus in a manner similar to the way that bacteriophage DNA became the provirus. But interestingly, even in his Nobel speech, it would be the “nucleic acid hybridization and infectious DNA experiments” that Temin gave pride of place over the enzyme itself as “actual proof of the existence of a DNA provirus.”124 In his book DNA: The Secret of Life, James Watson claimed that DNA, through the agency of the Human Genome Project, had “proved Darwin more right than Darwin himself would have dared dream.”125 He was right, of course, but I contend that this tale of two viruses extends the reach of Watson’s claim for the power of DNA in substantiating the work of earlier scientists significantly further. This molecule has indeed been biology’s “golden thread”—­to momentarily hark back to Dickens’s Tale—­a thread that proved right many other stalwarts of science, who dared to dream and to “pursue the unpopular” and so showed the world that despite beliefs to the contrary, some viruses could infect bacteria and others could cause cancer.126

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AFTERWORD It is a far, far better thing that I do, than I have ever done; it is a far, far better rest that I go to than I have ever known.” —Charles Dickens, A Tale of Two Cities

History does not end, but books must. To be sure, the history of either Rous sarcoma virus or the bacteriophages, not to mention the history of viruses more broadly, is far from over. They continue to unfold even as I conclude this book. Indeed, I sit proofreading these pages in a world dramatically changed by the COVID-­19 pandemic, the likes of which have never been witnessed before. This change has been sudden; not a hint of the dramatic devastation and upheaval of our lives existed even as recently as February 2020, when I turned in the final files for this book. Only consider how much change the viruses have wrought the world over since then. As regards the specific protagonists of this tale, in the more than half century that has followed the point at which I ended the last chapter, both them have gone on to far, far wider horizons and grander things than before. But these developments have been largely unrelated in any significant way to one another, and to claim any more parallels between the research trajectories of the viruses than have been shown already would be forced, no more legitimate than claims for the similarities between those of any randomly picked pair of viruses or

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groups of viruses. Providing as it did the conclusive evidence for the existence of something very like bacteriophage lysogeny in RSV, the discovery of reverse transcriptase is, I think, a fitting finishing point for the tale of these two viruses and so there I shall stop. The end of this tale brings me back to the underlying historiographic “why?” questions raised at the outset of this book: Why, why now, why viruses, and why RSV and bacteriophages in particular? To this list I should have probably added one more, “Why tell the story in this way?” which is the issue I shall address first, having given it only cursory attention in the introduction. As I declared at the outset, the perspective and approach in this book is that of an “internalist,” with a focus on the details of science in the works. Both this style of historical writing as well the terminology used to describe it are considered by many in the profession to be somewhat old-­fashioned. Already in the early 1990s the renowned historian Steven Shapin declared that the entire “discourse of ‘internalism’ and ‘externalism’ seems to have passed from the commonplace to the gauche,” and that historians of science, rather than engaging with the issue, had “turned our backs on it and wished it away.”1 The historian Donald Kelley, however, contended that the difference was meaningful in the context of our profession, and that neither wishing it away nor considering the matter “silly,”—­as Shapin did in his short but influential commentary on the scientific revolution—­would actually do away with it.2 Kelley went on to offer what I believe is a more fruitful way of considering the difference between, as he would have it, “the ‘intellectualist’ and ‘contextualist’ methods”—­t hat is to say, as a difference in the way that historians employ their sources. As is evident in this tale, the different primary sources—­published papers, private correspondences, and the like—­have been deployed in the service of the former, detailing as it does the history of the two viruses from the “inside.” I did not, by any means, ignore—­or even, I hope, give short shrift to—­outside influences, the social, institutional, national, and cultural forces that gave context to the words and ideas of the virologists. But for the most part I considered these influences in a unidirectional sense. What I mean is that I looked at their effects on the science, but not on the reciprocal effects of the scientific advances on these factors, as, for example, Angela Creager did in her history of tobacco mosaic virus.3 To undertake both tasks in a single book would have made the project too long and unwieldy, and the latter by itself was not where my sources led me. In taking the internalist approach, I may have picked what Michael

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Ruse recently suggested was the losing side of a battle; in fact, he went so far as to suggest that one reason that we hardly hear the terms any more in conversations about the state of the discipline is that “the externalists had won so decisively.”4 I will admit that in an uneven fight, I always harbor a sympathy for the underdog, but championing the underdog was not the main reason why I took the internalist, intellectualist approach to this history. For starters, even as he outed himself as “one of the worst of the lot” among the externalists, Ruse also admitted to wondering if the field as a whole had not lost something as a result of one side winning over the other. To that remark I would counter that I am not sure the internalists have lost decisively. At least not yet. For my part as a historian of science who came into the field having paid my scientific dues at the bench, I have no doubts whatsoever that my later-­chosen discipline would be greatly impoverished should either side win so completely as to silence or edge out the other. In this particular case, had it not been for my internalist’s perspective, I might have missed the compelling parallels between the research lives of the bacteriophages and tumor viruses altogether. I hope that readers will agree with me that the history has justified its writing, that this tale—­ like Rous’s Nobel Prize—­was long overdue, and that the details of all the discoveries, advances, and breakthroughs are interesting in their own right. The intrinsic interest value of the tale notwithstanding, there are also some broader reasons why I believe that this comparative history is relevant at this time. For one, viruses as a group pose some of the most pressing challenges to medical science, taking a toll not only on lives but also the quality of life. Since early 2020 COVID-­19 has made sure that viruses are on the front pages of newspapers and on television screens at all times, but over the past few decades we have had other scares due to outbreaks of acute viral infections—­SARS, bird flu, influenza (which remains a perennial problem), dengue, and Zika, to name a few examples. Less widely known perhaps, but with arguably wider societal and economic impact, are the viruses causing chronic infections. Cancers of different types—­including tumors—­are perhaps the best known among such diseases, and one in every six cancers in the world is linked to some sort of virus infection.5 Other examples abound; for instance, hepatitis B virus infections have been shown to be responsible for a “large proportion of chronic liver disease.”6 HIV/ AIDS is another example of a viral infection, which, though fatal, must be managed, especially since the development of azidothymidine Afterword

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(AZT)—­which staves off death but does not cure—­as a chronic disease. And while this book does not purport to shed any light on the specifics of the many diseases listed above, what it reveals about the nature of conducting viral research—­unexpected interdisciplinary links, for example, and the crossing-­over of techniques—­has implications of continuing relevance for the virology in particular, and medical research and even practice more broadly. History is important, and documenting it is essential if we are to learn from it. The specific protagonists of this tale, bacteriophages and cancer viruses, have both played vital roles in some of the fundamental biological discoveries of the twentieth century and beyond. Both in themselves and as models, these viruses continue to function as the objects of interest in investigations into a variety of problems in clinical medicine as well as basic biology. The development in 2006 of a successful and now virtually routine vaccine against the human papilloma virus—­ the causative agent not only of cervical cancers but also other genital cancers and possibly some cancers of the mouth—­is just one example of a direct product of cancer virus research. The use of bacteriophages as a therapeutic alternative against bacterial infections, especially in the face of increasing antibiotic resistance, has been regaining popularity in the last couple of decades.7 If researchers are to avoid reinventing the wheel in terms of efficiently devising and testing different approaches in the fight against infectious diseases, it behooves them to know about the history of the viruses they attempt to battle. Our job as historians is to provide these histories and perspectives. One more question to add to my aforementioned list would be, “What next?” especially in terms of where the history of viruses is going. As mentioned in the introduction, cancer viruses have already begun to garner interest among historians and philosophers of science, but I suspect that this tale together with other recent titles—­Robin Scheffler’s A Contagious Cause, Erling Norrby’s Nobel Prizes: Cancer, Vision and the Genetic Code, and Anna Marie Skalka’s Discovering Retroviruses—­ are just the opening acts.8 Meanwhile, the bacteriophage has been of interest to nonscientists for well nigh a century, beginning with the Pulitzer Prize–winning Arrowsmith within a decade of its discovery. The publication of Phage and the Origins of Molecular Biology, while not itself the output of historians of science, has given, and continues to give, the historical community much fodder for thought and discussion. Just as the December 2014 special issue of Studies in History and Philosophy of

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Biological and Biomedical Sciences on tumor viruses was the springboard for the histories of cancer virology now in print (including this one), so too, I hope, will the December 2020 special issue of Notes and Records: The Royal Society Journal of the History of Science on bacteriophages spur its participants and others to publish new books on their specific areas of expertise. The resurgence of interest in bacteriophage therapy is, I believe, an especially exciting area for consideration, but there are other fertile and interesting areas of phage research as well. Considering the viruses more generally, I also see a gap—­really just the Janus face of opportunity—­in the history of the development of techniques for virus cultivation. I have no doubt that such a book will be written, and soon, for it is an immensely important piece of the history of virus research and biology more broadly. Dare I hope that the future author is even now reading these words and getting inspired? Graduate students or postdocs reading this book, please take note!

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NOTES Preface 1. Neeraja Sankaran, “Frank Macfarlane Burnet and the Nature of the Bacteriophage, 1924–1937” (PhD diss., Yale University, 2006), 50n32. 2. Plutarch, Plutarch: Lives of the Noble Grecians and Romans, ed. Arthur Hugh Clough (Public Domain Books, 2011), Kindle ed. 3. John Moles, “Plutarch’s Lives,” review of Plutarch and the Historical Tradition, by Philip A. Stadter, Classical Review 43, no. 1 (1993): 30. 4. Sinclair Lewis, Arrowsmith (New York: Harcourt, Brace, 1925). 5. William C. Summers, “On the Origins of the Science in Arrowsmith: Paul de Kruif, Felix d’Herelle, and Phage,” Journal of the History of Medicine and Allied Sciences 46, no. 3 (1991): 315; Howard Markel, “Reflections on Sinclair Lewis’s Arrowsmith: The Great American Novel of Public Health and Medicine,” Public Health Reports 116, no. 4 (2001): 371–75. 6. Joshua Lederberg, “A Tribute to My Mentors,” in Gifted Young in Science: Potential through Performance, ed. Paul F. Brandwein and A. Harry Passow (Washington DC: National Science Teachers Association, 1988), 353–58. 7. Michael Cunningham, The Hours (New York: Farrar, Straus and Giroux, 1998).

Introduction Epigraph: Alexis Carrel, “Mechanism of the Formation and Growth of Malignant Tumors,” Annals of Surgery 82, no. 1 (1925): 4. 1. Charles Dickens, A Tale of Two Cities (Public Domain Books, 2010), 3, Kindle. 2. Sally Smith Hughes, The Virus: A History of the Concept (London: Heinemann Educational Books, 1977). 3. A. P. Waterson and Lise Wilkinson, An Introduction to the History of Virology (Cambridge: Cambridge University Press, 1978); David A. J. Tyrrell,

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“A New Science: What, Why, and How?” British Medical Journal 1, no. 6155 (1979): 45. 4. Gerald Geison, review of The Virus: A History of the Concept, by Sally Smith Hughes, Journal of the History of Medicine and Allied Sciences 33, no. 4 (1978): 562. 5. Tyrrell, “New Science,” 45. 6. See the bibliography for Wilkinson’s writings on the history of virus research. 7. Angela N. H. Creager, The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930–1965 (Chicago: University of Chicago Press, 2002). 8. Creager, Life of a Virus, 2–3, emphasis added. 9. Michael Ruse, review of Heredity Explored: Between Public Domain and Experimental Science, 1850–1930, edited by Staffan Müller-­Wille and Christina Brandt, Journal of the History of the Behavioral Sciences 53, no. 1 (2017): 105. 10. Donald R. Kelley, “Intellectual History and Cultural History: The Inside and the Outside,” History of the Human Sciences 15, no. 2 (2002): 2. 11. See the bibliography for a complete list of Dorothy Crawford’s books. 12. Daniel Kevles and Gerald Geison, “The Experimental Life Sciences in the Twentieth Century,” Osiris, 2nd Series, 10 (1995): 108–18. 13. Robin Wolfe Scheffler, A Contagious Cause: The American Hunt for Cancer Viruses and the Rise of Molecular Medicine (Chicago: University of Chicago Press, 2019); Erling Norrby, Nobel Prizes: Cancer, Vision and the Genetic Code (Hackensack, NJ: World Scientific, 2019); and Anna Marie Skalka, Discovering Retroviruses: Beacons in the Biosphere (Cambridge, MA: Harvard University Press, 2018). The manuscript for the third historical account, “Cancer Virus Hunters: From Chicken Tumors to the HPV Vaccine,” by Gregory Morgan, was under review at the time this book went to press. 14. Neeraja Sankaran, “When Viruses Were Not in Style: Parallels in the Histories of Chicken Sarcoma Viruses and Bacteriophages,” Studies in History and Philosophy of Biological and Biomedical Sciences 48 (2014): 189–99; Robin Wolfe Scheffler, “Managing the Future: The Special Virus Leukemia Program and the Acceleration of Biomedical Research,” Studies in History and Philosophy of Biological and Biomedical Sciences 48 (December 2014): 231–49; and Gregory J. Morgan, “Ludwik Gross, Sarah Stewart, and the 1950s Discoveries of Gross Murine Leukemia Virus and Polyoma Virus,” Studies in History and Philosophy of Biological and Biomedical Sciences 48 (2014): 200–209. 15. To get some idea of the variety and scope of bacteriophage research in the 1920s and 1930s, readers are referred to the comprehensive bibliographic analysis of publications pertaining to the topic by Hansjürgen

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Notes to Pages 6–9

Raettig: Bakteriophagie, 1917 bis 1956: zugleich ein Vorschlag zur Dokumentation wissenschaftlicher Literatur (Stuttgart: G. Fischer, 1958).

Chapter 1: Called or Recalled to Life Epigraphs: Peyton Rous, “A Sarcoma of the Fowl Transmissible by an Agent Separable from the Tumor Cells,” Journal of Experimental Medicine 13, no. 4 (April 1, 1911): 409. Félix d’Herelle, “Sur un microbe invisible antagoniste des bacilles dysentériques,” Comptes rendus de l’Académie des Sciences 145 (1917): 186; Félix d’Herelle, “On an Invisible Microbe That Is Antagonistic to the Dysentery Bacillus,” trans. William Summers, in Félix d’Herelle and the Origins of Molecular Biology, by William C. Summers (New Haven, CT: Yale University Press, 1999), 186. 1. Henceforth referred to as Rockefeller University or simply the Rockefeller. Originally founded in 1901 as the Rockefeller Institute for Medical Research, the institute first changed its name to the Rockefeller Institute to reflect its growing diversification. In 1965, under the aegis of its president Detlev Bronk, it began to offer graduate degrees in the life sciences and was renamed as the Rockefeller University. See the university’s website, https:// www.rucares.org/clinicalresearch/mission-­history. 2. Peyton Rous, “Acceptance of the Kober Medal for 1953,” Transactions of the Association of American Physicians 66, no. 66 (1953): 28; See also George Washington Corner, A History of the Rockefeller Institute, 1901–1953: Origins and Growth (New York: Rockefeller University Press, 1965), 216. 3. Peyton Rous, “A Transmissible Avian Neoplasm (Sarcoma of the Common Fowl),” Journal of Experimental Medicine 12, no. 5 (1910): 697. 4. Rous, “Sarcoma of the Fowl Transmissible,” 408. 5. Rous, “Transmissible Avian Neoplasm,” 705. 6. Rous, “Sarcoma of the Fowl Transmissible,” 397–99, 403, 409. 7. Rous reported these findings in published papers: “An Avian Tumor in Its Relation to the Tumor Problem,” Proceedings of the American Philosophical Society 51, no. 205 (1912): 204; Peyton Rous and James B. Murphy, “The Nature of the Filterable Agent Causing a Sarcoma of the Fowl,” Journal of the American Medical Association, 58, no. 25 (1912): 1938; and in periodic reports to the Rockefeller’s board of directors, specifically “Work of Peyton Rous and James B. Murphy, to 31 May, 1911” and “Work of Peyton Rous, James B. Murphy and W.H. Tytler,” January 10, 1912, both Rockefeller Institute: Reports for the Board of Directors, Peyton Rous Papers, American Philosophical Society, Philadelphia (hereafter cited as Rous Papers). These reports were quite variable in the level of their formality; some were not even typewritten.

Notes to Pages 11–15

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8. Rous and Murphy, “Nature of the Filterable Agent,” 1938. 9. Rous, “Avian Tumor in Its Relation,” 204–5. 10. Peyton Rous, James B. Murphy, and W. H. Tytler, “A Filterable Agent the Cause of a Second Chicken-­Tumor, an Osteochondrosarcoma,” Journal of the American Medical Association 59, no. 20 (1912): 1794; Peyton Rous, and Linda B. Lange, “The Characters of a Third Transplantable Chicken Tumor Due to a Filterable Cause: A Sarcoma of Intracanalicular Pattern,” Journal of Experimental Medicine 18, no. 6 (December 1, 1913): 651–64. 11. Peyton Rous and James B. Murphy, “On the Causation by Filterable Agents of Three Distinct Chicken Tumors,” Journal of Experimental Medicine 19, no. 1 (1914): 68. 12. James B. Murphy, “The Nature of the Filtrable Agent in Chicken Tumours,” in British Empire Cancer Campaign, Report of the International Conference on Cancer, London. 17th–20th July (Bristol: J. Wright, 1928), 33. 13. Rous and Murphy, “Nature of the Filterable Agent,” 1938. 14. Ton van Helvoort, “A Century of Research into the Cause of Cancer: Is the New Oncogene Paradigm Revolutionary?” History and Philosophy of the Life Sciences 21, no. 3 (1999): 300. 15. Rous to Christopher Andrewes, April 21, 1953, Andrewes, Christopher H., folder #3, 1943–1956, Series 1 (B: R77), Rous Papers . 16. Rous to Andrewes, April 21, 1953 Andrewes, folder #3, Rous Papers. 17. Peyton Rous, James B. Murphy, and W. H. Tytler, “The Relation between a Chicken Sarcoma’s Behavior and the Growth’s Filterable Cause,” Journal of the American Medical Association 58, no. 24 (June 1912): 1841; Rous and Murphy, “Nature of the Filterable Agent,” 1938. 18. Rous and Murphy, “Causation by Filterable Agents,” 68. 19. James B. Murphy to Waro Nakaharo, June 22, 1928, Folder Nakaharo, Waro Folder #2, 1928–1948, James B. Murphy Papers (B: M956), American Philosophical Society, Philadelphia (hereafter cited as Murphy Papers). The recipient of this letter, whose name is spelled Nakahara in other literature including an autobiographical essay, was Murphy’s laboratory assistant from 1918 to 1925. Waro Nakahara, “A Pilgrim’s Progress in Cancer Research, 1918 to 1974: Autobiographical Essay,” Cancer Research 34, no. 8 (1974): 1767–74. 20. Rous to Stephen Baker, October 4, 1930, Chicken tumor folder 1, 1930–1932, Rous Papers. 21. Rous to Andrewes, April 21, 1953, Andrewes, folder #3, Rous Papers. 22. Christopher Andrewes, “Francis Peyton Rous. 1879–1970,” Biographical Memoirs of Fellows of the Royal Society 17 (1971): 645–46. 23. Rous, letter to Greer Williams, November 4, 1958, Rous Papers. 24. Rous to Flexner, September 19, 1929, Rous, Peyton, folder #6, January

204

Notes to Pages 15–18

26, 1929–December 30, 1930, Simon Flexner Papers, American Philosophical Society, Philadelphia (hereafter cited as Flexner Papers). 25. Rous to Flexner, November 8, 1929, Flexner, folder #14, 1921–1926, Rous Papers. 26. Flexner to Rous, January 20, 1930, Rous, folder #6, Flexner Papers. 27. Joseph S. Fruton, “The Rockefeller Institute for Medical Research, an Essay Review,” Journal of the History of Medicine and Allied Sciences 21, no. 1 (1966): 71–77. 28. Rous to Joseph Fruton, May 4, 1966, Rous Papers. 29. Fruton to Rous, May 6, 1966, Rous Papers. 30. Rous to Andrewes, April 21, 1953, Andrewes folder #3, Rous Papers. 31. F. W. Twort, “An Investigation on the Nature of Ultra-­Microscopic Viruses,” Lancet 186, no. 4814 (1915): 1241, 1242. 32. Donna H. Duckworth, “‘Who Discovered Bacteriophage?’” Microbiology and Molecular Biology Reviews 40, no. 4 (1976): 794. 33. Twort, “Nature of Ultra-­Microscopic Viruses,” 1242, emphasis in original. 34. Fredrick W. Twort, “The Discovery of the ‘Bacteriophage,’” Lancet 205, no. 5303 (1925): 845; and Gavin Thomas, “Frederick William Twort: Not Just Bacteriophage,” Microbiology Today 41, no. 2 (2014): 72. 35. Carroll G. Bull, “Bacteriophage,” Physiological Reviews 5, no. 1 (1925): 95. 36. W.W. C. Topley, J. Wilson, and E. R. Lewis, “The Role of the Twort-­ d’Herelle Phenomenon in Epidemics of Mouse-­Typhoid,” Epidemiology & Infection 24, no. 1 (1925): 17–36; and Philip Hadley, “The Twort-­D’Herelle Phenomenon: A Critical Review and Presentation of a New Conception (Homogamic Theory) of Bacteriophage Action,” Journal of Infectious Diseases 42, no. 4 (1928): 263–434. 37. d’Herelle, “On an Invisible Microbe.” 38. Hans-­W. Ackermann, Mario Martin, Jean-­F. Vieu, and Pierre Nicolle, “Félix d’Hérelle: His Life and Work and the Foundation of a Bacteriophage Reference Center,” ASM News 48 (1982): 346; Summers, Félix d’Herelle and the Origins, 5. 39. Alain Dublanchet, “La vraie vie de Félix d’Hérelle avant la découverte du bactériophage,” Association des anciens élèves de l’Institut Pasteur 45, no. 175 (2003): 80–82. 40. William Summers, “Félix Hubert d’Herelle (1873–1949): History of a Scientific Mind,” Bacteriophage 6, no. 4 (2016), n.p., https://doi.org/10.1080 /21597081.2016.1270090. 41. Ackermann et al., “Félix d’Hérelle,” 347.

Notes to Pages 19–22

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42. Summers, Félix d’Herelle and the Origins, 5. 43. Félix d’Herelle, “Autobiographie,” unpublished manuscript, circa 1940s, Félix d’Herelle Collection, L’Institut Pasteur, Paris, France, all quoted passages my translations; Duckworth, “Who Discovered Bacteriophage,” 796. 44. Summers, Félix d’Herelle and the Origins, 11–47. 45. D’Herelle, “Autobiographie,” 277, 278, 278–79. 46. D’Herelle, “On an Invisible Microbe.” 47. Duckworth, “Who Discovered Bacteriophage,” 799–800. 48. Twort, “Nature of Ultra-­Microscopic Viruses,” 1243. 49. Félix d’Herelle, F. W. Twort, J. Bordet, A. Gratia, J. C. G. Ledingham, and J. W. McLeod, “Discussion on the Bacteriophage (Bacteriolysin),” British Medical Journal 2, no. 3216 (1922): 290. 50. D’Herelle, “On an Invisible Microbe,” 185, 186. 51. Félix d’Herelle, The Bacteriophage and Its Behavior, trans. George H. Smith (Baltimore: Williams and Wilkins, 1926): 21. 52. Thomas, “Frederick William Twort,” 72. Alan Varley, “Living Molecules or Autocatalytic Enzymes: The Controversy over the Nature of Bacteriophage, 1915–1925” (PhD diss., University of Kansas, 1986), 84. 53. Summers, Félix d’Herelle and the Origins, 61. 54. Duckworth, “Who Discovered Bacteriophage,” 797; 796. 55. Thomas, “Frederick William Twort,” 70. 56. Jules Bordet and Mihai Ciuca, “Remarques sur l’historique des recherches concernant la lyse microbienne transmissible,” Comptes rendus des séances de la Société de Biologie et de ses Filiales Paris 84 (1921): 745–47. 57. Fredrick Twort, “The Ultra-­Microscopic Viruses,” Lancet 198, no. 5108 (1921): 204. 58. Twort, “Discovery of the ‘Bacteriophage,’” 845. The review cited by Twort appeared in the March 14, 1925, issue of the journal. 59. Bordet and Ciuca, “Remarques sur l’historique,” 745, 747, as translated and quoted in Duckworth, “Who Discovered Bacteriophage,” 797. 60. Félix d’Herelle, “Sur l’historique du bactériophage,” Comptes rendus des séances de la Société de Biologie 84 (1921): 863–64. 61. Félix d’Herelle, F. W. Twort, J. Bordet, A. Gratia, J. C. G. Ledingham, and J. W. McLeod, “Discussion on the Bacteriophage (Bacteriolysin),” British Medical Journal 2, no. 3216 (1922): 289–93. 62. Ackermann et al., “Félix d’Hérelle,” 346–47. 63. Ilana Löwy, “Variances in Meaning in Discovery Accounts: The Case of Contemporary Biology,” Historical Studies in the Physical and Biological Sciences 21, no. 1 (1990): 87–121.

206

Notes to Pages 22–29

64. See Ton van Helvoort, “History of Virus Research in the Twentieth Century: The Problem of Conceptual Continuity,” History of Science 32, no. 2 (1994): 190–94; and Neeraja Sankaran, “When Viruses Were Not in Style: Parallels in the Histories of Chicken Sarcoma Viruses and Bacteriophages,” Studies in History and Philosophy of Biological and Biomedical Sciences 48 (2014): 192. 65. Louis Pasteur, “La rage,” in Oeuvres complètes de Pasteur, vol. 6, Maladies virulentes, virus-­vaccins et prophylaxie de la rage (Paris: Masson et Cie, 1933), 673, as quoted in Charles Galperin, “Le bactériophage, la lysogénie et son déterminisme génétique,” History and Philosophy of the Life Sciences 9, no. 2 (1987): 179; S. B. Wolbach, “The Filterable Viruses, a Summary,” Boston Medical and Surgical Journal 167, no. 13 (1912): 419. 66. Löwy, “Variances in Meaning,” 88. 67. Rous to Andrewes, April 21, 1953. 68. Rous to Greer Williams, November 4, 1958, Rous Papers. 69. “The International Cancer Conference,” British Medical Journal 2, no. 2599 (October 22, 1910): 1266–68. 70. Corner, History of the Rockefeller Institute, 216. 71. Eva Becsei-­Kilborn, “Going against the Grain: Francis Peyton Rous (1879–1970) and the Search for the Cancer Virus” (PhD diss., University of Illinois at Chicago, 2003), 95. 72. Simon Flexner and J. W. Jobling, “On Secondary Transplantation of a Sarcoma of the Rat,” Proceedings of the Society for Experimental Biology and Medicine 4, no. 1 (1906): 44–45. 73. Rous to Andrewes, April 21, 1953, Andrewes folder #3, Rous Papers; Becsei-­Kilborn, “Going against the Grain,” 104. The extensive correspondence between Rous and Flexner bears ample evidence of Flexner’s overall positive opinion of Rous’s theories about and work on the sarcoma agent. 74. Rous and Murphy, “Nature of the Filterable Agent,” 1938. 75. Peyton Rous, “Transmission of a Malignant New Growth by Means of a Cell-­Free Filtrate,” Journal of the American Medical Association 56, no. 21 (1911): 198; Rous, “Sarcoma of the Fowl Transmissible”; Rous and Murphy, “Nature of the Filterable Agent.” 76. Rous and Murphy, “Nature of the Filterable Agent,” 1938. 77. Rous, “Avian Tumor in Its Relation,” 204–205. 78. D’Herelle, “On an Invisible Microbe,” 186. 79. d’Herelle, Twort, Bordet, Gratia, Ledingham, and McLeod, “Discussion on the Bacteriophage (Bacteriolysin),” 290–91, 292. 80. Félix d’Herelle, Le bactériophage et son comportement (Paris: Masson et Cie, 1926); Williams & Wilkins Company, advertisement, The Bacteriophage and Its Behavior, by F. d’Herelle, Journal of Immunology 17, no. 4 (1929): 2.

Notes to Pages 29–32

207

81. Ton van Helvoort, “Research Styles in Virus Studies in the Twentieth Century: Controversies and the Formation of Consensus” (PhD diss., University of Limburg, 1993); Ton van Helvoort, “The Construction of Bacteriophage as Bacterial Virus: Linking Endogenous and Exogenous Thought Styles,” Journal of the History of Biology 27, no. 1 (Spring 1994); Sankaran, “When Viruses Were Not in Style.”

Chapter 2: Epochs of Incredulity and Belief Epigraphs: James B. Murphy to Waro Nakahara, June 22, 1928, Folder Nakaharo [sic], Waro #2, 1928–1948, James B. Murphy Papers (B: M956), American Philosophical Society, Philadelphia (hereafter cited as Murphy Papers). Jules Bordet, “Croonian Lecture: The Theories of the Bacteriophage,” Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character 107, no. 752 (1931): 404. 1. See Peyton Rous and James B. Murphy, “The Nature of the Filterable Agent Causing a Sarcoma of the Fowl,” Journal of the American Medical Association 58, no. 25 (1912): 1938; and Peyton Rous, “An Avian Tumor in Its Relation to the Tumor Problem,” Proceedings of the American Philosophical Society 51, no. 205 (1912): 204. 2. F. W. Twort, “An Investigation on the Nature of Ultra-­Microscopic Viruses,” Lancet 186, no. 4814 (1915): 1241–43; F. d’Herelle, “Sur un microbe invisible antagoniste des bacilles dysentériques,” Comptes rendus de l’Académie des Sciences 145 (1917): 373–75; Félix d’Herelle, F. W. Twort, J. Bordet, A. Gratia, J. C. G. Ledingham, and J. W. McLeod, “Discussion on the Bacteriophage (Bacteriolysin),” British Medical Journal 2, no. 3216 (1922): 290–91; F. W. Twort, “The Bacteriophage: The Breaking Down of Bacteria by Associated Filter-­Passing Lysins,” British Medical Journal 2, no. 3216 (1922): 293–97; and d’Herelle, Le bactériophage et son comportement (Paris: Masson et Cie, 1926). 3. “Peyton Rous—­Nobel Lecture: The Challenge to Man of the Neoplastic Cell,” accessed January 28, 2016, http://www.nobelprize.org/nobel _prizes/medicine/laureates/1966/rous-­lecture.html. 4. As quoted in Alan W. Varley, “Living Molecules or Autocatalytic Enzymes: The Controversy over the Nature of Bacteriophage, 1915–1925” (PhD diss., University of Kansas, 1986), 84. The person d’Herelle was referring to here was Émile Roux, a French physician, microbiologist, immunologist, a founding member of the Pasteur Institute in Paris, and its director from 1904 to 1933, not to be confused in any way with Peyton Rous. 5. Peyton Rous, Letter to Robert Parsons, August 22, 1967, Folder P-­Q , Nobel Prize—­congratulatory letters (1966–67), Series 1 (B: R77), Peyton Rous

208

Notes to Pages 32–34

Papers, American Philosophical Society, Philadelphia (hereafter cited as Rous Papers). 6. “The International Cancer Conference,” British Medical Journal 2, no. 2599 (October 22, 1910), 1266. 7. Roswell Park, “The Nature of the Cancerous Process,” Journal of the American Medical Association 37, no. 11 (1901): 672. 8. H. G. Plimmer, “The Parasitic Theory of Cancer,” British Medical Journal 2, no. 2241 (1903): 1511. 9. Henry Morris, “The Bradshaw Lecture on Cancer and Its Origin,” British Medical Journal 2, no. 2241 (1903): 1505–6. 10. James Ewing, “Cancer Problems,” Archives of Internal Medicine 1, no. 2 (1908): 180; 179. 11. James B. Murphy to Waro Nakaharo, June 22, 1928, Nakaharo, folder #2, Murphy Papers. 12. Jacob Furth, “The Making and Missing of Discoveries: An Autobiographical Essay,” Cancer Research 36, no. 3 (1976): 873. 13. James Henderson, “A View from a Center of a World,” in A Notable Career in Finding Out: Peyton Rous, 1879–1970, by James S. Henderson, Phillip D. McMaster, John G. Kidd, and Charles Huggins (New York: Rockefeller University Press, 1971), 11. 14. Christopher H. Andrewes, “Francis Peyton Rous. 1879–1970,” Biographical Memoirs of Fellows of the Royal Society 17 (1971): 643, 653. 15. C. C. Little, “James Bumgardner Murphy, August 4, 1884–August 24, 1950,” Biographical Memoirs of the National Academy of Science 38 (1960): 191. 16. Simon Flexner to James B. Murphy, April 26, 1923, Murphy, James B., folder 6, Jan 1923—­Dec 22, 1924, Simon Flexner Papers, American Philosophical Society, Philadelphia (hereafter cited as Flexner Papers). 17. Eva Becsei-­Kilborn, “Scientific Discovery and Scientific Reputation: The Reception of Peyton Rous’ Discovery of the Chicken Sarcoma Virus,” Journal of the History of Biology 43, no. 1 (2010): 112, 113–14. 18. Eva Becsei-­Kilborn, “Going against the Grain: Francis Peyton Rous (1879–1970) and the Search for the Cancer Virus” (PhD diss., University of Illinois at Chicago, 2003). 19. Rous to Andrewes, April 21, 1953, Andrewes, Christopher H. Folder #3, 1943–1956, Rous Papers. 20. Park, “Nature of the Cancerous Process,” 671. For further evidence regarding Park’s views on this matter based on a wide reading of the literature of his times, see also Roswell Park, “An Inquiry into the Etiology of Cancer, With Some Reference to the Latest Investigations of the Ital-

Notes to Pages 35–38

209

ian Pathologists,” American Journal of the Medical Sciences 115, no. 5 (1898): 503–19. 21. A. Fujinami and K. Inamoto, “Ueber Geschwülste bei japanischen Haushühnern, insbesondere über einen transplantablen Tumor,” Journal of Cancer Research and Clinical Oncology 14, no. 1 (1914): 94–119. In translation: “On tumors of the Japanese domestic fowl, particularly on a transplantable tumor.” At the time of the publication of this paper the journal was known as Zeitschrift für Krebsforschung. 22. Peyton Rous, James B. Murphy, and W. H. Tytler, “Transplantable Tumors of the Fowl: A Neglected Material for Cancer Research,” Journal of the American Medical Association 58, no. 22 (1912): 1683. 23. A. Fujinami and S. Hatano, “Contribution of the Pathology of Heterotransplantation of Tumor. A Duck Sarcoma from Chicken Sarcoma,” Gann 23 (1929): 67–75; and A. Fujinami and K. Suzue, “Contribution to the Pathology of Tumor Growth. Experiments in the Growth of Chicken Sarcoma in the Case of Heterotransplantation,” Transactions of the Japanese Pathological Society 18 (1928): 616–22. 24. “Cancer Research,” British Medical Journal 1, no. 2626 (1911): 1008. 25. Ewing, “Cancer Problems,” 180–81. 26. Peyton Rous, “A Sarcoma of the Fowl Transmissible by an Agent Separable from the Tumor Cells,” Journal of Experimental Medicine 13, no. 4 (1911): 409. 27. James B. Murphy, “James Ewing, 1866–1943,” Biographical Memoirs of the National Academy of Science 26 (1951): 48. 28. Willy Meyer, “Some Notes on Cancer with Special Reference to the Parasitic Theory,” American Journal of Cancer 8 (1924): 49. 29. Andrewes, “Francis Peyton Rous,” 644. 30. E. F. Bashford,“An Address Entitled ‘Are the Problems of Cancer Insoluble?’” British Medical Journal 2, no. 2345 (1905): 1509–10. 31. Charles Oberling, The Fight against Cancer (London: A. Deutsch, 1961), 33. 32. E. F. Bashford,“Is Cancer Infective?” Nature 91, no. 2282 (1913): 533. 33. Correspondence on “Chicken tumors,” 1913–1945, 14 folders, box 10, Rous Papers. 34. Stephen Baker to Rous, September 9, 1930, Chicken tumor folder 1, 1930–1932, Rous Papers. 35. Rous to Baker, October 4, 1930, Chicken tumor folder 1, 1930–1932, Rous Papers. 36. William E. Gye, “The Aetiology of Malignant New Growths,” Lancet 206, no. 5316 (1925): 109, 117.

210

Notes to Pages 38–42

37. Rous to Gye, July 24, 1925, Gye, William E., folder #1 1923–1925, Rous Papers. 38. The second paper is: J. E. Barnard, “The Microscopical Examination of Filterable Viruses, Associated with Malignant New Growths,” Lancet 206, no. 5316 (1925): 117–23. 39. Francis Carter Wood, “The New Cancer Germ,” New Republic, August 12, 1925, 310, 311. 40. Rous to Gye, July 24, 1925, Gye, folder #1, Rous Papers. 41. James B. Murphy,“Certain Etiological Factors in the Causation and Transmission of Malignant Tumors,” American Naturalist 60, no. 668 (1926): 230. Also quoted in a report on the meeting in the December 25, 1925, issue of the New York Times. 42. American Society for the Control of Cancer, Cancer Control: Report of an International Symposium Held under the Auspices of the American Society for the Control of Cancer, Lake Mohonk, New York, September 20-­24, 1926 (Chicago: Surgical Publishing, 1927), 327. 43. Thomas M. Rivers and Saul Benison. Tom Rivers: Reflections on a Life in Medicine and Science (Cambridge: MIT Press, 1967), vii. The specific person to dub Rivers the “father” was the Harvard-­based virologist John Enders, himself a recipient of the 1954 Nobel Prize in Physiology or Medicine for his contributions toward cultivating viruses. 44. T. M. Rivers, “Some General Aspects of Pathological Conditions Caused by Filterable Viruses,” American Journal of Pathology 4, no. 2 (1928): 111. Emphasis added. 45. Bordet, “Croonian Lecture,” 401. 46. Hansjürgen Raettig, Bakteriophagie, 1917 bis 1956: zugleich ein Vorschlag zur Dokumentation wissenschaftlicher Literatur (Stuttgart: G. Fischer, 1958); Varley, “Living Molecules or Autocatalytic Enzymes,” 84–135; Summers, Félix d’Herelle and the Origins of Molecular Biology (New Haven, CT: Yale University Press, 1999), 60–81. 47. Tamezo Kabeshima, “Sur un ferment d’immunité bactériolysant, du mécanisme d’immunité infectieuse intestinale, de la nature du dit ‘microbe filtrant bactériophage’ de d’Herelle,” Comptes rendus des séances de la Société de Biologie et des ses filiales Paris 83 (1920): 219–21. Translations mine. 48. Tamezo Kabeshima, “Sur le ferment d ‘immunité bactériolysant,” Comptes rendus des séances de la Société de Biologie et des ses filiales Paris 83 (1920): 471–73. 49. Jules Bordet and Mihai Ciuca, “Exsudats leucocytaires et autolyse microbienne,” Comptes rendus des séances de la Société de Biologie et des ses filiales Paris 83 (1920): 1293–95; Bordet and Ciuca, “Le bactériophage de d’Herelle,

Notes to Pages 42–46

211

sa production et son interprétation,” Comptes rendus des séances de la Société de Biologie 83 (1920): 1296–98. 50. Varley, “Living Molecules or Autocatalytic Enzymes,” 108; Ton van Helvoort, “Bacteriological and Physiological Research Styles in the Early Controversy on the Nature of the Bacteriophage Phenomenon,” Medical History 36, no. 3 (1992): 253; Summers, Félix d’Herelle and the Origins, 64. 51. Félix d’Herelle, “On an Invisible Microbe That Is Antagonistic to the Dysentery Bacillus,” trans. William Summers, in Summers, Félix d’Herelle and the Origins, 186. 52. “The Nobel Prize in Physiology or Medicine 1919,” The Nobel Prize, http://www.nobelprize.org/nobel_prizes/medicine/laureates/1919/. 53. Félix d’Herelle, Le bactériophage; son rôle dans l’immunité (Paris: Masson et Cie, 1921). 54. Félix d’Herelle, The Bacteriophage: Its Role in Immunity, trans. George H. Smith (Baltimore: Waverly, 1922): 165, 167, 169. 55. Much of the bench work on these experiments was most probably carried out by Ciuca, as Bordet himself was in the United States on a mission from the Free University of Brussels for much of this time. Varley, “Living Molecules or Autocatalytic Enzymes,” 110. 56. Bordet and Ciuca, “Le bactériophage de d’Herelle,” 1296–97. Translation mine. 57. André Lwoff, “Lysogeny,” Bacteriological Reviews 17, no. 4 (1953): 276. 58. Varley, “Living Molecules or Autocatalytic Enzymes,” 115. 59. Jules Bordet, “The Cameron Prize Lecture on Microbic Transmissible Autolysis,” British Medical Journal 1, no. 3240 (1923): 175. 60. Jules Bordet, “Concerning the Theories of the So-­Called Bacteriophage,” British Medical Journal 2, no. 3216 (1922): 296. Paper read by Gratia. 61. Summers, Félix d’Herelle and the Origins, 66. 62. Félix d’Herelle and G. Eliava, “Sur le serum antibactériophage,” Comptes rendus des séances de la Société de Biologie Paris 84 (1921): 719–20; Summers, Félix d’Herelle and the Origins, 69. 63. Jules Bordet and Mihai Ciuca, “Remarques sur l’historique des recherches concernant la lyse microbienne transmissible,” Comptes rendus des séances de la Société de Biologie et de ses filiales Paris 84 (1921): 745–47. 64. Félix d’Herelle, “Sur l’historique du bactériophage,” Comptes rendus des séances de la Société de Biologie 84 (1921): 863, 864. 65. Donna Duckworth, “‘Who Discovered Bacteriophage?’” Microbiology and Molecular Biology Reviews 40, no. 4 (1976): 798. 66. André Gratia, “Studies on the d’Herelle Phenomenon,” Journal of Experimental Medicine 34, no. 1 (1921): 121.

212

Notes to Pages 46–49

67. Bordet, “Cameron Prize Lecture,” 177–78. 68. Bordet and Ciuca, “Le bactériophage de d’Herelle.” 69. Varley, “Living Molecules or Autocatalytic Enzymes,” 118; Neeraja Sankaran, “Stepping-­Stones to One-­Step Growth: Frank Macfarlane Burnet’s Role in Elucidating the Viral Nature of the Bacteriophages,” Historical Records of Australian Science 19, no. 1 (2008): 93. 70. Jules Bordet, “Le problème de l’autolyse microbienne transmissible ou du bactériophage,” Annales de l’Institut Pasteur 39 (1925): 711–63, as interpreted by Lwoff in “Lysogeny,” 276. 71. Félix d’Herelle, The Bacteriophage and Its Behavior, trans. George Hathorn Smith (Baltimore: Williams and Wilkins, 1926), 209–11. 72. Alfons Billiau, “At the Centennial of the Bacteriophage: Reviving the Overlooked Contribution of a Forgotten Pioneer, Richard Bruynoghe (1881–1957),” Journal of the History of Biology (2016): 567–71. 73. Joseph Maisin and Richard Bruynoghe, “Adaptation du bactériophage,” Comptes rendus des séances de la Société de Biologie 84 (1921): 468–70. 74. Richard Bruynoghe, “Au sujet de la nature des bactériophages—­II,” Comptes rendus des séances de la Société de Biologie 85 (1921): 259, as translated in Billiau, “At the Centennial,” 567–68. 75. Joseph Maisin, and Richard Bruynoghe, “Au sujet du principe bactériophage et des anticorps,” Comptes rendus des séances de la Société de Biologie 84 (1921): 756. Translation mine. 76. R. Bruynoghe and R. Appelmans, “La neutralisation des bactériophages de provenance différente,” Comptes rendus des séances de la Société de Biologie 86 (1922): 98, as translated in Billiau, “At the Centennial,” 568. 77. R. Bruynoghe and J. Maisin, “Au sujet des microbes devenus résistants au principe bactériophage,” Comptes rendus des seances de la Société de Biologie 84 (1921): 847–48; Billiau, “At the Centennial,” 569. 78. R. Bruynoghe, “Contribution à l’étude de la nature des bactériophages,” Bulletin de l’Académie Royale de Médecine de Belgique 3 (1923), as translated in Billiau, “At the Centennial,” 570. 79. Billiau, “At the Centennial,” 576–77. 80. Alfons Billiau, “100 Years Ago: Discovery of the Bacteriophage(s) Part II: Richard Bruynoghe versus Jules Bordet in the Bacteriophage Debate,” Quarterly Newsletter of the Belgian Society for Microbiology, no. 11 (September 2015): 6–9. 81. Jean-­Pierre Gratia, “André Gratia: A Forerunner in Microbial and Viral Genetics,” Genetics 156, no. 2 (2000): 471–72. 82. André Gratia, in Félix d’Herelle, F. W. Twort, J. Bordet, A. Gratia, J. C. G. Ledingham, and J. W. McLeod, “Discussion on the Bacteriophage (Bacte-

Notes to Pages 49–52

213

riolysin),” British Medical Journal 2, no. 3216 (1922): 296. Nearly a decade later he reiterated this statement in French. See “André Gratia, Phénomène de Twort et bactériophagie,” Annales de l’Institut Pasteur 46 (1931): 219. 83. Gratia, “Studies on the d’Herelle Phenomenon,” 115; André Gratia, “Mis au point de quelques notions de bactériophagie,” Bruxelles-­Medical 11 (1931): 697. 84. Gratia, “André Gratia,” 472. 85. “Eugène Wollman (1883–1943)—­Notice Biographique,” Archives de l’Institut Pasteur, accessed February 24, 2017, https://webext.pasteur.fr /archives/wll0.html. 86. Eugène Wollman, “Recherches sur la bactériophagie (phénomène de Twort-­d’Herelle),” Annales de l’Institut Pasteur 39 (1925): 789; See also Charles Galperin, “Le bactériophage, la lysogénie et son déterminisme génétique,” History and Philosophy of the Life Sciences 9, no. 2 (1987): 179. Translation mine. 87. Eugène Wollman, “The Phenomenon of Twort-­d’Herelle and Its Significance,” Lancet 226, no. 5858 (1935): 1312. 88. Raettig, Bakteriophagie, 2. 89. F. M. Burnet, “A Method for the Study of Bacteriophage Multiplication in Broth,” British Journal of Experimental Pathology 10, no. 2 (1929): 109–15. 90. A. P. Waterson and Lise Wilkinson, An Introduction to the History of Virology (Cambridge: Cambridge University Press, 1978), 78. 91. Thomas M. Rivers, “The Nature of Viruses,” Physiological Reviews 12, no. 3 (1932): 423. 92. See, for example, the variety of suggestions enumerated by Burnet in “Method for the Study,” 109–10. 93. Bordet, “Croonian Lecture,” 404. 94. See Joseph Arkwright, “Variation,” in A System of Bacteriology in Relation to Medicine (London: His Majesty’s Stationery Office, 1930), 318. 95. Albert Claude and James B. Murphy, “Transmissible Tumors of the Fowl,” Physiological Reviews 13, no. 2 (1933): 261. 96. Bordet, “Cameron Prize Lecture,” 176; Claude and Murphy, “Transmissible Tumors of the Fowl,” 259. 97. James Ewing, “The Causal and Formal Genesis of Cancer,” in Report of the International Conference on Cancer. London, 17th–20th July, 1928, by British Empire Cancer Campaign (Bristol: J. Wright, 1928), 1, 12. 98. Bordet, “Cameron Prize Lecture,” 176. 99. Claude and Murphy, “Transmissible Tumors of the Fowl,” 261.

214

Notes to Pages 52–56

Chapter 3: What Was a Virus? Epigraphs: Louis Pasteur, “La rage,” in Oeuvres complètes de Pasteur, vol. 6, Maladies virulentes, virus-­vaccins et prophylaxie de la rage (Paris: Masson et Cie, 1933), 673, as quoted in Charles Galperin, “Le bactériophage, la lysogénie et son déterminisme génétique,” History and Philosophy of the Life Sciences no. 9, no. 2 (1987): 179. Translated, the quotation reads, “In summary, every virus is a microbe.” Earl B. McKinley,“A Concept of the Ultramicroscopic Virus Diseases and a Classification,” Science 76, no. 1977 (1932): 450. 1. Ilana Löwy, “Variances in Meaning in Discovery Accounts: The Case of Contemporary Biology,” Historical Studies in the Physical and Biological Sciences 21, no. 1 (1990): 87–121. 2. Felix d’Herelle, “The Nature of the Ultrafilterable Viruses,” Harvey Lectures 24 (1928): 51. 3. James Ewing to Peyton Rous, October 7, 1935, Ewing, James 1924-­1936, Series 1 (B: R77), Peyton Rous Papers, American Philosophical Society, Philadelphia (hereafter cited as Rous Papers). 4. William C. Summers, “Inventing Viruses,” Annual Review of Virology 1, no. 1 (2014): 26; Marian C. Horzinek,“The Birth of Virology,” Antonie van Leeuwenhoek 71, no. 1 (1997): 15. 5. Edward Jenner, “An Inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Discovered in Some of the Western Counties of England, Particularly Gloucestershire, and Known by the Name of The Cow Pox,” 1798, abridged and reprinted in Milestones in Microbiology, ed. Thomas D. Brock (Washington, DC: American Society for Microbiology, 1961), 121–25. 6. Summers, “Inventing Viruses,” 26. 7. S. B. Wolbach, “The Filterable Viruses, a Summary,” Boston Medical and Surgical Journal 167, no. 13 (1912): 419. 8. F. M. Burnet, “Virology as an Independent Science Lecture II: The Substance of Virology,” Medical Journal of Australia 2, no. 22 (1953): 841. 9. Lute Bos, “Beijerinck’s Work on Tobacco Mosaic Virus: Historical Context and Legacy,” Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 354, no. 1383 (1999): 684, emphasis in original. 10. Peyton Rous, letter to James Ewing, October 3, 1935. 11. Thomas M. Rivers and Saul Benison, Tom Rivers: Reflections on a Life in Medicine and Science (Cambridge: MIT Press, 1967), 88. Emphasis added. 12. Thomas Rivers, preface to Filterable Viruses, ed. T. M. Rivers (Baltimore: Williams & Wilkins, 1928), ix; See Ton van Helvoort, “When Did Virology Start?” ASM News 62, no. 3 (1996): 142, for a discussion on the beginnings of virology.

Notes to Pages 57–60

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13. Rivers and Benison, Tom Rivers, 116. 14. Bos, “Beijerinck’s Work,” 676. 15. Angela Creager, The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930–1965 (Chicago: University of Chicago Press, 2002), 5; Heinz Fraenkel-­Conrat, “Portraits of Viruses: Tobacco Mosaic Virus,” Intervirology 15, no. 4 (1981): 177. 16. Creager, Life of a Virus, 9; W. M. Stanley, “Isolation of a Crystalline Protein Possessing the Properties of Tobacco-­Mosaic Virus,” Science 81, no. 2113 (1935); G. A. Kausche, E. Pfankuch, and H. Ruska, “Die Sichtbarmachung von pflanzlichem Virus im Übermikroskop,” Naturewissenschaften 27, no. 18 (1939): 292–99; Fraenkel-­Conrat, “Portraits of Viruses,” 177. 17. Adolf Mayer,“Over de Mozaikziekte van de Tabak; Voorloopige Mededeeling,” Tijdschrift voor Landbouwkunde 2 (1882). 18. Adolf Mayer, “Over de in Nederland dikwijls voorkommende Mozaikziekte der Tabak,” Landbouwkundig Tijdschrift (1885), as cited in Lise Wilkinson, “The Development of the Virus Concept as Reflected in Corpora of Studies on Individual Pathogens 3. Lessons of the Plant Viruses—­Tobacco Mosaic Virus,” Medical History 20, no. 2 (1976): 114. 19. Adolf Mayer, “Concerning the Mosaic Disease of Tobacco,” Phytopathology Classics 7 (1942): 20, 24. 20. C. Chamberland, “Sur un filtre donnant de l’eau physiologiquement pure,” Compte rendu hebdomadaire des séances de l’Académie des Sciences 99 (1884): 247–48; A. P. Waterson and Lise Wilkinson, An Introduction to the History of Virology (Cambridge: Cambridge University Press, 1978): 15–17. 21. Dmitrii M. Iwanowski, “On the Mosaic Disease of the Tobacco Plant,” Phytopathology Classics 7 (1942): 27–30. Emphasis in original translation. The name of this researcher has been variously spelled in English by different authors as: Ivanovski (Wilkinson, 1976; van Helvoort, 1991); Ivanovsky (Bos, 1999); Ivanowski (Lechevalier, 1972; Duckworth, 1976), and Iwanowsky (Rivers and Benison, 1967). 22. Emile Roux, and Alexandre Yersin, “Contribution à l’étude de la diphthérie,” Annales de l’Institut Pasteur, Paris 2 (1888) as quoted in Wilkinson, “3. Lessons of the Plant Viruses,” 115. 23. Beijerinck, Über ein Contagium vivum fluidum als Ursache der Fleckenkrankheit der Tabaksblätter,” Verhandelingen der Koninklyke akademie van Wettenschappen te Amsterdam 5 (1898): 3–21; “Über ein Contagium vivum fluidum als Ursache der Fleckenkrankheit der Tabaksblatter,” Centralblatt fur Bacteriologie und Parasitenkunde, part 2, 5 (1899): 27–33. 24. Dmitrii M. Iwanowski,“Mozaichnaia bolezn tabaka” (PhD diss., Uni-

216

Notes to Pages 60–62

versity of Warsaw, 1902), as quoted in H. Lechevalier, “Dmitri Iosifovich Ivanovski (1864–1920),” Bacteriological Reviews 36, no. 2 (1972): 140. 25. As cited in Bos, “Beijerinck’s Work,” 678. 26. Martinus W. Beijerinck, “Concerning a Contagium Vivum Fluidum as the Cause of the Mosaic Disease of Tobacco Leaves” (1899), translated by Thomas D. Brock, in Milestones in Microbiology: 1546 to 1940, by Thomas D. Brock (Washington, DC: American Society for Microbiology Press, 1961), 155, 154. At the time, contagium fixum was the description given to a bacterium that was a “fixed,” or organized, living particle. 27. Albert J. Kluyver, “Beijerinck, The Microbiologist,” in Martinus Willem Beijerinck: His Life and His Work, ed. Gerrit Van Iterson, L. E. den Dooren de Jong, and Albert Jan Kluyver (1940, repr., Dordrecht: Springer, 2013), 119. 28. Bos, “Beijerinck’s Work,” 678. 29. Creager, Life of a Virus, 23, emphasis added. 30. Dmitrii M. Iwanowski, “Über die Mosaikkrankheit der Tabakspflanze,” Zentralblatt für Bakteriologie Parasitenkunde, Infektionskrankheiten und Hygiene 7, no. 4 (1901): 148, as quoted in Lechevalier, “Dmitri Iosifovich Ivanovski,” 141. 31. Dmitrii M. Iwanowski, “Über die Mosaikkrankheit der Tabakspflanze,” Zeitschrift für Pflanzenkrankheit und Pflanzenschutz 13 (1903): 1–41, as quoted in Horzinek, “Birth of Virology,” 19–20. 32. Iwanowski, “Über die Mosaikkrankheit,” as quoted in Lechevalier, “Dmitri Iosifovich Ivanovski,” 140–41. 33. Friedrich Löffler and Paul Frosch, “Report of the Commission for Research on the Foot-­and-­Mouth Disease,” in Milestones in Microbiology: 1546 to 1940, ed. and trans. Thomas D. Brock (Washington, DC: American Society for Microbiology, 1961), 152. Emphasis added. 34. Beijerinck, “Concerning a Contagium Vivum Fluidum as the Cause,” 37. 35. Fredrick A. Murphy, “Historical Perspective: What Constitutes Discovery (of a New Virus)?” in Advances in Virus Research, vol. 95, edited by Margaret Kielian, Karl Maramorosch, and Thomas Mettenleiter (New York: Academic Press, 2016): 199. 36. Creager, Life of a Virus, 45. 37. See Chamberland, “Sur un filtre donnant.” 38. Murphy, “What Constitutes Discovery,” 198. 39. E. Roux, “Sur les microbes dits ‘invisibles,’” Bulletin de l’Institut Pasteur 1 (1903): 7, as quoted in Charles Galperin, “Le bactériophage, la lysogénie,” 180. Translation mine.

Notes to Pages 62–66

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40. Peyton Rous, “A Sarcoma of the Fowl Transmissible by an Agent Separable from the Tumor Cells,” Journal of Experimental Medicine 13, no. 4 (1911): 397–411; Peyton Rous, “Transmission of a Malignant New Growth by Means of a Cell-­Free Filtrate,” Journal of the American Medical Association 56, no. 21 (1911): 198. 41. Walter E. King, F. W. Baeslack, and George L. Hoffmann, “Studies on the Virus of Hog Cholera,” Journal of Infectious Diseases 12, no. 2 (1913): 206. 42. Thomas M. Rivers, “Viruses,” Journal of the American Medical Association 92, no. 14 (1929): 1147. 43. Thomas M. Rivers, ed., Filterable Viruses (Baltimore: Williams & Wilkins, 1928). 44. Rivers and Benison, Tom Rivers, 116. 45. Stuart Mudd, “Filters and Filtration,” in Rivers, ed., Filterable Viruses, 55, 74. 46. Rivers and Benison, Tom Rivers, 116–17. 47. F. W. Twort, “An Investigation on the Nature of Ultra-­Microscopic Viruses,” Lancet 186, no. 4814 (1915): 1241–43. 48. Ton van Helvoort, “What Is a Virus? The Case of Tobacco Mosaic Disease,” Studies in History and Philosophy of Science 22, no. 4 (1991): 570. 49. Charles Joseph Singer, The Development of the Doctrine of Contagium Vivum, 1500–1750; A Preliminary Sketch (N.p.: Privately printed, 1913), 3. 50. Ludwik Fleck, Genesis and Development of a Scientific Fact, ed. T. J. Trenn and Robert K. Merton, trans. T. J. Trenn and F. Bradley (Chicago: University of Chicago Press, 1979), 24. 51. Ludwik Fleck,“Crisis in Science,” in Cognition and Fact: Materials on Ludwik Fleck, ed. Robert S. Cohen and Thomas Schnelle (Boston: D. Reidel, 1986), 155. 52. Ilana Löwy, “Ludwik Fleck on the Social Construction of Medical Knowledge,” Sociology of Health & Illness 10, no. 2 (1988): 135. 53. Fleck, Genesis and Development, 23, 25, 39, emphasis in original; Stig Brorson, “Ludwik Fleck on Proto-­Ideas in Medicine,” Medicine, Health Care and Philosophy 3, no. 2 (2000): 148. 54. Ton van Helvoort, “Bacteriological and Physiological Research Styles in the Early Controversy on the Nature of the Bacteriophage Phenomenon,” Medical History 36, no. 3 (1992): 243–44. 55. Nicola Mößner, “Thought Styles and Paradigms: A Comparative Study of Ludwik Fleck and Thomas S. Kuhn,” Studies in History and Philosophy of Science Part A 42, no. 3 (2011): 421. 56. Gerald Geison, “The Protoplasmic Theory of Life and the Vitalist-­ Mechanist Debate,” Isis 60, no. 3 (1969): 273–92.

218

Notes to Pages 66–70

57. Beijerinck, “Concerning a Contagium Vivum Fluidum as the Cause,” 154; Lechevalier, “Dmitri Iosifovich Ivanovski,” 141. 58. Kluyver, “Beijerinck, the Microbiologist,” 120. The name of the academy in Dutch is Koninklijke Nederlandse Akademie van Wetenschappen. 59. Thomas Henry Huxley, On the Physical Basis of Life (New Haven, CT: Charles C. Chatfield, 1870), 5. 60. van Helvoort, “What Is a Virus?” 558. 61. van Helvoort, “What Is a Virus?” 595. 62. Laurent Loison, Jean Gayon, and Richard M. Burian, “The Contributions—­and Collapse—­of Lamarckian Heredity in Pasteurian Molecular Biology: 1. Lysogeny, 1900–1960,” Journal of the History of Biology 50, no. 1 (2017): 9. 63. Lechevalier, “Dmitri Iosifovich Ivanovski,” 141. 64. Karen-­Beth Scholthof, “Tobacco Mosaic Virus: The Beginnings of Plant Virology,” Plant Health Instructor, 1997, https://doi.org/10.1094/PHI -­I-­2000-­1010-­01. 65. Albert Fred Woods, “Observations on the Mosaic Disease of Tobacco,” Washington DC: Bureau of Plant Industry, USDA, 1902, http://agris.fao .org/agris-­search/search.do?recordID=US201300010315; Scholthof, “Beginnings of Plant Virology.” 66. B. D. Harrison and T. M. A. Wilson, “Milestones in the Research on Tobacco Mosaic Virus,” Philosophical Transactions: Biological Sciences 354, no. 1383 (1999): 522. Helen A. Purdy, “Immunologic Reactions with Tobacco Mosaic Virus,” Proceedings of the Society for Experimental Biology and Medicine 25, no. 8 (1928): 702–3; H. H. McKinney,“Mosaic Diseases in the Canary Islands, West Africa and Gibraltar,” Journal of Agricultural Research 39 (1929): 577–78; Francis O. Holmes, “Local Lesions in Tobacco Mosaic,” Botanical Gazette 87, no. 1 (1929): 39–55. 67. Scholthof, “Beginnings of Plant Virology.” 68. Peyton Rous and James Murphy, “The Nature of the Filterable Agent Causing a Sarcoma of the Fowl,” Journal of the American Medical Association 58, no. 25 (1912): 1938. 69. Felix d’Herelle, “On an Invisible Microbe That Is Antagonistic to the Dysentery Bacillus,” trans. William Summers, in Felix d’Herelle and the Origins of Molecular Biology, by William C. Summers (New Haven, CT: Yale University Press, 1999), 185–86. 70. As quoted by Kluyver in Gerrit Van Iterson et al., Martinus Willem Beijerinck, 21. Although the biography was in English, the authors quoted d’Herelle’s speech in its original French. Translation mine. 71. Martinus W. Beijerinck, “Pasteur En de Ultramicrobiologie,” Chemicsh

Notes to Pages 70–74

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Weekblad 19 (1922): 525–27; Van Iterson et al., Martinus Willem Beijerinck, 43. 72. Félix d’Herelle, “Bacteriophage: A Living Colloidal Micell,” in Colloid Chemistry: Theoretical and Applied, vol. 2, Biology and Medicine, ed. Jerome Alexander (New York: Chemical Catalog, 1928). 73. Bos, “Beijerinck’s Work,” 682. 74. Wendell M. Stanley, “The Nature of Viruses,” Transactions of the New York Academy of Sciences 1, no. 2, series II (1938): 21. 75. Rivers and Benison, Tom Rivers, 115–16. 76. Jean Witz, “A Reappraisal of the Contribution of Friedrich Loeffler to the Development of the Modern Concept of Virus,” Archives of Virology 143, no. 11 (1998): 2262; M. H. Van Regenmortel,“Nature of Viruses,” Encyclopedia of Virology, 3rd ed., edited by Brian W. J. Mahy and Marc H. V. Van Regenmorte (Amsterdam: Academic Press, 2008): 399; Murphy, “What Constitutes Discovery,” 199. 77. L. Bos,“The Embryonic Beginning of Virology: Unbiased Thinking and Dogmatic Stagnation,” Archives of Virology 140, no. 3 (1995): 618. 78. Bos, “Beijerinck’s Work,” 683. 79. Gregor Mendel, “Versuche über Plflanzen-­Hybriden,” Verhandlungen des naturforschenden Vereines in Brünn 4 (1865): 3–47; Hugo de Vries,“Sur la loi do disjonction des hybrides,” Comptes rendus de l’Académie des Sciences 130 (1900): 845–47; “Sur les unités des caractères spécifiques et leur application à l’étude des hybrides,” Revue générale de botanique 12, no. 119001 (1900): 259–71; Carl Correns, “G. Mendel’s Regel über das Verhalten der Nachkommenschaft der Rassenbastarde,” Berichte der Deutschen Botanischen Gesellschaft 18, no. 4 (1900): 158–68; Eric Tschermak, “Ueber künstliche Kreuzung bei Pisum sativum,” Berichte der Deutschen Botanischen Gesellschaft 18 (1900): 232–49. 80. Robert C. Olby, “Mendel, Mendelism and Genetics,” Mendelweb, 1997, accessed November 20, 2017, http://www.mendelweb.org/MWolby .html. 81. As quoted in van Iterson et al., Beijerinck: His Life and His Work, 46. 82. Bos, “Beijerinck’s Work,” 683. 83. Carl Correns, “G. Mendel’s Law,” Genetics 35, no. 5 (1950), 39. 84. Robert Olby, and Peter Gautrey, “Eleven References to Mendel before 1900,” Annals of Science 24, no. 1 (1968): 7, 19. 85. H. A. Allard, “A Review of Investigations of the Mosaic Disease of Tobacco, Together with a Bibliography of the More Important Contributions,” Bulletin of the Torrey Botanical Club 41, no. 9 (1914): 438. 86. Helen Purdy, “Attempt to Cultivate an Organism from Tomato Mosaic,” Botanical Gazette 81, no. 2 (1926): 210.

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Notes to Pages 74–76

87. Rivers, “Nature of Viruses,” 423. 88. Lily E. Kay, The Molecular Vision of Life: Caltech, the Rockefeller Foundation, and the Rise of the New Biology (New York: Oxford University Press, 1992), 16. 89. Scott Podolsky, “The Role of the Virus in Origin-­of-­Life Theorizing,” Journal of the History of Biology 29, no. 1 (1996): 85, 83. 90. Robert Kohler, “The Enzyme Theory and the Origin of Biochemistry,” Isis 64, no. 2 (June 1973): 185. 91. d’Herelle, “Bacteriophage: A Living Colloidal Micell.” 92. John Northrop, “Chemical Nature and Mode of Formation of Pepsin, Trypsin and Bacteriophage,” Science 86, no. 2239 (1937): 480.

Chapter 4: Romancing the Phage Epigraphs: Burnet, diary letter to Linda Druce, November 19, 1926, Series 2, folder 10; F. Macfarlane Burnet, diary letter to Linda Druce, October 30, 1927, Series 2, folder 10, both Frank Macfarlane Burnet Collection, University of Melbourne Archives, Melbourne, Australia (hereafter cited as Burnet Collection). 1. Hansjürgen Raettig, Bakteriophagie, 1917 bis 1956: zugleich ein Vorschlag zur Dokumentation wissenschaftlicher Literatur, part I, Einfürung Sachregister Stichwortverzeichnis (Stuttgart: G. Fischer, 1958), 2, fig. 1. 2. Burnet, diary letter to Linda Druce, January 15, 1926, Series 2, folder 10, Burnet Collection. 3. F. Macfarlane Burnet, Changing Patterns: An Atypical Autobiography (Melbourne: Heinemann, 1968), 52. 4. Burnet published a total of twenty-­eight papers on bacteriophage research published between 1924 and 1937. Burnet, Changing Patterns, 4. 5. Frank Fenner, “Frank Macfarlane Burnet as I Knew Him,” Immunology and Cell Biology 86, no. 1 (January 2008): 22; “Nomination Archive: Frank M Burnet,” The Nobel Prize, https://www.nobelprize.org/nomination/archive /show_people.php?id=1464. 6. Peter C. Doherty,“Burnet Oration: Living in the Burnet Lineage,” Immunology and Cell Biology 77, no. 2 (April 1999): 168. 7. Christopher H. Andrewes, “Francis Peyton Rous. 1879–1970,” Biographical Memoirs of Fellows of the Royal Society 17 (1971): 646. 8. This multivolume series was titled A System of Bacteriology in Relation to Medicine, and published by His Majesty’s Stationery Office. Burnet’s contribution was published in volume 7 of the series. Burnet, “Bacteriophage and Cognate Phenomena,” in A System of Bacteriology in Relation to Medicine (London: His Majesty’s Stationery Office, 1930), 463–509.

Notes to Pages 77–82

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9. Burnet, diary letter to Linda Druce, October 30, 1926, Series 1, folder 101, Burnet Collection. 10. Jacques Bronfenbrenner, “The Virus Diseases of Bacteria—­ Bacteriophagy,” in Filterable Viruses, ed. Tom Rivers (Baltimore: Williams & Wilkins, 1928), 373. 11. Burnet, personal diary, January 29, 1922—­April 17, 1924, Series 2, folder 7, Burnet Collection. 12. Burnet, Changing Patterns, 49. Burnet began his residency in pathology sometime in 1923. 13. Burnet, 1965, as quoted in Christopher Sexton, Burnet: A Life (Melbourne: Oxford University Press, 1999), 43. 14. Burnet, diary entry, Feb 4, 1924. The reference here is to Charles Halliley Kellaway, an Australian medical researcher and administrator, who at the time of this diary entry was the director of the Walter and Eliza Hall Institute of Medical Research, where Burnet was undertaking his residency in pathology. 15. Burnet, “The Nature of the Acquired Resistance to Bacteriophage Action,” Journal of Pathology and Bacteriology 28, no. 3 (1925): 407–18; Burnet, “The Conditions Governing the Appearance of Tâches Vièrges in Bacteriophage Activity,” Journal of Pathology and Bacteriology 28, no. 3 (1925): 419–26. 16. F. M. Burnet,“Bacteriophage Phenomena in Their Relation to the Antigenic Structure of Bacteria” (PhD diss., London University, 1928); Neeraja Sankaran, “Setting Patterns: The Atypical Choices That Shaped the Career of Sir Frank Macfarlane Burnet in Twentieth-­Century Australia,” Korean Journal for the History of Science 35, no. 2 (2013): 345. 17. Burnet, “Nature of Acquired Resistance,” 415. 18. Burnet, “Conditions Governing Appearance,” 419. 19. Burnet, diary letter to Linda Druce, March 10, 1927, Series 2, folder 10, Burnet Collection. 20. F. M. Burnet, “A Method for the Study of Bacteriophage Multiplication in Broth,” British Journal of Experimental Pathology 10, no. 2 (1929): 109. 21. Although he made these points in his earlier publications, d’Herelle’s fullest exposition of this mechanism was put forth in his 1926 monograph, Le bactériophage et son comportement (Paris: Masson et Cie, 1926). 22. Burnet, “Method for the Study,” 109–10; 114. Emphasis in original. 23. Bacteriophage concentration at any given point in time was estimated based on the number of plaques produced when a fixed volume of solution (1 drop) was plated along with a standard concentration of bacterial cells.

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Notes to Pages 82–87

24. Burnet, interview with Christopher Sexton, 1985, Series 18, Tape 6, Burnet Collection. 25. Burnet, “Method for the Study,” 114, 109–10. 26. Robert Doerr, “Die Bakteriophagen,” Klinische Wochenschrift 1, no. 30 (1922): 1489–95; “Über Bakteriophagen,” Schweizerische medizinische Wochenschrift 53 (1923): 1009. 27. Oskar Bail, “Der Stand und die Ergebnisse der Bakteriophagenforschung,” Deutsche Medizinische Wochenschrift 51, no. 1 (1925): 13–16; Alan W. Varley, “Early Bacteriophage Research: The Contribution of Frank MacFarlane Burnet” (master’s thesis, University of Kansas, 1981), 49; Claas Kirchhelle, “The Forgotten Typers: The Rise and Fall of Weimar Bacteriophage-­ Typing (1921–1935),” Notes and Records: The Royal Society Journal of the History of Science 74, no. 4 (2020): 539–65. 28. Bail, “Der Stand und die Ergebnisse,” as summarized in Burnet, “Method for the Study,” 110. 29. Philip Hadley, “The Twort-­D’Herelle Phenomenon: A Critical Review and Presentation of a New Conception (Homogamic Theory) of Bacteriophage Action,” Journal of Infectious Diseases 42, no. 4 (1928): 421. Hadley’s contributions to theories of the bacteriophage, especially in promoting the notion that it represented a stage in the bacterial life cycle has been examined thoroughly by the historian Olga Amsterdamska in “Stabilizing Instability: The Controversy over Cyclogenic Theories of Bacterial Variation during the Interwar Period,” Journal of the History of Biology 24, no. 2 (1991): 191–222. 30. Jacques Bronfenbrenner, “The Bacteriophage: Present Status of the Question of Its Nature and Mode of Action,” in Newer Knowledge of Bacteriology and Immunology, edited by Edwin Oakes Jordan and I. S. Falk (Chicago: University of Chicago Press, 1928), 525–56. 31. Burnet, “Method for the Study,” 110. 32. Burnet, “Method for the Study,” 109–10. 33. Jules Bordet, “Croonian Lecture: The Theories of the Bacteriophage,” Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character 107, no. 752 (1931): 398–417. 34. J. C. G. Ledingham, “Discussion,” Transactions of the Royal Society of Tropical Medicine and Hygiene 26, no. 5 (March 23, 1933), 418, following the report of a paper by Burnet, “Recent Work on the Biological Nature of Bacteriophages.” 35. Emory Ellis and Max Delbrück, “The Growth of Bacteriophage,” Journal of General Physiology 22, no. 3 (1939): 365–84.

Notes to Pages 87–89

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36. Thomas Anderson, “Electron Microscopy of Phages,” in Phage and the Origins of Molecular Biology, ed. John Cairns, Gunther S. Stent, and James D. Watson (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory of Quantitative Biology, 1966), 63. 37. For a more detailed account of this argument see Neeraja Sankaran, “Stepping-­Stones to One-­Step Growth: Frank Macfarlane Burnet’s Role in Elucidating the Viral Nature of the Bacteriophages,” Historical Records of Australian Science 19, no. 1 (2008): 92–93. 38. Ellis and Delbrück, “Growth of Bacteriophage,” 365, emphasis added. 39. Ellis and Delbrück, “Growth of Bacteriophage”; Max Delbrück, “Adsorption of Bacteriophage under Various Physiological Conditions of the Host,” Journal of General Physiology 23, no. 5 (1940): 631–42; Max Delbrück, “The Growth of Bacteriophage and Lysis of the Host,” Journal of General Physiology 23, no. 5 (1940): 643–60. 40. William Summers, “How Bacteriophage Came to Be Used by the Phage Group,” Journal of the History of Biology 26, no. 2 (1993): 261–62, 265. 41. F. M. Burnet and Margot McKie, “Observations on a Permanently Lysogenic Strain of B. entiritidis Gaertner,” Australian Journal for Experimental Biology and Medical Science 6, no. 4 (1929): 277–84. Although this paper has two authors, I will, for the most part, use only Burnet’s name in discussions of the main ideas, because he was the principal investigator and primary author. Although his research assistant and coauthor Margot McKie contributed to the design and execution of the experiments, she did not publish any independent papers on bacteriophage. Burnet is the only person whose name was attached to all the papers on the subject to emerge from his laboratory, and so it is fair to assume that he was responsible for the arguments and reasoning that were developed in this body of work. 42. Burnet, “Cognate Phenomena,” 485–87, 494–95. 43. Burnet, diary letter to Linda Druce, November 19, 1926, Series 2, folder 10, Burnet Collection. 44. Burnet and McKie, “Observations on a Permanently Lysogenic Strain,” 277; 279. 45. Burnet and McKie, “Observations on a Permanently Lysogenic Strain,” 280–82, 284, 282. 46. F. Macfarlane Burnet and Dora Lush, “Induced Lysogenicity and Mutation of Bacteriophage within Lysogenic Bacteria,” Australian Journal for Experimental Biology and Medicine Sciences 14, no. 1 (1936): 37. 47. Charles Galperin, “Le bactériophage, la lysogénie et son déterminisme génétique,” History and Philosophy of the Life Sciences 9, no. 2 (1987): 197. Translation mine.

224

Notes to Pages 90–94

48. Burnet and McKie, “Observations on a Permanently Lysogenic Strain,” 283. 49. André Lwoff, “Lysogeny,” Bacteriological Reviews 17, no. 4 (1953): 277. 50. See Eugène Wollman, “À propos de la note de MM. Bordet et Ciuca,” Comptes rendus des séances de la Société de Biologie 83 (1920): 1478–79. For a detailed reprise of the contributions of the Wollmans to lysogeny see Jean Gayon and Richard M. Burian, “Eugène et Élisabeth Wollman: la question de la lysogénie,” in L’invention de la régulation génétique: Les Nobel 1965 (Jacob, Lwoff, Monod) et le modèle de l’opéron dans l’histoire de la biologie, ed. Laurent Loison and Michel Morange (Paris: Rue d’Ulm/Presses de l’École normale supérieure, 2017). 51. The results of these experiments were published in a series of memoirs that appeared in the Annales de l’Institut Pasteur from 1925 to 1940. 52. Galperin, “Le bactériophage, la lysogénie,” 179 (Wollman appears to have begun this monograph in 1937), 183–204; Gayon and Burian, “Eugène et Élizabeth Wollman.” 53. Jules Bordet, “Le problème de l’autolyse microbienne transmissible ou du bactériophage,” Annales de l’Institut Pasteur 39 (1925), as quoted in Lwoff, “Lysogeny,” 276. 54. Jules Bordet and Ernest Renaux, “L’autolyse microbienne transmissible ou le bactériophage,” Annales de l’Institut Pasteur 42 (1928): 1283–335, as quoted in Lwoff, “Lysogeny,” 276. 55. Galperin, “Le bactériophage, la lysogénie,” 184. Translations mine. 56. Eugène Wollman, “The Phenomenon of Twort-­d’Herelle and Its Significance,” Lancet 226, no. 5858 (1935): 1312; See also Eugène Wollman and Élisabeth Wollman, “Sur la transmission ‘parahéréditaire’ de caractères chez les bactéries,”Comptes rendus des séances de la Société de Biologie 93 (1925): 1568–69. 57. Burnet and Lush, “Induced Lysogenicity,” 37. 58. Wollman, “Phenomenon of Twort-­d’Herelle and and Its Significance,” 1312. 59. André Lwoff, “Lysogeny,” Bacteriological Reviews 17, no. 4 (1953): 277. Emphasis in original. 60. Elie Wollman, “Bacterial Conjugation,” in Cairns, Stent, and Watson, Phage and the Origins of Molecular Biology, 216–17. 61. Angela N. H. Creager, “Adaptation or Selection? Old Issues and New Stakes in the Postwar Debates over Bacterial Drug Resistance,” Studies in History and Philosophy of Biological and Biomedical Sciences 38, no. 1 (2007): 160. 62. Joseph Arkwright, “Variation,” in A System of Bacteriology in Relation to Medicine, vol. 1 (London: His Majesty’s Stationery Office, 1930), 319.

Notes to Pages 95–98

225

63. Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty, “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types,” Journal of Experimental Medicine 79, no. 2 (1944): 137–58; Ralf Dahm, “Friedrich Miescher and the Discovery of DNA,” Developmental Biology 278, no. 2 (2005): 274–88. 64. James D. Watson and Francis H. C. Crick, “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid,” Nature 171, no. 4356 (1953): 737–38; Watson and Crick, “Genetical Implications of the Structure of Deoxyribonucleic Acid,” Nature 171, no. 4361 (1953): 964–67. 65. Melvin Cohn, “Burnet, Lysogeny, and Creativity,” in Walter and Eliza Hall Institute of Medical Research Annual Review 1978–79, special volume, A Tribute to Sir Macfarlane Burnet (Melbourne: Walter and Eliza Hall Institute of Medical Research, 1979), 9. 66. Burnet, Changing Patterns, 41, 62. 67. See Christopher H. Andrewes, “Fifty Years with Viruses,” Annual Reviews in Microbiology 27, no. 1 (1973): 1–3; William E. Gye and Christopher H. Andrewes, “A Study of the Rous Fowl Sarcoma No. 1: I. Filterability,” British Journal of Experimental Pathology 7, no. 2 (1926): 81–87. 68. William Joseph Elford, “A New Series of Graded Collodion Membranes Suitable for General Bacteriological Use, Especially in Filterable Virus Studies,” Journal of Pathology and Bacteriology 34, no. 4 (1931): 505–21. 69. William J. Elford and Christopher H. Andrewes, “The Sizes of Different Bacteriophages,” British Journal of Experimental Pathology 13, no. 5 (1932): 446–56; W. J. Elford, “Centrifugation Studies: I. Critical Examination of a New Method as Applied to the Sedimentation of Bacteria, Bacteriophages and Proteins,” British Journal of Experimental Pathology 17, no. 5 (1936): 399– 422; W. J. Elford and Christopher H. Andrewes, “Centrifugation Studies: II. The Viruses of Vaccinia, Influenza and Rous Sarcoma,” British Journal of Experimental Pathology 17, no. 5 (1936): 422–30. 70. Andrewes, “Fifty Years with Viruses,” 3. 71. Christopher Andrewes to Peyton Rous, January 28, 1933, Andrewes folder #1, 1929–1935, Series 1 (B: R77), Peyton Rous Papers, American Philosophical Society, Philadelphia (hereafter cited as Rous Papers). 72. D. A. J. Tyrrell, “Christopher Howard Andrewes. 7 June 1896–31 December 1987,” Biographical Memoirs of Fellows of the Royal Society 37 (1991): 39. 73. F. M. Burnet, “The Bacteriophages,” Biological Reviews 9, no. 3 (1934): 332–50. 74. Neeraja Sankaran, “Frank Macfarlane Burnet and the Nature of the Bacteriophage, 1924–1937” (PhD diss., Yale University, 2006), 9.

226

Notes to Pages 99–100

75. Burnet and Lush, “Induced Lysogenicity.” 76. Joshua Lederberg, ed., Papers in Microbial Genetics: Bacteria and Bacterial Viruses (Madison: University of Wisconsin Press, 1952), xviii. 77. Burnet, Changing Patterns, 59. 78. Andrewes to Rous, February 18, 1936, Andrewes, folder #2, 1936– 1940, Rous Papers. 79. Gye to Rous, June 25, 1937, Gye. William E., folder #3, 1935–1937, Rous Papers. 80. Eduard Kellenberger, “History of Phage Research as Viewed by a European,” FEMS Microbiology Reviews 17, no. 1–2 (1995): 10. 81. Burnet, “The Seven Ages of Virology,” February 14, 1972, Typescript of lecture, Series # MS098: Sir F. Macfarlane Burnet Records, 1928–1975, Adolph Basser Library, Australian Academy of Science, Canberra, Australia; M. Schlesinger, “Zur Frage der chemischen Zusammensetzung der Bakteriophagen,” Biochemische Zeitschrift 273 (1934): 306–11; Schlesinger, “The Feulgen Reaction of the Bacteriophage Substance,” Nature 138, no. 3490 (1936): 508. 82. Gunther S. Stent, Molecular Biology of Bacterial Viruses (San Francisco: W. H. Freeman, 1963), 21. 83. Max Delbrück, preface to Bacteriophages, by Mark Adams (New York: Interscience, 1959). 84. Burnet, interview with Christopher Sexton (1985), Series 18, Tape 6, Burnet Collection. 85. Burnet, Changing Patterns, 53. 86. Ton van Helvoort, “The Controversy between John H. Northrop and Max Delbrück on the Formation of Bacteriophage: Bacterial Synthesis or Autonomous Multiplication?” Annals of Science 49, no. 6 (1992): 545–75. 87. John H. Northrop, “Crystalline Pepsin: I. Isolation and Tests of Purity,” Journal of General Physiology 13, no. 6 (1930): 739–66; John. H. Northrop, and Moses Kunitz, “Crystalline Trypsin: I. Isolation and Tests of Purity,” Journal of General Physiology 16, no. 2 (1932): 267–94; Moses Kunitz and John Northrop, “Crystalline Chymo-­Trypsin and Chymo-­Trypsinogen: I. Isolation, Crystallization, and General Properties of a New Proteolytic Enzyme and Its Precursor,” Journal of General Physiology 18, no. 4 (1935): 433–58. 88. John H. Northrop, “Chemical Nature and Mode of Formation of Pepsin, Trypsin and Bacteriophage,” Science 86, no. 2239 (1937): 480. 89. F. M. Burnet, “From Bacteriophage to Influenza Virus,” in Immunity and Virus Infection, edited by Victor A. Najjar (New York: Wiley, 1959), 165.

Notes to Pages 100–103

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Chapter 5: Reawakenings Epigraphs: James B. Murphy, “Discussion of Some Properties of the Causative Agent of a Chicken Tumor,” Transactions of the Association of American Physicians 46 (1931): 187. Christopher H. Andrewes,“Viruses in Relation to the Aetiology of Tumours,” Lancet 224, no. 5786 (1934): 123. 1. Rous to Andrewes, April 21, 1953, Andrewes, Christopher H., Folder #3, 1943–1956, Series 1 (B: R77), Peyton Rous Papers American Philosophical Society, Philadelphia (hereafter cited as Rous Papers). 2. Carol Moberg, “James B. Murphy, the Rous Sarcoma Agent, and Origins of Modern Cell Biology,” in Creating a Tradition of Biomedical Research: Contributions to the History of the Rockefeller University, ed. Darwin Stapleton (New York: Rockefeller University Press, 2004), 261. Information about the endowment as cited: Henry J. James Jr., July 22, 1915, Henry Rutherford Bequest for Cancer Research, folder 1913–1949, box 5, Endowment 1913–1949, RG 216.3, Rockefeller University Archives, Rockefeller Archive Center, Sleepy Hollow, NY. 3. C. C. Little, “James Bumgardner Murphy, August 4, 1884–August 24, 1950,” Biographical Memoirs of the National Academy of Science 38 (1960): 186. 4. Rous to Simon Flexner, November 8, 1929, Flexner, Simon, folder #14, 1921–1926, Rous Papers. 5. James B. Murphy and Peyton Rous, “The Behavior of Chicken Sarcoma Implanted in the Developing Embryo,” Journal of Experimental Medicine 15, no. 2 (1912): 119–32. 6. Moberg, “Murphy, Sarcoma Agent and Origins,” 260; Arthur M. Silverstein, “The Lymphocyte in Immunology: From James B. Murphy to James L. Gowans,” Nature Immunology 2, no. 7 (2001): 569. 7. Little, “James Bumgardner Murphy,” 187. 8. As recounted in Rous, letter to Flexner, November 8, 1929, Flexner, folder #12, 1924-­1935, Rous Papers; Peyton Rous, “A Sarcoma of the Fowl Transmissible by an Agent Separable from the Tumor Cells,” Journal of Experimental Medicine 13, no. 4 (1911): 399. 9. Silverstein, “Lymphocyte in Immunology,” 569–70. 10. For a complete list of papers see Murphy’s bibliography provided in Little, “James Bumgardner Murphy,” 194–99. The monograph was published as: James B. Murphy, The Lymphocyte in Resistance to Tissue Grafting, Malignant Disease, and Tuberculous Infection: An Experimental Study, Rockefeller Institute for Medical Research, 1926, https://qspace.library.queensu.ca/bitstream /handle/1974/136/murphy01.htm. 11. Ton van Helvoort, “Bacteriological and Physiological Research Styles

228

Notes to Pages 104–107

in the Early Controversy on the Nature of the Bacteriophage Phenomenon,” Medical History 36, no. 3 (1992): 243–70. 12. Jules Bordet and Mihai Ciuca, “Exsudats leucocytaires et autolyse microbienne,” Comptes rendus des séances de la Société de Biologie 83 (1920): 1293–95. 13. The Rous archive at the American Philosophical Society in Philadelphia contains extensive holdings of his correspondence with with both Andrewes (five folders) ranging from 1928 to 1968, two years before Rous’s death, and Gye (also five folders) from 1923 through 1951, the year before before Gye’s death. 14. Christopher H. Andrewes, “William Ewart Gye. 1884–1952,” Obituary Notices of Fellows of the Royal Society 8, no. 22 (1953): 422. 15. Gye to Rous, November 8, 1923, Gye, William E., folder #1, 1923–1925, Rous Papers, emphasis in original (underscored in a handwritten letter). 16. Rous to Gye, December 27, 1923, Gye, folder #1, Rous Papers. 17. William E. Gye, “The Aetiology of Malignant New Growths,” Lancet 206, no. 5316 (1925): 109, 110, 111, 112, 114. 18. Andrewes, “William Ewart Gye,” 426. 19. Gye, “Aetiology of Malignant New Growths,” 112–14, 117. 20. Rous to Andrewes, April 21, 1953. 21. Neeraja Sankaran and Ton van Helvoort, “Andrewes’s Christmas Fairy Tale: Atypical Thinking about Cancer Aetiology in 1935,” Notes and Records: the Royal Society Journal of the History of Science 70, no. 2 (2016): 175–201. 22. Rous to Flexner, July 24, 1925, Simon Flexner Papers, American Philosophical Society, Philadelphia (hereafter cited as Flexner Papers). 23. Murphy to Flexner, July 26, 1925, Flexner Papers. 24. Murphy to Nobel Prize Committee, January 18, 1926, Folder 1 (1925/1926), Nobel Prize Committee, 1926–1939, James B. Murphy Papers, (B: M956), American Philosophical Society, Philadelphia (hereafter cited as Murphy Papers). 25. James B. Murphy, “Certain Etiological Factors in the Causation and Transmission of Malignant Tumors,” American Naturalist 60, no. 668 (1926): 232–33. 26. James B. Murphy, “Observations on the Etiology of Tumors: As Evidenced by Experiments with a Chicken Sarcoma,” Journal of the American Medical Association 86, no. 17 (1926): 1271. 27. Murphy, “Observations on the Etiology,” 1270–71. 28. “Report of Dr. Murphy,” April 9, 1927, RG 439: box 3, vol. 15, Rockefeller University Archives. 29. James B. Murphy, Oscar M. Helmer, and Ernest Sturm, “Association

Notes to Pages 107–112

229

of the Causative Agent of a Chicken Tumor with a Protein Fraction of the Tumor Filtrate,” Science 68, no. 1749 (1928): 18–19. 30. James B. Murphy, “The Nature of the Filtrable Agent in Chicken Tumours,” in British Empire Cancer Campaign, Report of the International Conference on Cancer, London. 17th–20th July (Bristol: J. Wright, 1928), 34, 36. 31. For a detailed analysis of the broad trends in, and different influences on, the attitudes toward cancer viruses see Angela Creager and Jean-­Paul Gaudillière, “Experimental Technologies and Techniques of Visualisation: Cancer as a Viral Epidemic, 1930–1960,” in Heredity and Infection: The History of Disease Transmission, ed. Ilana Löwy and Jean-­Paul Gaudillière (London: Routlege, 2001). 32. Ewing to Murphy, April 14, 1926; and Murphy to Ewing, April 15, 1926, both Ewing, James, Folder #1, 1925–1928, Murphy Papers. 33. Albert Claude and James B. Murphy, “Transmissible Tumors of the Fowl,” Physiological Reviews 13, no. 2 (1933): 259. 34. Murphy to Ewing, February 7, 1935, Ewing, folder #3, 1934-­1935, Murphy Papers. 35. James Ewing, “The General Pathological Conception of Cancer,” Canadian Medical Association Journal 33, no. 2 (August 1935): 130. 36. Claude and Murphy, “Transmissible Tumors of the Fowl,” 259. 37. Murphy, “Nature of the Filtrable Agent,” 33. 38. Creager and Gaudillière, “Experimental Technologies,” 206–7. 39. Thomas F. Glick and Antoni Roca Rosell, “Francesc Duran Reynals (Barcelona, 1899-­New Haven, USA, 1958): Virus and Cancer: A Controversial Theory,” Contributions to Science 1, no. 1 (1999): 87–98. 40. “The Nobel Prize in Physiology or Medicine 1974,” The Nobel Prize, http://www.nobelprize.org/nobel_prizes/medicine/laureates/1974/. See also Albert Claude, “The Coming of Age of the Cell,” Science 189, no. 4201 (1975): 433–35. 41. Glick and Rosell, “Francesc Duran Reynals,” 89–90. 42. Francesc Duran-­Reynals, “Report to the Junta para Ampliación de Estudios,” June 14, 1926, as quoted/cited in Glick and Rosell, “Francesc Duran-­Reynals,” 90. The review cited by Duran-­Reynals refers to the following article: Francesc Duran-­Reynals, “Resumen crítico sobre el problema de la lisis bacteriana transmisible (fenómeno de Twort-­D’Hérelle) y sobre los principios filtrantes y contagiosos en general,” Revista Médica de Barcelona 5 (1926): 469–90; Alexis Carrel, “Mechanism of the Formation and Growth of Malignant Tumors,” Annals of Surgery 82, no. 1 (1925): 4. 43. F. Duran-­Reynals, and James B. Murphy, “Properties of the Causative

230

Notes to Pages 113–116

Agent of a Chicken Tumor I. The Specific Fixation by Tissues of Susceptible Animals,” Journal of Experimental Medicine 50, no. 3 (1929). 44. Duran-­Reynals to Murphy, November 22, 1928, Duran-­Reynals, Francisco, 1926–1939, Murphy Papers. “Ch. T. 1 was the shorthand for Chicken Tumor 1.” 45. Murphy to Duran-­Reynals, December, 16, 1938, Murphy Papers. 46. Glick and Rosell, “Francesc Duran Reynals,” 91. 47. Christian de Duve and George E. Palade, “Albert Claude, 1899–1983,” Nature 304, no. 5927 (1983): 588; Claude, “Coming of Age of the Cell,” 433. 48. Ilana Löwy, “Variances in Meaning in Discovery Accounts: The Case of Contemporary Biology,” Historical Studies in the Physical and Biological Sciences 21, no. 1 (1990): 100. 49. See bibliography for a detailed list of publications on the Rous sarcoma agent in which Claude was a coauthor. 50. Murphy to Claude, June 28, 1947, Claude, Albert Folder #3, 1947, Murphy Papers. 51. Albert Claude, Keith Porter, and Edward G. Pickels, “Electron Microscope Study of Chicken Tumor Cells,” Cancer Research 7, no. 7 (1947): 429. 52. James B. Murphy, Ernest Sturm, Albert Claude, and Oscar M. Helmer, “Properties of the Causative Agent of a Chicken Tumor III. Attempts at Isolation of the Active Principle,” Journal of Experimental Medicine 56, no. 1 (1932): 103. 53. John H. Northrop, “Chemical Nature and Mode of Formation of Pepsin, Trypsin and Bacteriophage,” Science 86, no. 2239 (1937): 480. 54. Murphy, “Discussion of Some Properties,” 186. 55. Fred Griffith, “Types of Pneumococci Obtained from Cases of Lobar Pneumonia,” Reports on Public Health and Medical Subjects, no. 13 (1922): 1–13; Griffith, “The Influence of Immune Serum on the Biological Properties of Pneumococci,” Reports of the Local Government Board on Public Health and Medical Subjects, no. 18 (1923): 1–13. 56. Fred Griffith, “The Significance of Pneumococcal Types,” Epidemiology & Infection 27, no. 2 (1928): 113–59; Griffith, “Serological Races of Pneumococci,” in A System of Bacteriology in Relation to Medicine, vol. 2 (London: His Majesty’s Stationery Office, 1930), 201–25; Pierre-­Olivier Méthot, “Bacterial Transformation and the Origins of Epidemics in the Interwar Period: The Epidemiological Significance of Fred Griffith’s ‘Transforming Experiment,’” Journal of the History of Biology 49, no. 2 (2016): 311–58. 57. Oswald Avery, Colin M. MacLeod, and Maclyn McCarty, “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types,” Journal of Experimental Medicine 79, no. 2 (1944): 137–58.

Notes to Pages 117–119

231

58. J. Lionel Alloway, “The Transformation in Vitro of R Pneumococci into S Forms of Different Specific Types by the Use of Filtered Pneumococcus Extracts,” Journal of Experimental Medicine 55, no. 1 (1932): 91–99. 59. Murphy, “Discussion of Some Properties,” 186–87; emphasis added. 60. Murphy, Sturm, Claude, and Helmer, “Properties of the Causative Agent of a Chicken Tumor III,” 105. 61. Andrewes to Murphy, April 25, 1931; Murphy to Andrewes, May 12, 1931, Folder Andrewes, C. H., 1930–1932, Murphy Papers. The paper Andrewes has requested was James B. Murphy, O. M. Helmer, Albert Claude, and Ernest Sturm, “Observations Concerning the Causative Agent of a Chicken Tumor,” Science 73, no. 1888 (1931): 266–68. 62. Andrewes to Murphy, May 22, 1931; emphasis in original. The paper by Arthur Boycott, “The Transition from Live to Dead: The Nature of Filtrable Viruses,” Journal of the Royal Society of Medicine 22, no. 1 (1928): 55–69, offers a good snapshot of the ideas Andrewes was referring to. 63. See, for example, Claude and Murphy, “Transmissible Tumors of the Fowl.” 64. Andrewes, “Viruses in Relation to the Aetiology of Tumours,” 120. 65. John H. Northrop, “Chemical Nature and Mode of Formation of Pepsin, Trypsin and Bacteriophage,” Science 86, no. 2239 (1937): 480. 66. See Gerrit van Iterson, L. E. den Dooren de Jong, and Albert Jan Kluyver, Martinus Willem Beijerinck: His Life and His Work (1940, repr., Dordrecht: Springer, 2013), 92. 67. Oswald Avery to Roy T. Avery, May 13, 1943, Oswald T. Avery papers, Rockefeller University Faculty (FA230), Rockefeller Archive Center, Tarrytown, NY. 68. Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty, “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types,” Journal of Experimental Medicine 79, no. 2 (1944): 155, 138. 69. Murphy to Ewing, December 3, 1935, Ewing, folder #4, 1935-­1949, Murphy Papers. 70. Richard E. Shope, “A Transmissible Tumor-­like Condition in Rabbits,” Journal of Experimental Medicine 56, no. 6 (1932): 793–802; and Shope, “A Filtrable Virus Causing a Tumor-­like Condition in Rabbits and Its Relationship to Virus Myxomatosum,” Journal of Experimental Medicine 56, no. 6 (1932): 803–22. 71. Christopher Andrewes, “Francis Peyton Rous. 1879–1970,” Biographical Memoirs of Fellows of the Royal Society 17 (1971): 648; “Richard Edwin Shope, 1901–1966,” Biographical Memoirs 50 (1979): 361. 72. Rous to Andrewes, April 21, 1953, Andrewes, folder #3, Rous Papers.

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Notes to Pages 119–125

73. James Ewing, “The Causal and Formal Genesis of Cancer,” in Report of the International Conference on Cancer. London, 17th–20th July, 1928, by British Empire Cancer Campaign (Bristol: J. Wright, 1928), 12. 74. Peyton Rous and J. W. Beard, “A Virus-­Induced Mammalian Growth with the Characters of a Tumor (the Shope Rabbit Papilloma) I. The Growth on Implantation within Favorable Hosts,” Journal of Experimental Medicine 60, no. 6 (1934): 701; “A Virus-­Induced Mammalian Growth with the Characters of a Tumor (the Shope Rabbit Papilloma) III. Further Characters of the Growth; General Discussion,” Journal of Experimental Medicine 60 (1934): 763. 75. Simon Flexner to Rous, August 14, 1934, Flexner, folder #10, 1933-­ 1934, Rous Papers. 76. Andrewes to Rous, May 15, 1934; November 19, 1934, Andrewes, folder #1, 1929-­1935, Rous Papers. 77. Rous to Andrewes, November 25, 1935, Andrewes, folder #1, Rous Papers. 78. Andrewes to Rous, November 19, 1934, Andrewes, folder #1, Rous Papers. 79. Murphy to Ewing, January 31, 1935, Ewing, folder #3, Murphy Papers. Eugene Opie was an American pathologist and contemporary of Rous and Murphy at the Rockefeller. 80. Ewing to Murphy, February 11, 1935, Ewing, folder #3, Murphy Papers. 81. Ewing, “Causal and Formal Genesis,” 1. 82. Ewing, “General Pathological Conception,” 134; 132. 83. Andrewes to Rous, October 1, 1935, Andrewes, folder #1, Rous Papers. 84. Andrewes to Ewing, September 17, 1935, Ewing, James, folder 1924– 1936, Rous Papers. 85. Ewing, “General Pathological Conception,” 129; Andrewes to Ewing, September 17, 1935, Ewing, 1924–1936, Rous papers. 86. Ewing to Murphy, September 28, 1935, Ewing, folder #3, Murphy Papers; and Ewing to Rous, September 28, 1935, Rous Papers. 87. Murphy to Ewing, October 10, 1935, Ewing, folder #3; December 3, 1935, Ewing, folder #4, Murphy Papers. 88. Rous to Andrewes, continuation of letter to Andrewes, begun on November 25, 1935, Andrewes, folder #1, Rous Papers. 89. Rous to Ewing, October 3, 1935, and Ewing’s response to Rous, October 7, 1935, Ewing, James, folder 1924–1936, Rous Papers. 90. Murphy to Ewing, December 3, 1935, Ewing, folder #4, Murphy Papers. 91. Andrewes, “Viruses in Relation to Aetiology,” 117.

Notes to Pages 125–129

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92. Boycott in J. A. Murray et al., “Discussion on Experimental Production of Malignant Tumours,” Proceedings of the Royal Society of London (1933): 292. 93. Andrewes, “Viruses in Relation to Aetiology,” 123; Andrewes to Rous, January 28, 1933, Andrewes, folder #1, Rous Papers. 94. Andrewes to Rous, April 11, 1933, Andrewes, folder #1, Rous Papers. 95. Rous to Andrewes, March 20, 1933, Andrewes, folder #1, Rous Papers. 96. Rous to Andrewes, November 24, 1935, Andrewes, folder #1, Rous Papers; Peyton Rous, “The Virus Tumors and the Tumor Problem,” Harvey Lectures 31 (1936): 112. 97. Andrewes to Rous, December 6, 1935, Andrewes, folder #1, Rous Papers. 98. See Sankaran and van Helvoort, “Andrewes’s Christmas Fairy Tale,” for a detailed analysis and complete version of the fairy story. 99. F. M. Burnet and Margot McKie, “Observations on a Permanently Lysogenic Strain of B. Entiritidis Gaertner,” Australian Journal for Experimental Biology and Medical Science 6, no. 4 (1929): 277–84. 100. Andrewes, “Viruses in Relation to Aetiology,” 118–19. 101. Christopher Andrewes, “Latent Virus Infections and Their Possible Relevance to the Cancer Problem (President’s Address),” Proceedings of the Royal Society of Medicine 33, no. 2 (1939): 77. 102. Rous to Andrewes, January 11, 1936, Andrewes, folder #1, Rous Papers. 103. Rous to Andrewes, January 11, 1936, Andrewes, folder #1, Rous Papers. 104. Gye to Rous, December 9, 1935, and Rous’s response on December 28, 1935, Gye, folder #3, 1935–1937, Rous Papers. 105. Sankaran and van Helvoort, “Andrewes’s Christmas Fairy Tale,” 186–92. 106. Jacob Furth and Donald Metcalf, “An Appraisal of Tumor-­Virus Problems,” Journal of Chronic Diseases 8, no. 1 (1958): 89.

Chapter 6: What Viruses Became Epigraphs: James Ewing, letter to Peyton Rous, October 7, 1935, Folder—­ Ewing, James, 1924–1936, Peyton Rous Papers, American Philosophical Society, Philadelphia (hereafter cited as Rous Papers). André Lwoff, “The Concept of Virus,” Journal of General Microbiology 17, no. 2 (1957): 252. 1. Jacob Furth and Donald Metcalf, “An Appraisal of Tumor-­Virus Problems,” Journal of Chronic Diseases 8, no. 1 (1958): 88. The contributions of all

234

Notes to Pages 129–135

scientists named herein are discussed in this chapter and details about each will be provided in context rather than in this footnote. 2. Lwoff, “Concept of Virus,” 239; Material quoted from F. C. Bawden, and N. W. Pirie, “Virus Multiplication Considered as a Form of Protein Synthesis,” in The Nature of Virus Multiplication, 2nd Symposium of the Society for General Microbiology, edited by P. Fildes and W. E. Van Heyningen (Oxford: Cambridge University Press, 1953), 21. 3. Thomas M. Rivers, and Saul Benison, Tom Rivers: Reflections on a Life in Medicine and Science: An Oral History Memoir Prepared by Saul Benison (Cambridge: MIT Press, 1967), 77. 4. Earl B. McKinley, “The Filterable Viruses,” Scientific Monthly 32, no. 5 (1931): 403. 5. F. C. Bawden, “Some Reflexions on Thirty Years of Research on Plant Viruses,” Annals of Applied Biology 58, no. 1 (1966): 2. 6. Karen-­Beth Scholthof, personal communication, January 19, 2017. 7. Francis O. Holmes, “Local Lesions in Tobacco Mosaic,” Botanical Gazette 87, no. 1 (1929): 39–55; W. M. Stanley, “Isolation of a Crystalline Protein Possessing the Properties of Tobacco-­Mosaic Virus,” Science 81, no. 2113 (June 28, 1935): 644–45; and M. Schlesinger, “Zur Frage der chemischen Zusammensetzung der Bakteriophagen,” Biochemische Zeitschrift 273 (1934): 306–11; F. C. Bawden, N. W. Pirie, J. D. Bernal, and I. Fankuchen, “Liquid Crystalline Substances from Virus-­Infected Plants,” Nature 138, no. 3503 (1936): 1051–52. 8. Dmitrii Ivanowski, “On the Mosaic Disease of the Tobacco Plant,” Phytopathology Classics, 7 (1942): 27–30; Martinus W. Beijerinck, “Concerning a Contagium Vivum Fluidum as Cause of the Spot Disease of Tobacco-­Leaves,” Phytopathological Classics 7 (1942): 33–52; Friedrich Löffler and Paul Frosch, “Report of the Commission for Research on the Foot-­and-­Mouth Disease,” in Milestones in Microbiology: 1546 to 1940, ed. and trans. Thomas D. Brock (Washington, DC: American Society for Microbiology, 1961), 149–53. 9. William Joseph Elford, “A New Series of Graded Collodion Membranes Suitable for General Bacteriological Use, Especially in Filterable Virus Studies,” Journal of Pathology and Bacteriology 34, no. 4 (1931): 505–21. 10. William J. Elford and Christopher H. Andrewes, “The Sizes of Different Bacteriophages,” British Journal of Experimental Pathology 13, no. 5 (1932): 446–56; “Estimation of the Size of a Fowl Tumour Virus by Filtration through Graded Membranes,” British Journal of Experimental Pathology 16, no. 1 (1935): 61–66. 11. Elford and Andrewes, “Sizes of Different Bacteriophages,” 446. 12. Elford and Andrewes, “Estimation of the Size of a Fowl Tumour Virus,” 61.

Notes to Pages 135–138

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13. Waro Nakahara, “A Pilgrim’s Progress in Cancer Research, 1918 to 1974: Autobiographical Essay,” Cancer Research 34, no. 8 (1974): 1768. For a snapshot of Nakahara’s earlier view see his article “The Filterable Entity Transmitting Chicken Sarcoma,” in Colloid Chemistry: Theoretical and Applied, vol. 2, Biology and Medicine, ed. Jerome Alexander (New York: Chemical Catalog, 1928), 907–10. 14. Christopher H. Andrewes, “William Joseph Elford. 1900–1952,” Obituary Notices of Fellows of the Royal Society 8, no. 21 (1952): 152. Of the sixty-­one publications from 1926–1952 listed in the bibliography provided at the end of this obituary (pp. 155–57), more than two dozen publications between 1932–1937 focus on the topic of ultramicroscopic analysis and size determination of different viruses. 15. Stuart Mudd, “Filters and Filtration,” in Filterable Viruses, ed. Thomas M. Rivers (Baltimore: Williams & Wilkins, 1928), 55. 16. “The Svedberg: Facts,” The Nobel Prize, https://www.nobelprize .org/nobel_prizes/chemistry/laureates/1926/svedberg-­facts.html. 17. The Svedberg and Herman Rinde, “The Ultra-­Centrifuge, a New Instrument for the Determination of Size and Distribution of Size of Particle in Amicroscopic Colloids,” Journal of the American Chemical Society 46, no. 12 (1924): 2677. 18. See the bibliography of Elford’s publications appended to Andrewes, “William Joseph Elford,” 156–57, 153. 19. New York Times, June 24, 1937; Angela N. H. Creager, The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930–1965 (Chicago: University of Chicago Press, 2002), 79. 20. W. J. Elford, “Centrifugation Studies: I. Critical Examination of a New Method as Applied to the Sedimentation of Bacteria, Bacteriophages and Proteins,” British Journal of Experimental Pathology 17, no. 5 (1936): 414; M. Schlesinger, “Die Bestimmung von Teilchengrösse und spezifischem Gewicht des Bakteriophagen durch Zentrifugierversuche,” Zeitschrift für Hygiene und Infektionskrankheiten 114, no. 1 (1932): 161–76. 21. Creager, Life of a Virus, 81; 79; W. M. Stanley, “An Evaluation of Methods for the Concentration and Purification of Influenza Virus,” Journal of Experimental Medicine 79, no. 3 (1944): 255–66; Stanley, “The Size of Influenza Virus,” Journal of Experimental Medicine 79, no. 3 (1944): 267–83; Stanley, “The Preparation and Properties of Influenza Virus Vaccines Concentrated and Purified by Differential Centrifugation,” Journal of Experimental Medicine 81, no. 2 (1945): 193–218. 22. J. C. G. Ledingham, and W. E. Gye, “On the Nature of the Filterable Tumour-­Exciting Agent in Avian Sarcomata,” Lancet 225, no. 5816 (1935):

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Notes to Pages 138–140

377; W. J. Elford, and C. H. Andrewes, “Centrifugation Studies: II. The Viruses of Vaccinia, Influenza and Rous Sarcoma,” British Journal of Experimental Pathology 17, no. 5 (1936): 422–30. 23. J. C. G. Ledingham in discussion following Burnet, “Recent Work on the Biological Nature of Bacteriophages,” Transactions of the Royal Society of Tropical Medicine and Hygiene 26, no. 5 (1933): 418. 24. Gye to Rous, June 25, 1937, Gye, William E., folder #3, 1935–1937, Rous Papers. 25. Albert Claude, and James B. Murphy, “Transmissible Tumors of the Fowl,” Physiological Reviews 13, no. 2 (1933): 246–75. 26. Carol L. Moberg, “James B. Murphy, the Rous Sarcoma Agent, and Origins of Modern Cell Biology,” in Creating a Tradition of Biomedical Research: Contributions to the History of the Rockefeller University, ed. Darwin Stapleton, 259–70 (New York: Rockefeller University Press, 2004), 262. 27. Albert Claude, “Preparation of an Active Agent from Inactive Tumor Extracts,” Science 85, no. 2203 (1937): 294–95; “Fractionation of Chicken Tumor Extracts by High Speed Centrifugation,” American Journal of Cancer 30, no. 4 (1937): 742–45; “Properties of the Causative Agent of a Chicken Tumor XIII: Sedimentation of the Tumor Agent, and Separation from the Associated Inhibitor,” Journal of Experimental Medicine 66, no. 1 (1937): 59–72; “Concentration and Purification of Chicken Tumor I Agent,” Science 87, no. 2264 (1938): 467–68. 28. Moberg, “James B. Murphy,” 262. 29. W. M. Stanley, “Isolation of a Crystalline Protein,” 644. 30. “Crystals Isolated at Princeton Believed Unseen Disease Virus,” New York Times, June 28, 1935. 31. For a comprehensive treatment of the history of TMV in general and Stanley’s contributions in particular, see the work of Angela Creager: “Wendell Stanley’s Dream of a Free-­Standing Biochemistry Department at the University of California, Berkeley,” Journal of the History of Biology 29, no. 3 (1996): 331–60; The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930–1965 (Chicago: University of Chicago Press, 2002); and Angela Creager, Karen-­Beth G. Scholthof, Vitaly Citovsky, and Herman B. Scholthof, “Tobacco Mosaic Virus: Pioneering Research for a Century,” Plant Cell 11, no. 3 (1999): 301–8. 32. Lily E. Kay, “W. M. Stanley’s Crystallization of the Tobacco Mosaic Virus, 1930–1940,” Isis 77, no. 3 (1986): 450. 33. Bawden, Pirie, Bernal, and Fankuchen, “Liquid Crystalline Substances,” 1051–52; F. C. Bawden and N. W. Pirie, “A Plant Virus Preparation in a Fully Crystalline State,” Nature 141, no. 3568 (1938): 513–14.

Notes to Pages 140–142

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34. F. C. Bawden, “Musings of an Erstwhile Plant Pathologist,” Annual Review of Phytopathology 8, no. 1 (1970): 3. 35. Wendell M. Stanley, “The Nature of Viruses,” Transactions of the New York Academy of Sciences 1, no. 2, series 2 (1938): 22. 36. “The Nobel Prize in Chemistry 1946,” https://www.nobelprize.org /nobel_prizes/chemistry/laureates/1946/. 37. “Nomination Archive: Nomination for Nobel Prize in Chemistry, 1939,” The Nobel Prize, https://www.nobelprize.org/nomination/archive /show.php?id=9491; “Nomination Archive: Nomination for Nobel Prize in Physiology or Medicine, 1939,” The Nobel Prize, https://www.nobelprize .org/nomination/archive/show.php?id=6743; “Nomination Archive: Wendell M Stanley,” The Nobel Prize, https://www.nobelprize.org/nomination /archive/show_people.php?id=8731. 38. Kay, “Stanley’s Crystallization,” 450; see also Kay’s rather more detailed description of the different views of life over the first half of the twentieth century in Lily E. Kay, The Molecular Vision of Life: Caltech, the Rockefeller Foundation, and the Rise of the New Biology (New York: Oxford University Press, 1992): 104–16. 39. Heinz Fraenkel-­Conrat, “Portraits of Viruses: Tobacco Mosaic Virus,” Intervirology 15, no. 4 (1981): 178. 40. F. C. Bawden,“Crystallography and Plant Viruses,” Nature 149, no. 3777 (1942): 321. 41. Robert Hooke, Micrographia, or, Some Physiological Descriptions of Minute Bodies Made by Magnifying Glasses (1665; repr., New York: Cosmo Classics, 2007). 42. V. K. Zworykin, “An Electron Microscope for the Research Laboratory,” Science 92, no. 2377 (1940): 53. 43. Ton van Helvoort and Neeraja Sankaran, “How Seeing Became Knowing: The Role of the Electron Microscope in Shaping the Modern Definition of Viruses,” Journal of the History of Biology 52, no. 1 (2019): 125–60. 44. T. F. Anderson, “The Application of the Electron Microscope to Biology,” Collecting Net 17 (1942): 4. 45. John H. Reisner, “An Early History of the Electron Microscope in the United States,” Advances in Electronics and Electron Physics, vol. 73, Aspects of Charged Particle Optics, edited by Peter W. Hawkes (Burlington, VT: Elsevier, 1989), 134. 46. Gabor, “Preface,” in Ladislaus Marton, Early History of the Electron Microscope (San Francisco: San Francisco Press, 1968), vi. 47. Björn A. Afzelius, “Half a Century of Electron Microscopy: The Early Years,” Ultrastructural Pathology 2, no. 3 (1981): 309–11.

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Notes to Pages 142–145

48. As quoted in Ladislaus Marton, “Early Application of Electron Microscopy to Biology,” Ultramicroscopy 1, no. 3 (1976): 282. 49. Reisner, “Early History of the Electron Microscope.” 50. Hans R. Gelderblom and Detlev H. Krüger, “Helmut Ruska (1908– 1973): His Role in the Evolution of Electron Microscopy in the Life Sciences, and Especially Virology,” Advances in Imaging and Electron Physics 182 (2014): 19. 51. Bodo von Borries, Ernst Ruska, and Helmut Ruska, “Bakterien und Virus in Übermikroskopischer Aufnahme,” Klinische Wochenschrift 17, no. 27 (1938): 921–25; Helmut Ruska, Bodo v. Borries, and Ernst Ruska, “Die Bedeutung der Übermikroskopie für die Virusforschung,” Archives of Virology 1, no. 1 (1939/1940): 155–69. 52. Nicolas Rasmussen, Picture Control: The Electron Microscope and the Transformation of Biology in America, 1940–1960 (Stanford, CA: Stanford University Press, 1999), 219. 53. H. Ruska, “Die Sichtbarmachung der Bakteriophagen Lyse im Übermikroskop,” Naturewissenschaften 28 (1940): 45. 54. Salvador E. Luria, Max Delbrück, and Thomas F. Anderson, “Electron Microscope Studies of Bacterial Viruses,” Journal of Bacteriology 46, no. 1 (1943): 57; Thomas Anderson, “Electron Microscopy of Phages,” in Phage and the Origins of Molecular Biology, ed. John Cairns, Gunther S. Stent, and James D. Watson (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory of Quantitative Biology, 1966). 55. Anderson, undated transcript of Men and Molecules program, Thomas Anderson Papers, American Philosophical Society, Philadelphia. Men and Molecules was a radio documentary program broadcast in the 1960s and 1970s by the American Chemical Society news service. Anderson’s interview was part of an eight-­program series titled “New Directions in Cancer Research,” which, according to the September 3, 1962, issue of Chemical and Engineering News began broadcasting on October 15, 1962. 56. Robley Williams, S. J. Kass, and C. A Knight, “Structure of Shope Papilloma Virus Particles,” Virology 12, no. 1 (September 1, 1960): 48–58; Anderson, Men and Molecules interview. 57. Robley C. Williams, “The Role of the Electron Microscope in Virus Research,” in International Review of Cytology, vol. 6, edited by G. H. Bourne and J. F. Danielli (Amsterdam: Academic Press, 1957), 146–47; See also William N. Takahashi and Mamoru Ishii, “An Abnormal Protein Associated with Tobacco Mosaic Virus Infection,” Nature 169, no. 4297 (1952): 419–20; Robert V. Rice, Paul Kaesberg, and Mark A. Stahmann, “The Breaking of Tobacco Mosaic Virus Using a New Freeze Drying Method,” Biochimica et Biophysica

Notes to Pages 145–146

239

Acta 11, no. 3 (July 1953): 337–43; Roger G. Hart, “Electron-­Microscopic Evidence for the Localization of Ribonucleic Acid in the Particles of Tobacco Mosaic Virus,” Proceedings of the National Academy of Sciences 41, no. 5 (1955): 261–64; and Heinz Fraenkel-­Conrat and Robley C. Williams, “Reconstitution of Active Tobacco Mosaic Virus from Its Inactive Protein and Nucleic Acid Components,” Proceedings of the National Academy of Sciences 41, no. 10 (1955): 690–98. 58. Leon Dmochowski, “Viruses and Tumors in the Light of Electron Microscope Studies: A Review,” Cancer Research 20, no. 7 (1960): 1003–4, emphasis added. 59. Greer Williams, Virus Hunters (New York: Knopf, 1959), 89. 60. For a detailed analysis of this aspect of the electron microscopy of viruses see Helvoort and Sankaran, “How Seeing Became Knowing.” 61. Ladislaus Marton, “Alice in Electronland,” American Scientist 31, no. 3 (1943): 254. 62. Carol Moberg, “The Electron Microscope Enters the Realm of the Intact Cell,” Journal of Experimental Medicine 181, no. 3 (1995): 831–37. 63. F. M. Burnet, “Virology as an Independent Science Lecture II: The Substance of Virology,” Medical Journal of Australia 2, no. 22 (1953): 843; A. H. Doermann, “Intracellular Growth of Bacteriophage,” Carnegie Institution of Washington Yearbook 47 (1948): 176–82; Doermann, “The Eclipse in the Bacteriophage Life Cycle,” in Phage and the Origins of Molecular Biology, 79. 64. Albert Claude, “The Coming of Age of the Cell,” Science 189, no. 4201 (1975): 433–35; Moberg, “James B. Murphy”; Keith Porter, Albert Claude, and Ernest F. Fullam, “A Study of Tissue Culture Cells by Electron Microscopy,” Journal of Experimental Medicine 81, no. 3 (1945): 233–46; Albert Claude, Keith Porter, and Edward G. Pickels, “Electron Microscope Study of Chicken Tumor Cells,” Cancer Research 7, no. 7 (1947): 421–30. 65. Helmut Ruska, Bodo von Borries, and Ernst Ruska, “Übermikroskopie für die Virusforschung,” Archives of Virology 1, no. 1 (1939/1940): 167; emphasis in original. 66. McKinley, “Filterable Viruses,” 404. 67. S. Gard, “Nobel Prize in Physiology or Medicine 1954: Award Ceremony Speech,” The Nobel Prize, https://www.nobelprize.org/nobel_prizes /medicine/laureates/1954/press.html. 68. Beijerinck, “Concerning a Contagium Vivum Fluidum as Cause of the Spot Disease,” 39. 69. Dmitrii Iwanowski, “Über die Mosaikkrankheit der Tabakspflanze,” Zeitschrift für Pflanzenkrankheit und Pflanzenschutz 13 (1903): 1–41, as quoted

240

Notes to Pages 146–149

in L. Bos, “The Embryonic Beginning of Virology: Unbiased Thinking and Dogmatic Stagnation,” Archives of Virology 140, no. 3 (1995): 614. 70. Rivers and Benison, Tom Rivers, 116. 71. T. M. Rivers, “Filterable Viruses: A Critical Review,” Journal of Bacteriology 14, no. 4 (1927): 228. 72. G. M. Findlay, “A Note on the Cultivation of the Virus of Fowl-­Pox,” British Journal of Experimental Pathology 9, no. 1 (1928): 28–29; Rivers and Benison, Tom Rivers, 146. 73. Rivers and Benison, Tom Rivers, 144. 74. Frank Macfarlane Burnet, The Use of the Developing Egg in Virus Research, Medical Research Council Special Reports 220 (Medical Research Council, 1936), 3. 75. John Enders, Thomas H. Weller, and Frederick C. Robbins, “The Cultivation of the Poliomyelitis Viruses in Tissue Culture,” in Nobel Lectures, Physiology or Medicine, 1942–1962 (Amsterdam: Elsevier, 1964), 457. 76. F. W. Twort, “An Investigation on the Nature of Ultra-­Microscopic Viruses,” Lancet 186, no. 4814 (1915): 1243. 77. Felix d’Herelle, “Sur un microbe invisible antagoniste des bacilles dysentériques,” Comptes rendus de l’Académie des Sciences 145 (1917): 373–75; d’Herelle detailed his experimental protocols in his monograph The Bacteriophage and Its Behavior, trans. George Hathorn Smith (Baltimore: Williams & Wilkins, 1926), 116. 78. F. M. Burnet, “A Method for the Study of Bacteriophage Multiplication in Broth,” British Journal of Experimental Pathology 10, no. 2 (1929): 109– 15; Emory L. Ellis and Max Delbrück, “The Growth of Bacteriophage,” Journal of General Physiology 22, no. 3 (1939). 79. Heinz Fraenkel-­Conrat, “Portraits of Viruses: Tobacco Mosaic Virus,” Intervirology 15, no. 4 (1981): 177. 80. Holmes, “Local Lesions,” 54–55. 81. John Tooze, “A Seminal Assay in Plant Virology,” Trends in Biochemical Sciences 4, no. 4 (1979): 96; Karen-­Beth G. Scholthof, “Making a Virus Visible: Francis O. Holmes and a Biological Assay for Tobacco Mosaic Virus,” Journal of the History of Biology 47, no. 1 (2014): 107. 82. Peyton Rous and James B. Murphy, “Tumor Implantations in the Developing Embryo: Experiments with a Transmissible Sarcoma of the Fowl,” Journal of the American Medical Association 56, no. 10 (1911): 741, 740. 83. Burnet, Use of the Developing Egg, 4. With reference to Alice M. Woodruff and Ernest W. Goodpasture, “The Susceptibility of the Chorio-­Allantoic Membrane of Chick Embryos to Infection with the Fowl-­Pox Virus,” American Journal of Pathology 7, no. 3 (1931): 209–225..

Notes to Pages 149–152

241

84. Burnet, Use of the Developing Egg, 16–17; Burnet, “Propagation of the Virus of Epidemic Influenza on the Developing Egg,” Medical Journal of Australia 2, no. 20 (1935): 687–89; Burnet, “From Bacteriophage to Influenza Virus,” in Immunity and Virus Infection, edited by V. A. Najjar (New York: Wiley, 1959), 173. 85. “Nomination Archive: Ernest W Goodpasture,” The Nobel Prize, https://www.nobelprize.org/nomination/archive/show_people. php?id=3545; “Nomination Archive: Nomination for Nobel Prize in Physiology or Medicine, 1949,” The Nobel Prize, https://www.nobelprize.org /nomination/archive/show.php?id=9360. 86. F. Macfarlane Burnet, “The Influence of a Great Pathologist: A Tribute to Ernest Goodpasture,” Perspectives in Biology and Medicine 16, no. 3 (1973): 334, 338–39. According to the editor’s note (p. 333), this paper was intended as the first Goodpasture Lecture at Vanderbilt University in 1969, which did not happen because Burnet was unable to attend. 87. The phrase “tissue culture” is a misnomer for the technique because it is individual cells rather than contiguous tissues that are cultivated, as has been frequently noted by various researchers in the field—­see, for example, Joseph F. Morgan, “Tissue Culture Nutrition,” Bacteriological Reviews 22, no. 1 (1958): 20; H. E. Swim, “Microbiological Aspects of Tissue Culture,” Annual Review of Microbiology 13, no. 1 (1959): 141—­but the label has stuck nevertheless. 88. P. Mortimer, “How Monolayer Cell Culture Transformed Diagnostic Virology: A Review of a Classic Paper and the Developments That Stemmed from It,” Reviews in Medical Virology 19, no. 4 (2009): 241. The specific paper reviewed is John F. Enders, Thomas H. Weller, and Frederick C. Robbins, “Cultivation of the Lansing Strain of Poliomyelitis Virus in Cultures of Various Human Embryonic Tissues,” Science 109, no. 2822 (1949): 85–87. 89. Well beyond the scope of this book, readers are referred to the excellent work by Hannah Landecker for a detailed history of this subject: “Technologies of Living Substance: Tissue Culture and Cellular Life in Twentieth Century Biomedicine” (PhD diss., Massachusetts Institute of Technology, 1999); and Culturing Life: How Cells Became Technologies (Cambridge, MA: Harvard University Press, 2007). 90. Ross G. Harrison, “Observations of the Living Developing Nerve Fiber,” Anatomical Record 1, no. 5 (1907): 116. He published a far more detailed treatment of the method and its applications in 1910: “The Outgrowth of the Nerve Fiber as a Mode of Protoplasmic Movement,” Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 9, no. 4 (1910): 787–846. For a detailed historical account of Harrison’s experiments and their implica-

242

Notes to Pages 152–153

tions see also Hannah Landecker, “New Times for Biology: Nerve Cultures and the Advent of Cellular Life in Vitro,” Studies in History and Philosophy of Biological and Biomedical Sciences 33, no. 4 (2002): 667–94. 91. Edna Steinhardt, C. Israeli, and R. A. Lambert, “Studies on the Cultivation of the Virus of Vaccinia,” Journal of Infectious Diseases 13, no. 2 (1913): 294–300; Edna Steinhardt and Robert A. Lambert, “Studies on the Cultivation of the Virus of Vaccinia. II,” Journal of Infectious Diseases 14, no. 1 (1914): 87–92; C. Kling and C. Levaditi, “Studies of Acute Epidemic Poliomyelitis,” Annales de l’Institut Pasteur 27, no. 9 (1913): 718–46. 92. Hannah Landecker, “Building ‘A New Type of Body in Which to Grow a Cell’: Tissue Culture at the Rockefeller Institute, 1910–1914,” in Creating a Tradition of Biomedical Research: Contributions to the History of the Rockefeller University, ed. Darwin Stapleton (New York: Rockefeller University Press, 2004), 151; J. A. Witkowski, “Alexis Carrel and the Mysticism of Tissue Culture,” Medical History 23, no. 3 (1979): 279; “Dr Alexis Carrel and Tissue Culture,” Journal of the American Medical Association, 252, no. 1 (1984): 44, emphasis in original. 93. Jacob Furth, “The Making and Missing of Discoveries: An Autobiographical Essay,” Cancer Research 36, no. 3 (1976): 873. 94. Alexis Carrel, “Tissue Cultures in the Study of Viruses,” in Filterable Viruses, ed. Thomas Rivers (Baltimore: Williams & Wilkins, 1928), 97–109. 95. Rivers and Benison, Tom Rivers, 115. 96. Witkowski, “Alexis Carrel and the Mysticism of Tissue Culture,” 296. 97. Carrel, “Tissue Culture and Viruses,” 97–98. 98. Alexis Carrel and Montrose T. Burrows, “Cultivation in Vitro of Malignant Tumors,” Journal of Experimental Medicine 13, no. 5 (1911): 571–75. 99. Alexis Carrel, “Some Conditions of the Reproduction in Vitro of the Rous Virus,” Journal of Experimental Medicine 43, no. 5 (1926): 647, emphasis added. 100. Enders, Robbins, and Weller, “Cultivation of the Poliomyelitis Viruses,” 450. 101. Thomas H. Weller, and Frederick C. Robbins, “John Franklin Enders, February 10, 1897–September 8, 1985,” Biographical Memoirs of the National Academy of Science 60 (1991): 53–55; Enders, Robbins, and Weller, “Cultivation of Poliomyelitis Viruses,” 449. 102. Mark Henderson, “Frederick Chapman Robbins,” Lancet 362, no. 9391 (2003): 1245; Enders, Robbins, and Weller, “Cultivation of the Poliomyelitis Viruses,” 450. 103. George O. Gey, “An Improved Technic for Massive Tissue Culture,” American Journal of Cancer 17, no. 3 (1933): 752–56.

Notes to Pages 154–156

243

104. Thomas H. Weller, “As It Was and as It Is: A Half-­Century of Progress,” Journal of Infectious Diseases 159, no. 3 (1989): 379. 105. Hans-­Jörg Rheinberger,“From Microsomes to Ribosomes: ‘Strategies’ of ‘Representation,’” Journal of the History of Biology 28, no. 1 (1995): 72–79 106. Warren Weaver, “Molecular Biology,” in The Rockefeller Foundation Annual Report, ed. Raymond B. Fosdick, Norma S. Thompson, Wilbur Sawyer, Alan Gregg, Warren Weaver, Joseph H. Willits, and David H. Stevens (1938): 39–44, 203–20. 107. W. T. Astbury, “Adventures in Molecular Biology,” Harvey Lectures 46 (1950): 3. It is worth mentioning that Astbury was the head of the laboratory at the University of Leeds where, in 1951, his research fellow Elwyn Beighton produced X-­ray photographs of DNA virtually identical to the famous 1953 photographs from Rosalind Franklin’s laboratory that was, unbeknown to her, the catalyst for Watson and Crick’s proposition of the double helix structure of DNA—­see Kersten Hall, “William Astbury and the Biological Significance of Nucleic Acids, 1938–1951,” Studies in History and Philosophy of Biological and Biomedical Sciences 42, no. 2 (2011): 119–20. 108. Creager, Life of a Virus, 185. 109. Angela N. H. Creager, “The Paradox of the Phage Group: Essay Review,” Journal of the History of Biology 43, no. 1 (2010): 183, in reference to John Cairns, Gunther S. Stent, and James D. Watson, eds., Phage and the Origins of Molecular Biology (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory of Quantitative Biology, 1966). 110. André Lwoff, “The Progphage and I,” in Cairns, Stent, and Watson, Phage and the Origins of Molecular Biology, 79. 111. John C. Kendrew, “How Molecular Biology Started,” Scientific American 216, no. 3 (1967): 141. 112. Kay, “Stanley’s Crystallization,” 450. 113. Angela Creager and Jean-­Paul Gaudillière, “Experimental Technologies and Techniques of Visualisation: Cancer as a Viral Epidemic, 1930– 1960,” in Heredity and Infection: The History of Disease Transmission, ed. Ilana Löwy and Jean-­Paul Gaudillière (London: Routlege, 2001), 224. 114. Gladys Kostyrka, “La place des virus dans le monde vivant” (PhD diss., Sorbonne, 2018).

Chapter 7: Knitting Done Epigraphs: André Lwoff, “Interaction among Virus, Cell, and Organism,” Science 152, no. 3726 (1966): 1216. Howard M. Temin, “The Control of Cellular

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Notes to Pages 156–160

Morphology in Embryonic Cells Infected with Rous Sarcoma Virus in Vitro,” Virology 10, no. 2 (1960): 196. 1. André Lwoff, “The Prophage and I,” in Phage and the Origins of Molecular Biology, ed. John Cairns, Gunther S. Stent, and James D. Watson (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory of Quantitative Biology, 1966), 97. 2. F. M. Burnet and Margot McKie, “Observations on a Permanently Lysogenic Strain of B. entiritidis Gaertner,” Australian Journal for Experimental Biology and Medical Science 6, no. 4 (1929): 282–84; Eugène Wollman, “The Phenomenon of Twort-­d’Herelle and Its Significance,” Lancet 226, no. 5858 (1935): 1312–14; Eugène Wollman and Elisabeth Wollman, “Les ‘phases’ des bactériophages (facteurs lysogènes),” Comptes rendus des séances de la Société de biologie 124 (1937): 931–34. 3. Melvin Cohn, “Burnet, Lysogeny and Creativity,” Walter and Eliza Hall Institute of Medical Research Annual Review 1978–79, special volume, A Tribute to Sir Macfarlane Burnet (Melbourne: Walter and Eliza Hall Institute of Medical Research, 1979), 9; André Lwoff, “From Protozoa to Bacteria and Viruses: Fifty Years with Microbes,” Annual Reviews in Microbiology 25, no. 1 (1971): 14. 4. Oswald Avery, Colin M. MacLeod, and Maclyn McCarty, “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types,” Journal of Experimental Medicine 79, no. 2 (1944): 137–58. 5. André Lwoff, “Lysogeny,” Bacteriological Reviews 17, no. 4 (1953): 330. 6. W. C. Summers, “How Bacteriophage Came to Be Used by the Phage Group,” Journal of the History of Biology 26, no. 2 (1993): 255–67. 7. Lwoff, “From Protozoa to Bacteria and Viruses,” 14–15. 8. Lwoff, “Lysogeny”; Lwoff, “The Concept of Virus,” Journal of General Microbiology 17, no. 2 (1957): 256. 9. Lwoff, “Prophage and I,” 97. 10. Édouard Chatton and André Lwoff, “Imprégnation, par diffusion argentique, de l’infraciliature des Ciliés marins et d’eau douce, après fixation cytologique et sans dessiccation,” Comptes Rendus de la Société de Biologie 104 (1930): 834–36. 11. Laurent Loison, Jean Gayon, and Richard M. Burian, “The Contributions—­and Collapse—­of Lamarckian Heredity in Pasteurian Molecular Biology: 1. Lysogeny, 1900–1960,” Journal of the History of Biology 50, no. 1 (2017): 30; Gladys Kostyrka and Neeraja Sankaran, “From Obstacle to Lynchpin: The Evolution of the Role of Bacteriophage Lysogeny in Defining and Understanding Viruses,” Notes and Records: The Royal Society Journal of the History of Science 74, no. 4 (2020): 599–623.

Notes to Pages 160–163

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12. Lwoff, “Prophage and I,” 88–89. 13. As cited in Charles Galperin, “Le bactériophage, la lysogénie et son déterminisme génétique,” History and Philosophy of the Life Sciences 9, no. 2 (1987): 212. 14. See also Loison, Gayon, and Burian, “Contributions—­and Collapse,” 30. 15. Lwoff, “Prophage and I,” 89. See also Andre Lwoff, André Lwoff, une autobiographie: itinéraire scientifique d’un prix Nobel, ed. Laurent Loison (Paris: Hermann, 2017). 16. Michel Morange, “What History Tells Us III. André Lwoff: From Protozoology to Molecular Definition of Viruses,” Journal of Biosciences 30, no. 5 (2005): 592. 17. Lwoff, “Prophage and I,” 89. 18. A. Lwoff, “Cycle du bactériophage chez une bactérie lysogène,” Bulletin of the World Health Organization 6, nos. 1–2 (1952): 251. Translation mine. 19. Lwoff, “Lysogeny,” 279. 20. Lwoff, “Prophage and I,” 90. 21. André Lwoff and A. Gutmann, “La perpétuation endomicrobienne du bactériophage chez un Bacillus megatherium lysogène,” Comptes rendus hebdomadaires des séances de l’Académie des Sciences 229 (1949): 789–91; “La libération de bactériophages par la lyse d’une bactérie lysogène,” Comptes rendus hebdomadaires des séances de l’Académie des Sciences 230 (1950): 155. 22. Alfred D. Hershey and J. Bronfenbrenner, “Bacterial Viruses: Bacteriophages,” in Viral and Rickettsial Infections of Man, ed. Thomas M. Rivers, 2nd ed. (Philadelphia: J. B. Lippincott Company, 1948), 197. 23. Wolfgang Joklik, in Microbiology—­A Centenary Perspective, ed. Wolfgang Joklik, Lars G. Ljungdahl, and Alison D. O’Brien (Washington, DC: American Society for Microbiology, 1999), 471. 24. Herman J. Muller, “The Production of Mutations by X-­Rays,” Proceedings of the National Academy of Sciences of the United States of America 14, no. 9 (1928): 714–26. 25. André Lwoff, Louis Siminovitch, and Niels Kjeldgaard, “Induction de la lyse bactériophagique de la totalité d’une population microbienne lysogène,” Comptes rendus hebdomadaires des séances de l’Académie des Sciences 231 (1950): 191. In its original French the passage reads as follows: “La lyse de la souche lysogène n’est donc vraisemblablement pas l’effet direct du rayonnement U.V.” 26. André Lwoff and A. Gutmann, “Investigations on a Lysogenic Bacillus megatherium,” in Papers on Bacterial Viruses, ed. Gunther S. Stent (Boston: Little, Brown, 1960), 318.

246

Notes to Pages 163–166

27. Lwoff, “Lysogeny,” 280. 28. Loison, Gayon, and Burian, “Contributions—­and Collapse,” 26; Gladys Kostyrka, “La place des virus dans le monde vivant” (PhD diss., Sorbonne, 2018), 416–17. 29. Lwoff, “Lysogeny,” 272; emphasis in original. 30. F. Macfarlane Burnet, Changing Patterns: An Atypical Autobiography (Melbourne: Heinemann, 1968), 53. 31. Lwoff, “Lysogeny,” 280–81. 32. Richard Shope, “Immunization of Rabbits to Infectious Papillomatosis,” Journal of Experimental Medicine 65, no. 2 (1937): 219–31; John Kidd and Peyton Rous, “A Transplantable Rabbit Carcinoma Originating in a Virus-­ Induced Papilloma and Containing the Virus in Masked or Altered Form,” Journal of Experimental Medicine 71, no. 6 (1940): 813–38. 33. Richard Shope, “‘Masking,’ Transformation and Interepidemic Survival of Animal Viruses,” in Viruses 1950, ed. Max Delbrück (Pasadena: California Institute of Technology, 1950), 80. 34. Milislav Demerec, “Foreword to Symposium on Viruses,” Cold Spring Harbor Symposia on Quantitative Biology 18 (1953): v. 35. Max Delbrück, “Introductory Remarks about the Program” Cold Spring Harbor Symposia on Quantitative Biology 18 (1953): 1. Emphasis in original. 36. Lwoff, “Concept of Virus,” 239. 37. William Summers,“Inventing Viruses,” Annual Review of Virology 1, no. 1 (2014): 25; See also M. H. V. van Regenmortel, “Viruses Are Real, Virus Species Are Man-­Made, Taxonomic Constructions,” Archives of Virology 148, no. 12 (2003): 2481–88, for a fuller discussion of this issue. 38. Lwoff, “Concept of Virus,” 244. 39. Lwoff, “Lysogeny,” 270, 323. 40. Lwoff, “Concept of Virus,” 246. The phrase “Lipmann system,” perhaps less familiar today, refers to the system of enzymes living organisms posses for conducting their metabolic functions. See also H. Chantrenne, “For the 25th Anniversary of ~ P,” in Current Aspects of Biochemical Energetics: Fritz Lipmann Dedicatory Volume, ed. Nathan O. Kaplan and Eugene P. Kennedy (New York: Academic Press, 1966); Fritz Lipmann, “A Long Life in Times of Great Upheaval,” Annual Review of Biochemistry 53, no. 1 (1984): 1–34. 41. Lwoff, “Concept of Virus,” 239. Pons asinorum, literally translated from Latin as the “asses’s bridge” is a reference to Euclid’s law about the isosceles triangles, thus named for the difficulty that students had in grasping the concept. See Dictionary.com, s.v. “pons asinorum,” accessed October 20, 2018, https://www.dictionary.com/browse/pons-­asinorum. Lwoff introduced the

Notes to Pages 167–171

247

viruses as a pons asinorum, adding, “I suspect that some of you have come to see how I behave on the asses’ bridge.” 42. Nadine Peyrieras and Michel Morange, “The Study of Lysogeny at the Pasteur Institute (1950–1960): An Epistemologically Open System,” Studies in History and Philosophy of Biological and Biomedical Sciences 33, no. 3 (2002): 425. 43. Lwoff, “Lysogeny,” 324. 44. Christopher Andrewes to Peyton Rous, December 6, 1935, Andrewes, Christopher H., folder 1, 1929–35, Series 1, Peyton Rous Papers, American Philosophical Society, Philadelphia (hereafter cited as Rous Papers); Andrewes, “Latent Virus Infections and Their Possible Relevance to the Cancer Problem (President’s Address),” Proceedings of the Royal Society of Medicine 33, no. 2 (1939): 75–86. See also Neeraja Sankaran and Ton van Helvoort, “Andrewes’s Christmas Fairy Tale: Atypical Thinking about Cancer Aetiology in 1935,” Notes and Records: the Royal Society Journal of the History of Science 70, no. 2 (2016): 175–201. 45. Ronald T. Javier and Janet S. Butel, “The History of Tumor Virology,” Cancer Research 68, no. 19 (2008): 7693–706. 46. Howard Temin, “The DNA Provirus Hypothesis: The Establishment and Implications of RNA-­Directed DNA Synthesis,” in The Nobel Lectures: Physiology or Medicine, 1971–1980, edited by Jan Lindsten (London: World Scientific, 1992), 245–63. The specific primary publications that he was referring to in this comment are: Avery, MacLeod, and McCarty, “Studies on the Chemical Nature”; James D. Watson and Francis H. C. Crick, “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid,” Nature 171, no. 4356 (1953): 737–38; Lwoff, “Lysogeny”; Emory L. Ellis and Max Delbrück, “The Growth of Bacteriophage,” Journal of General Physiology 22, no. 3 (1939): 365–84; John F. Enders, Thomas H. Weller, and Frederick C. Robbins, “Cultivation of the Lansing Strain of Poliomyelitis Virus in Cultures of Various Human Embryonic Tissues,” Science 109, no. 2822 (1949): 85–87; and Renato Dulbecco and Marguerite Vogt, “One-­Step Growth Curve of Western Equine Encephalomyelitis Virus on Chicken Embryo Cells Grown in Vitro and Analysis of Virus Yields from Single Cells,” Journal of Experimental Medicine 99, no. 2 (1954): 183–99. 47. Renato Dulbecco, interview by Shirley K. Cohen, September 9, 1998, Caltech Oral Histories, http://oralhistories.library.caltech.edu/26/1/OH _Dulbecco_R.pdf. 48. Renato Dulbecco, “Reactivation of Ultra-­Violet-­Inactivated Bacteriophage by Visible Light,” Nature 163, no. 4155 (1949): 949–50. 49. Renato Dulbecco, “The Plaque Technique and the Development of

248

Notes to Pages 171–174

Quantitative Animal Virology,” in Phage and the Origins of Molecular Biology, ed. John Cairns, Gunther S. Stent, and James D. Watson (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory of Quantitative Biology, 1966), 287–88. 50. Dulbecco, interview by Cohen, September 10, 1998. See also Daniel Kevles, “Renato Dulbecco and the New Animal Virology: Medicine, Methods, and Molecules,” Journal of the History of Biology 26, no. 3 (1993): 409–42, for a more detailed account of Dulbecco’s life in science. 51. Renato Dulbecco and Marguerite Vogt, “Some Problems of Animal Virology as Studied by the Plaque Technique,” Cold Spring Harbor Symposia on Quantitative Biology 18 (1953): 273–79. 52. Dulbecco, interview by Cohen, September 10, 1998. 53. Dulbecco, “Plaque Technique,” 287–91. 54. Dulbecco, interview by Cohen, September 10, 1998. 55. Renato Dulbecco, “Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus,” Proceedings of the National Academy of Sciences 38, no. 8 (1952): 747–52; Dulbecco and Vogt, “One-­Step Growth Curve.” 56. John W. Drake, and James F. Crow, “Recollections of Howard Temin (1934–1994),” Genetics 144, no. 1 (1996): 2. 57. Dulbecco, “Plaque Technique,” 290, 291. 58. Rubin, “Quantitative Tumor Virology,” in Phage and the Origins of Molecular Biology, ed. John Cairns, Gunther S. Stent, and James D. Watson (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory of Quantitative Biology, 1966), 292–93. See also Ludwik Gross, “Pathogenic Properties, and ‘Vertical’ Transmission of the Mouse Leukemia Agent,” Proceedings of the Society for Experimental Biology and Medicine 78, no. 1 (1951): 342–48. 59. Drake and Crow, “Recollections,” 2. 60. Rubin, “Quantitative Tumor Virology,” 294. 61. E. V. Keogh, “Ectodermal Lesions Produced by the Virus of Rous Sarcoma,” British Journal of Experimental Pathology 19, no. 1 (1938): 1–11. 62. Temin, “DNA Provirus Hypothesis,” 246; Robert A. Manaker and Vincent Groupé, “Discrete Foci of Altered Chicken Embryo Cells Associated with Rous Sarcoma Virus in Tissue Culture,” Virology 2, no. 6 (1956): 838–40. 63. Rubin, “Quantitative Tumor Virology,” 295; Howard Temin and Harry Rubin, “Characteristics of an Assay for Rous Sarcoma Virus and Rous Sarcoma Cells in Tissue Culture,” Virology 6, no. 3 (1958): 669–88. 64. Howard M. Temin and Harry Rubin, “A Kinetic Study of Infection of Chick Embryo Cells in Vitro by Rous Sarcoma Virus,” Virology 8, no. 2 (1959): 209–22. 65. Temin, “DNA Provirus Hypothesis,” 246.

Notes to Pages 174–177

249

66. Angela Creager and Jean-­Paul Gaudillière, “Experimental Technologies and Techniques of Visualisation: Cancer as a Viral Epidemic, 1930– 1960,” in Heredity and Infection: The History of Disease Transmission, ed. Ilana Löwy and Jean-­Paul Gaudillière (London: Routlege, 2001), 226. 67. The earliest report on the composition of RNA was a report by R. Bather, “The Nucleic Acid of Partially Purified Rous No. 1 Sarcoma Virus,” British Journal of Cancer 11, no. 4 (1957): 611–19. A few years later, Lionel Crawford, a member of Dulbecco’s own team, provided more definitive conformation of this fact using sophisticated separation techniques: L. V. Crawford, and E. M. Crawford, “The Properties of Rous Sarcoma Virus Purified by Density Gradient Centrifugation,” Virology 13, no. 2 (1961): 227–32. 68. Francis H. C. Crick, “On Protein Synthesis,” Symposia of the Society for Experimental Biology 12 (1958): 152–53, emphasis in original. 69. James D. Watson, The Double Helix: A Personal Account of the Discovery of the Structure of DNA (1968, repr., New York: Norton, 1980), 90. Here Watson claims to have sketched out this idea in the early 1950s even before he and Crick proposed their double helix structure for DNA. 70. Dulbecco, interview by Cohen, September 10, 1998. 71. Ludwik Gross, “A Filterable Agent, Recovered from Ak Leukemic Extracts, Causing Salivary Gland Carcinomas in C3H Mice,” Proceedings of the Society for Experimental Biology and Medicine 83, no. 2 (1953): 414–21; “The Fortuitous Isolation and Identification of the Polyoma Virus,” Cancer Research 36, no. 11 (1976): 4195. See also Daniel Kevles, “Pursuing the Unpopular: A History of Courage. Viruses. and Cancer,” in Hidden Histories of Science, ed. Robert B. Silvers (New York: New York Review of Books, 1995), 81–84; Gregory J. Morgan, “Ludwik Gross, Sarah Stewart, and the 1950s Discoveries of Gross Murine Leukemia Virus and Polyoma Virus,” Studies in History and Philosophy of Biological and Biomedical Sciences 48 (December 2014): 200–209. 72. Renato Dulbecco and G. Freeman, “Plaque Production by the Polyoma Virus,” Virology 8, no. 3 (1959): 396–97; See Renato Dulbecco, “From the Molecular Biology of Oncogenic DNA Viruses to Cancer,” Science 192, no. 4238 (1976): 437. 73. Harry M. Weaver, “Foreword,” Cancer Research 20, no. 5, part 1 (1960): 671. 74. Christopher Andrewes, “The Bearing of Recent Work on the Virus Theory of Cancer,” British Medical Journal 1, no. 4645 (1950): 81–85; Andrewes, “Discussion of Dr. Luria’s Paper,” Cancer Research 20, no. 5, part 1 (1960): 189. 75. Peyton Rous, “Opening Remarks,” Cancer Research 20, no. 5, part 1 (1960): 672.

250

Notes to Pages 177–179

76. Peyton Rous, “Summary of Informal Discussions,” Cancer Research 20, no. 5, part 1 (1960): 707. 77. Rous, “Opening Remarks,” 672; Rous, “Summary of Informal Discussions,” 707. 78. Salvador Luria, “Viruses, Cancer Cells, and the Genetic Concept of Virus Infection,” Cancer Research 20, no. 5, part 1 (1960): 686, 679. 79. Andrewes, “Discussion of Dr. Luria’s Paper,” 689, 690. 80. Andrewes, “Discussion of Dr. Luria’s Paper,” 691; Salvador E. Luria, General Virology (New York: Wiley, 1953), 362. 81. Rous, “Summary of Informal Discussions,” 707. 82. Andrewes, “Discussion of Dr. Luria’s Paper,” 689. 83. Renato Dulbecco, “A Consideration of Virus-­Host Relationship in Virus-­Induced Neoplasia at the Cellular Level,” Cancer Research 20, no. 5, part 1 (1960): 751–61. 84. Dulbecco, “Consideration of Virus-­Host Relationship,” 756, emphasis added; Renato Dulbecco and G. Freeman, “Plaque Production by the Polyoma Virus,” Virology 8, no. 3 (1959): 396. 85. André Lwoff, “Tumor Viruses and the Cancer Problem: A Summation of the Conference,” Cancer Research 20, no. 5, part 1 (1960): 820–29. Although he did not immediately switch his interests to cancer research, Lwoff spent the last four years of his research career from 1968 until his retirement as the director of Institute for Scientific Research on Cancer (now known as the Institut André Lwoff) in Villejuif, outside Paris. 86. Peyton Rous, “Viruses and Tumors,” in Virus Diseases, ed. Thomas M. Rivers (Ithaca, NY: Cornell University Press, 1943), 161. 87. Lwoff, “Tumor Viruses and the Cancer Problem,” 820. 88. Rous, “Comments,” Cancer Research 20, no. 5, part 1 (1960): 830. 89. J. D. Smith, G. Freeman, M. Vogt, and R. Dulbecco, “The Nucleic Acid of Polyoma Virus,” Virology 12, no. 2 (1960): 195; Marguerite Vogt and Renato Dulbecco, “Virus-­Cell Interaction with a Tumor-­Producing Virus,” Proceedings of the National Academy of Sciences of the United States of America 46, no. 3 (1960): 369. 90. David Gillespie and Sol Spiegelman, “A Quantitative Assay for DNA-­ RNA Hybrids with DNA Immobilized on a Membrane,” Journal of Molecular Biology 12, no. 3 (1965): 829–42; Heiner Westphal and Renato Dulbecco, “Viral DNA in Polyoma-­ and SV40-­Transformed Cell Lines,” Proceedings of the National Academy of Sciences of the United States of America 59, no. 4 (1968): 1158–65; J. Sambrook, H. Westphal, P. R. Srinivasan, and R Dulbecco, “The Integrated State of Viral DNA in SV40-­Transformed Cells,” Proceedings of the National Academy of Sciences of the United States of America 60, no. 4 (1968):

Notes to Pages 179–183

251

1288–95. For a fuller and richly deserved treatment of the importance of the hybridization techniques described in, readers are referred to Susie Fisher’s in-­depth analysis: “Not Just ‘a Clever Way to Detect Whether DNA Really Made RNA’: The Invention of DNA–RNA Hybridization and Its Outcome,” Studies in History and Philosophy of Science Part C: Studies in History and Philosophy of Biological and Biomedical Sciences 53 (October 2015): 40–52. 91. Drake and Crow, “Recollections,” 2. 92. Dulbecco, interview by Cohen, September 10, 1998. 93. Howard M. Temin, “The Interaction of Rous Sarcoma Virus and Cells in Vitro” (PhD diss., California Institute of Technology, 1960), 47–49; Harry Rubin and Howard M. Temin, “A Radiological Study of Cell-­Virus Interaction in the Rous Sarcoma,” Virology 7, no. 1 (1959): 75–91. 94. Dulbecco, interview by Cohen, September 10, 1998. 95. Renato Dulbecco, “Howard M. Temin. 10 December 1934–9 February 1994,” Biographical Memoirs of Fellows of the Royal Society 41 (1995): 475–76. 96. Temin, “Control of Cellular Morphology,” 196. 97. Harry Rubin, “Quantitative Relations between Causative Virus and Cell in the Rous No. 1 Chicken Sarcoma,” Virology 1, no. 5 (1955): 445–73; Rubin, “The Early History of Tumor Virology: Rous, RIF, and RAV,” Proceedings of the National Academy of Sciences 108, no. 35 (2011): 14394. 98. Rubin and Temin, “Radiological Study,” 90. 99. Harrison Echols, Operators and Promoters: The Story of Molecular Biology and Its Creators, ed. Carol A. Gross (Berkeley: University of California Press, 2001), 299–300. 100. See Temin, “DNA Provirus Hypothesis,” 248; Rubin, “Early History of Tumor Virology,” 14394. 101. Rous to Harry Rubin, November 6, 1958, Rubin, Harry Folder #2, 1953–1964, Rous Papers. 102. Harry Rubin and Howard M. Temin, “Infection with the Rous Sarcoma Virus in Vitro,” Federation Proceedings 17, no. 4 (1958): 1003. 103. Harry Rubin, Ardra Cornelius, and Lois Fanshier, “The Pattern of Congenital Transmission of an Avian Leukosis Virus,” Proceedings of the National Academy of Sciences 47, no. 7 (1961): 1068; See also Rubin, “Early History of Tumor Virology,” 14394. 104. “Howard M. Temin: Biographical,” The Nobel Prize, https://www .nobelprize.org/prizes/medicine/1975/temin/biographical/. 105. David Baltimore, “Thinking about Howard Temin,” Genes and Development 9, no. 11 (1995): 1304. 106. Temin, “Control of Cellular Morphology,” 197.

252

Notes to Pages 184–189

107. Howard Temin, “Cancer and Viruses,” Engineering and Science 23, no. 4 (1960): 24. 108. Susie Fisher, “Not Beyond Reasonable Doubt: Howard Temin’s Provirus Hypothesis Revisited,” Journal of the History of Biology 43, no. 4 (2010): 669, 673. 109. Howard M. Temin, “Separation of Morphological Conversion and Virus Production in Rous Sarcoma Virus Infection,” Cold Spring Harbor Symposia on Quantitative Biology 27 (1962): 414; Temin, “Further Evidence for a Converted, Non-­Virus-­Producing State of Rous Sarcoma Virus-­Infected Cells,” Virology 20, no. 2 (June 1, 1963): 235–45. 110. James A. Marcum, “Experimental Series and the Justification of Temin’s DNA Provirus Hypothesis,” Synthese 154, no. 2 (January 2007): 268–71. 111. E. Reich, R. M. Franklin, A. J. Shatkin, and E. L. Tatum, “Effect of Actinomycin D on Cellular Nucleic Acid Synthesis and Virus Production,” Science 134, no. 3478 (1961): 556. 112. Howard M. Temin, “The Effects of Actinomycin D on Growth of Rous Sarcoma Virus in Vitro,” Virology 20, no. 4 (1963): 578–79, 582. 113. Howard M. Temin,“Homology between RNA from Rous Sarcoma Virus and DNA from Rous Sarcoma Virus-­Infected Cells,” Proceedings of the National Academy of Sciences of the United States of America 52, no. 2 (1964): 328. 114. Howard M. Temin, “Nature of the Provirus of Rous Sarcoma,” in International Conference on Avian Tumor Viruses: Proceedings, edited by Joseph Willis Beard, National Cancer Institute Monograph 17 (Washington, DC: U.S. Department of Health, Education, and Welfare, Public Health Service, National Cancer Institute, 1964), 557–70. 115. See James A. Marcum, “From Heresy to Dogma in Accounts of Opposition to Howard Temin’s DNA Provirus Hypothesis,” History and Philosophy of the Life Sciences 24, no. 2 (2002): 165–92, for a full discussion of the reception of Temin’s hypothesis. 116. Van Rensselaer Potter, “Biochemical Perspectives in Cancer Research,” Cancer Research 24, no. 7 (1964): 1090. 117. Rubin, “Early History of Tumor Virology,” 14394. 118. Fisher, “Not Beyond Reasonable Doubt,” 686. 119. Drake and Crow, “Recollections,” 3–4. 120. Dulbecco, “Howard M. Temin,” 476, 121. Rubin, “Early History of Tumor Virology,” 14394, with reference to John P. Bader, “The Role of Deoxyribonucleic Acid in the Synthesis of Rous Sarcoma Virus,” Virology 22, no. 4 (1964): 462–68; and John P. Bader, “The Requirement for DNA Synthesis in the Growth of Rous Sarcoma and Rous-­ Associated Viruses,” Virology 26, no. 2 (1965): 253–61.

Notes to Pages 189–193

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122. Reverse transcriptase was discovered independently by David Baltimore at MIT; and Temin himself in collaboration with a new colleague Satoshi Mizutani. Baltimore, “RNA-­Dependent DNA Polymerase in Virions of RNA Tumour Viruses,” Nature 226, no. 5252 (June 27, 1970): 1209–11. Howard M. Temin and Satoshi Mizutani, “Viral RNA-­Dependent DNA Polymerase: RNA-­Dependent DNA Polymerase in Virions of Rous Sarcoma Virus,” Nature 226, no. 5252 (1970): 1211–13. 123. As quoted by Daniel Kevles, “Howard Temin: Rebel of Evidence and Reason,” in Rebels, Mavericks, and Heretics in Biology, ed. Oren Harman and Michael Dietrich (New Haven, CT: Yale University Press, 2008), 260. 124. Temin, “DNA Provirus Hypothesis,” 252. 125. James D. Watson and Alfred Berry, DNA: The Secret of Life, 1st ed. (New York: Alfred A. Knopf, 2003), 218. 126. Charles Dickens, A Tale of Two Cities (Public Domain Books 2010), 131, Kindle; Kevles, “Pursuing the Unpopular.”

Afterword Epigraph: Charles Dickens, A Tale of Two Cities (Public Domain Books 2010), 237, Kindle. 1. Steven Shapin, “Discipline and Bounding: The History and Sociology of Science as Seen through the Externalism-­Internalism Debate,” History of Science 30, no. 90 (1992): 333–34. 2. Steven Shapin, The Scientific Revolution (Chicago: University of Chicago Press, 1996), 9; Donald R. Kelley, “Intellectual History and Cultural History: The Inside and the Outside,” History of the Human Sciences 15, no. 2 (2002): 3. 3. Angela N. H. Creager, The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930–1965 (Chicago: University of Chicago Press, 2002). 4. Michael Ruse, “Review of Heredity Explored: Between Public Domain and Experimental Science, 1850–1930, by Staffan Müller-­Wille and Christina Brandt,” Journal of the History of the Behavioral Sciences 53, no. 1 (2017): 105. 5. Robin Scheffler, interview by Carrie Adams, May 16, 2019, https:// pressblog.uchicago.edu/2019/05/16/5-­questions-­for-­robin-­wolfe-­scheffler -­author-­of-­a-­contagious-­cause-­t he-­american-­hunt-­for-­cancer-­viruses-­and -­t he-­rise-­of-­molecular-­medicine.html. 6. Siobhán M. O’Connor, Christopher E. Taylor, and James M. Hughes, “Emerging Infectious Determinants of Chronic Diseases,” Emerging Infectious Diseases 12, no. 7 (2006): 1051. 7. Karen Ho, “Bacteriophage Therapy for Bacterial Infections: Rekindling a Memory from the Pre-­Antibiotics Era,” Perspectives in Biology and Medicine

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Notes to Pages 193–198

44, no. 1 (2001): 1–16; Nina Chanishvili, “Phage Therapy—­History from Twort and d’Herelle through Soviet Experience to Current Approaches,” in Advances in Virus Research, vol. 83, Bacteriophages, Part B, edited by Waclaw Szybalski and Malgorzata Lobocka, (San Diego: Academic Press, 2012), 3–40; William C. Summers, “The Strange History of Phage Therapy,” Bacteriophage 2, no. 2 (2012): 130–33; Xavier Wittebole, Sophie De Roock, and Steven M. Opal, “A Historical Overview of Bacteriophage Therapy as an Alternative to Antibiotics for the Treatment of Bacterial Pathogens,” Virulence 5, no. 1 (2014): 226–35; Zhabiz Golkar, Omar Bagasra, and Donald Gene Pace, “Bacteriophage Therapy: A Potential Solution for the Antibiotic Resistance Crisis,” Journal of Infection in Developing Countries 8, no. 2 (2014): 129–36. 8. Robin Wolfe Scheffler, A Contagious Cause: The American Hunt for Cancer Viruses and the Rise of Molecular Medicine (Chicago: University of Chicago Press, 2019); Erling Norrby, Nobel Prizes: Cancer, Vision and the Genetic Code (Hackensack, NJ: World Scientific, 2019); and Anna Marie Skalka, Discovering Retroviruses: Beacons in the Biosphere (Cambridge, MA: Harvard University Press, 2018).

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INDEX Ahrens, Eva, ix Allard, H. A., 76 American Association of Cancer Research (Potter’s lecture, 1964), 191–92 American Cancer Society (1959 symposium), 179–83 American Phage Group, 89, 98, 158, 162 American Zoological Society (1925 meeting), 43 Anderson, Thomas, 89–90, 144 Andrewes, Christopher H.: American Cancer Society symposium (1959), 179–81; analogy for tumor causation, 130–32; “Christmas fairy story for oncologists,” 130–32; criticism of virus as cellular mutation, 180–81; correspondence with Murphy, 120-­123; correspondence with Rous, 128–30; early work at NIMR, 99–101; ideas about transmissible mutagens, 120–22; papilloma virus, 126, 127–28; photographs of, 121, 123; quoted, 104; relationship with Rous, 20, 37, 100, 101, 107, 109; size determination of viruses by filtration, 138–39; symbiosis, 130–31; three musketeers of viral theory of tumor etiology, 107; virology, 60; work on bacteriophages, 99, 138; work with Elford, 138–40 Andrewes, Sir Frederick, 99 anlage, 94–95, 167–68

Arkwright, Joseph, 98 Arrowsmith (Lewis), xi, 27, 198 Astbury, William, 157 asymptomatic carriers, 92 Avery, Oswald, 119, 120, 123–24, 161, 172 avian leukosis, 188 avian sarcomas, 12–13; illustration, 15 avian tumor virus conference (Duke University 1964), 191–92 Bacillus megatherium, 163, 164 bacteria contrasted with viruses, 67–72 bacterial lysis, 22 bacteriology, 67–72 Le Bactériophage (d’Herelle), 46, 84 bacteriophage: Burnet’s work on, 79–95, 103; cultivation of, 151; discovery of, 21–26; early reactions of scientific community to, 44–53; early research on, 21–28; 84–86; and genetics, 94; growth and multiplication of, 86–90; nature of, 44–53; overview, 4–5; secretion theory, 161–62; under the electron microscope, 146; the Wollmans’ work on, 95–97 Bail, Oskar, 88 Baker, Stephen, 41 Baltimore, David, 188–89, 193, 254n122 Barnard, Joseph E., 42, 100, 108 Bashford, Ernest, 39, 40 Bawden, Frederick, 135, 136, 142, 143 Bechhold, Heinrich, 101

289

Beijerinck, Martinus: contagium vivum fluidum, 68, 70–77; contrasted with Mendel, 75–76; on filtration of viruses, 65, 137; on obligate parasitism, 148–49; reception in scientific community, 73–75; work on tobacco mosaic virus (TMV), 62–64, 123 Benison, Saul, 67, 74, 154 Benzer, Seymour, 174 Bernal, John D., 142 Bertillon, Georges, 23–24 Billiau, Alfons, 50 Bordet, Jules: on bacteriophagy, 54–55; bacteriophagy as nutritive vitiation, 47–48, 55–56; lysogeny, discovery of, 49–50; on mutations, 55; Nobel Prize, 27, 46; opposition to d’Herelle, 46–47, 160; opposition to viral theory of bacteriophage, 44–45, 51, 114; photograph of, 54; quoted, 33 Bos, Lute, 59, 74, 75 Boycott, Arthur, 122, 129, 139 Bronfenbrenner, Jacques, 82, 88, 166 Bruynoghe, Richard, 50–51 Bull, Carroll, 22 Burian, Richard M., 97 Burnet, Frank Macfarlane: anlage, 94–94, 167; bacteriophage multiplication, 86–90, 150–53; d’Herelle’s influence on, 82–86; early research, 79–82; golden age of virology, 99–103; influenza virus assay technique, 139–40; illustrations of, 81, 83; lysogeny, the problem of, 90–95; lysogeny, early model for, 97, 130–31, 161, 167–169; on theories of bacteriophage, 53; on virology, 58–60; virus as microorganism, 135; work with Andrewes, 100 cancer: clinical treatment of, 39–41; parasitic theory of causation, 35–36, 38–39; theories of causation, 34–36, 39–40; viral theory of causation, 30, 104–112, 114, 117, 118, 120 Carrel, Alexis, 3–4, 116, 154–55

290

carriers, asymptomatic, 92 Carroll, Lewis, 147 Chatton, Edouard, 163 Ciuca, Mihai, 27, 47–49, 52, 107 Claude, Albert: cell biology, 147–48; correspondence with Flexner, 117; Nobel Prize, 115; viral theory, 118; use of centrifugation, 141; work on sarcoma agent in Murphy’s lab, 117–19, 141 Cohn, Melvin, 99, 161 Cold Spring Harbor symposium (1953), 169-­71, 175-­76 Cole, Rufus, 120 contagion theory, 67–68 A Contagious Cause (Scheffler), 8, 198 contagium vivum fluidum, 63–64, 68, 70–77 Corner, George, 19 Correns, Carl, 76 COVID-­19, 195, 197 Crawford, Dorothy, 7 Crawford, Lionel, 187 Creager, Angela, 7, 61, 66, 140, 177, 196 Crick, Francis, 172, 177–78 Crow, James, 192–93 crystallography, 141–43 Cunningham, Michael, xi–xii Dale, Sir Henry, 99 de Duve, Christian, 148 Delbrück, Max: bacteriophage growth, 89–90, 151; debate with Northrup, 102–3; denial of lysogeny, 162; and gene duplication, 91; influence on Lwoff, 161; molecular biology, 158; Phage and the Origins of Molecular Biology, 158, 162–63; provirus, 169–70; single-­burst experiment, 89, 158; Temin’s dissertation defense, 185–86 Demerec, Milislav, 169 de Vries, Hugo, 64, 75 d’Herelle, Félix: acknowledgement of Beijerinck, 73–75; background and early work, 23–26; bacteriophage, clinical implications of, 26, 28; bacteriophage, name, 25–26;

A Tale of Two Viruses

bacteriophage, nature of, 25, 47–48; Le Bactériophage, 46, 84; conflict with Bordet, 46–48; discovery of bacteriophage, 23–25; 31–32, 150; influence on Burnet, 82–86; opposition to, 34, 45–46, 48–50, 57–58; photograph of, 20; quoted, 11; support for, 50; Twort-­d’Herelle phenomenon, 27–28, 52 Dickens, Charles, ix, 5, 195 Discovering Retroviruses (Skalka), 8, 198 Dmochowski, Leon, 146 DNA: actionomycin D, effect on, 90–91; and genes, 158; as material basis of heredity, 119–20, 158, 167; in bacteriophage, 101; provirus, 189; and transforming principle, 119; tumor viruses, 178, DNA provirus hypothesis, 183, 186–93 DNA: The Secret of Life (Watson), 193 Doerr, Robert, 88 Doherty, Peter, 80 Drake, John, 184 Druce, Linda, 79, 80, 92 Dublanchet, Alain, 23 Duckworth, Donna, 26 Duke University, avian tumor virus conference (1964), 191–92 Dulbecco, Renato: American Cancer Society symposium (1959), 179, 181; Nobel Prize, 193; plaque-­counting assay for the animal viruses, 174–75; polyoma virus, 183; quantitative turn in animal virology, 175; rus, tumor virology, 177–78; switch to animal virology, 174–78; on Temin’s work, 184, 185, 193; work on bacteriophages, 172–74, 183 Duran-­Reynals, Francesc, 115–17 dysentery, 23–24 Eagles, George, 150 Earle, Wilton, 175 Echols, Harrison, 187 E. coli, 50 electron microscopy, 143–48; impact on virology, 143, 146–48

Index

Elford, William, 99, measurement of virus size, 100; 138–39 Ellis, Emory, 90, 91, 151, 158 Enders, John, 148, 155–56, 172, 179 enzyme theory of life, 78, enzyme theory of tumor causation, 113–15, 122–24 etiology of tumors: controversy concerning viral theory, 126–33; Duran-­Reynals’ reaction to viral theory, 115–17; Ewing’s ideas about, 35–36, 39–40, 55, 114–15, 125–29; enzyme theory, 114–15, 122–24; lessons from lysogeny, 171–78; Murphy’s reaction to viral theory, 112–15; Rous and rabbit papilloma virus, 124–26; theories of, 30, 34–36, 39–40; and transmissible mutagens, 119–22; viral theories, 30, 104–12, 114, 117, 118, 120 Ewing, James: on Andrewes’s analogy of causation, 132; correspondence with Murphy, 114–15, 124; correspondence with Rous, 58; Harvey Lecture (1908), 35–36; on the nature of viruses, 136; opposition to viral theory, 55, 114, 129; parasites, 39; quoted, 134; sarcoma virus discovery, 59; on Shope papilloma virus, 127–28; theories of cancer causation, 35–36, 39–40, 55, 114–15, 125–29 externalist approach, historical scholarship, 196–97 Fankuchen, Isidor, 142 filterable viruses, 13–15, 61–62, 65–67 Filterable Viruses (Rivers), 82, 154 filtration of viruses, 65–67 Fisher, Susie, 190, 192 Fleck, Ludwik, 68–70 Flexner, Simon, 12, 30, 59, 105, 141; correspondence with Murphy, 37, 109; correspondence with Rous 17–19, 28, 109, 125–26; role at Rockefeller Institute, 12, 30, 105 Flügge, Carl, 68

291

focus assay for tumor viruses, 177 foot-­and-­mouth disease of cattle, 64–65, 71 Fraenkel-­Conrat, Heinz, 61, 143, 151 Frosch, Paul, 64–65, 71, 72 Fruton, Joseph, 19, 28 Furth, Jacob, 37, 132–35, 154 Gabor, Dennis, 145 Galperin, Charles, 97 Gard, Sven, 148 Gasser, Herbert Spencer, 131 Gaudillière, Jean-­Paul, 177 Gautrey, Peter, 76 Gayon, Jean, 97 Geison, Gerald, 8 genes: bacteriophages as, 94; gene theory of tumor causation, 131; genetic theory of lysogeny, 97; viruses as, 94. See also DNA The Genesis and Development of a Scientific Fact (Fleck), 68 Gey, George, 156 glassy plaques, 23, 24–25 “glassy transformation,” 22 Goodpasture, Ernest, 135, 151–53 Gordon, Mervyn, 99 Gratia, André, 48, 49, 51–52, 56, 86 Griffith, Fred, 119 Gye, William E.: centrifugation, 140; correspondence with Rous, 107–8; identification of virus sarcoma agent as virus, 108–12; Murphy’s reaction to, 112–15; publication in Lancet, 42; sarcoma research at NIMR, 41–43, 100; support for Rous’s hypotheses, 41–42 Hadley, Philip, 88 Haerens, Hubert Augustin Félix. See d’Herelle, Félix Harrison, Ross, 153 Harvey Lectures: Ewing, 35–36; Rous, 126, 129, 132 Henderson, James, 37 Hershey, Alfred, 166

292

historical scholarship, 196–97 Holmes, Francis O., 151 Hooke, Robert, 143 The Hours (Cunningham), xi–xii Hughes, Sally Smith, 6 Huxley, Thomas, 70 internalist approach, historical scholarship, 196–97 An Introduction to the History of Virology (Waterson and Wilkinson), 6 Ivanovsky, Dmitri: chemical viewpoint, 71; disagreement with Beijerinck’s views, 64; filtration use of, 62–63, 137; nature of tobacco mosaic agent, 63, 64, 71; tobacco mosaic virus, discovery of, 62–63 Jenner, Edward, 58 Joklik, Wolfgang, 166 Journal of Experimental Medicine, 37 Kabeshima, Tameza, 45–46 Kay, Lily, 77, 142–43 Kellaway, Charles Halliley, 84 Kellenberger, Eduard, 101 Kelley, Donald, 196 Kendall, Arthur, 150 Kendrew, John, 158 Keogh, E. V., 176 Kevles, Dan, 8 kinetosomes, 163–64 Kjeldgaard, Niels, 166, 168 Koch, Robert, 61 Kuhn, Thomas, 68 Kunkel, Louis, 74, 140 Lancet, publication of Gye’s research, 42 Lederberg, Joshua, xi, 100–101 Ledingham, J. C. G. (John), 89, 140, 150 Levi, Giuseppe, 172–73 Levi-­Montalcini, Rita, 173 Lewis, Sinclair, xi, 27 life, nature of, 77–78 The Life of a Virus (Creager), 7 Lives (Plutarch), xi

A Tale of Two Viruses

Loeffler, Friedrich, 64–65, 71, 72, 75, 137 Löwy, Ilana, 29, 57 Luria, Salvador, 173, 179–80 Lwoff, André: American Cancer Society symposium (1959), 179, 181–83; anlage, 95; ciliates, work on, 164; defining “virus,” 135–36; early work, 162–64; genetic continuity, 163, 164, 167; kinetosomes, work on, 163–64; lysogeny, experimental evidence for, 165–67; lysogeny, investigations, 161–69; on nutritive vitiation, 47; photograph of, 165; quoted, 134, 160; relationship with Eugène Wollman, 163–64; Society for General Microbiology lecture, 170; on viral theory of cancer etiology, 171–72; virus, definition of, 170–71; viruses, ideas about role in tumor formation, 171–72 lyophilization, 106 lysogeny: analogy to tumor viruses, by Andrewes, 130–31; analogy to tumor viruses, by Lwoff, 170–71; Bordet/ Ciuca introduction of term, 49–50; Burnet and problem of lysogeny, 90–95; early hypotheses, 160–62; ideas and work of Eugène and Élisabeth Wollman on, 95–99; influence on cancer research, 178–83; life cycle of bacteriophage, 97; Lwoff and experimental evidence, 162–69; prophage hypothesis and provirus formation, 167–71, 186; reactions to in France, 95–99; Temin and DNA provirus hypothesis, 183–93; and tumor etiology, 171–78 Manaker, Robert A., 177 Marton, Ladislaus, 147 Mayer, Adolf, 61–62, 71 McKie, Margot, 95, 167 McKinley, Earl B., 136, 148; quoted, 57 Mendel, Gregor, 75–76, 94 Metcalf, Donald, 132–35 Meyer, Willy, 39–40

Index

Micrographia (Hooke), 143 microscopy, electron, 143–48 Moberg, Carol, 141 molecular biology, 157–59 Monod, Jacques, 164 Morange, Michel, 171 Morris, Henry, 35 Mortimer, Philip, 153 Mrs. Dalloway (Woolf), xi–xii Mudd, Stuart, 66–67, 139 Murphy, Fred, 65–66 Murphy, James B.: and “chicken tumor agent,” 117; enzyme theory of tumor causation, 114–15; lymphocyte work, 106–7; on mutations, 55; opposition to viral theory of tumor etiology, 43, 56, 111–14, 127, 138; on pathology, 59–60; photograph of, 113; quoted, 33, 104; reaction to Gye, 109, 112–15; relationship with Rous, 14, 16–20, 36–38, 105–6; on transmissible mutagens, 119–22; on tumor agent as mutagen, 119–20; on use of term “virus” for sarcoma agent, 117–19, 128–29; and virus cultivation, 151–52; work with Claude, 117–19 mutagens. See transmissible mutagens mutations, 55 Nakahara, Waro, 138 National Institute of Medical Research (NIMR), 41–42, 99–100, 102, 107 Nobel Prizes: Cancer, Vision and the Genetic Code (Norrby), 8, 198 Noguchi, Hideyo, 150 Norrby, Erling, 198 Northrop, John, 78, 103, 118, 122–23, 142 nutritive vitiation, 47–48, 55–56 Oberling, Charles, 40 obligate parasitism, 148–50 Olby, Robert, 75, 76 Opie, Eugene, 127 Palade, George, 148 papilloma virus, Shope, 59, 124–27

293

parasitic theory of cancer causation, 35–36, 38–39 Paris cancer conference (1910), 34–36 Park, Roswell, 38 Pasteur, Louis, 29, 58, 61; quoted, 57 Pasteur Institute, 23, 45, 66, 95–99, 116; illustration, 20 Peyrieras, Nadine, 171 Phage and the Origins of Molecular Biology, 158, 162–63, 198 Pirie, Norman, 135, 142 plaque-­counting assay for animal viruses, 175–76 Plimmer, H. G., 35, 38 Plutarch, xi Pneumococcus, 119 Podolsky, Scott, 78 polyoma virus, 178 Porter, Keith, 147–48 Potter, Van Rensselaer, 192 primary sources in historical scholarship, 196 prophages, 166–67, 181 prophage hypothesis, 167–71 proto-­ideas, 69 provirus, 181; and lysogeny, 186; polyoma virus, 178, 183; and prophage, 171. See also DNA provirus Prudden, T. Mitchell, 16, 29 Purdy, Helen, 76 rabbit papilloma virus, 124–27 Raettig, Hansjürgen, 45, 79 Rasmussen, Nicolas, 145 reverse transcriptase, 154n122, 193 Rheinberger, Hans-­Jörg, 157 Rivers, Thomas Milton: Filterable Viruses, 82, 154–55; on “filtrable viruses” (terminology), 66–67; history of viral theory, 44, 149–50; virology, 59, 74, 135; “virus,” defined, 53 RNA, 177–78, 188, RNA virus, 190–91 Robbins, Frederick, 148, 156, 172 Rockefeller Foundation, 157 Rockefeller Institute: Arrowsmith (Lewis),

294

xi; formation of, 203n1; Murphy’s lab and chicken sarcoma agent, 116–17; Peyton Rous’ role at, 12, 30; Simon Flexner’s role at, 12, 30, 105 Rous, Peyton: American Cancer Society symposium (1959), 179, 181–83; on Andrewes’s fairytale, 131–32; correspondence with Andrewes 128–30; correspondence with Gye, 100, 101, 107–8; correspondence with Ewing, 128–30; early research on chicken sarcoma virus, 12–21, 29–30, 59; early views of virus, 31; and Gye’s experiments, 41–44; Harvey Lecture, 126, 129, 132; Nobel Prize, 44; photograph of, 14; quoted, 11; rabbit papilloma virus, work on, 124–26; relationship with Andrewes, 106; relationship with Gye, 41–42, 109–10; relationship with Murphy, 16–20, 36–38, 105; tumor causation hypothesis, 34–36, 39–41; use of term “virus,” 16–17; work on virus cultivation, 30–31, 151–52 Roux, Émile, 25, 34, 62, 66 Rubin, Harry: American Cancer Society symposium (1959), 179, 181; criticism of Temin, 192, 193; focus assay for tumor viruses, 177; on RNA of RSV genome, 186–88; tumor virus cultivation, 176–77; work with Temin, 184–87 Ruse, Michael, 7, 196–97 Ruska, Ernst, 144–45 Ruska, Helmut, 145 Rutherford, Henry, 105 Sabin, Florence, 105 sarcomas: avian sarcomas, 12–13, 15; causative agents, 15–17; maintenance and supply of tissue, 41 Scheffler, Robin, 198 Schlesinger, Martin, 101, 102, 140 Scholthof, Karen-­Beth, 136 secretion theory, of bacteriophages by lysogenic bacteria, 161–62

A Tale of Two Viruses

Shapin, Steven, 196 Shiga bacillus, 31. See also dysentery Shigella, 50 Shope, Richard, 124–25, 168–69; American Cancer Society symposium (1959), 179 Shope rabbit papilloma virus, 59, 124–27 Siminovitch, Lou, 166, 168 Singer, Charles, 67–68 Skalka, Anna Marie, 198 Spiegelman, Sol, 183 Stanley, Wendell, 74, 135, 140, 141–43, 179 Stent, Gunther, 101–2 The Structure of Scientific Revolutions (Kuhn), 68 Studies in History and Philosophy of Biological and Biomedical Sciences, 8 Summers, William, 4, 45, 46, 91, 170 Sumner, James, 142 Svedberg, Theodor, 139, 142 Szigmondy, Richard, 139 taches vierges, 23 A Tale of Two Cities (Dickens), ix, x, 5, 195 Tatum, Edward, 190 TB (tuberculosis), 40 Temin, Howard: actinomycin D experiments, 191–92; analogy of tumor viruses to lysogeny, 183–93; biochemistry of RSV replication, 188–91; discovery of reverse transcriptase, 193; dissertation defense, 185–88; DNA provirus hypothesis 186, 189–93; focus assay for tumor viruses (with Rubin), 177; hybridization experiments, 191–92; Nobel Prize, 192; quoted, 160; reception to ideas and work, 191–93; viral etiology of cancer, 184; work at Dulbecco’s lab, 177, 181; work with Rubin, 177 thought styles, 69 Through the Looking-­Glass (Carroll), 147 tissue culture, 153–54, 242n87 TMV (tobacco mosaic virus): centrifugation, 140; chemical theory v.

Index

bacteriological theory, 70–71; contagium vivum fluidum, 72–73; crystallization, 141–43; discovery of, 61–64; under electron microscopy, 146; The Life of a Virus (Creager), 7; as model in virology, 61, 62–63, 237n31; and virus cultivation, 151 transmissible mutagens, 119–22, 141 tumors, causative factors. See etiology of tumors Twort, Frederick: early work on bacteriophages (transmissible lysis), 21–22; “glassy transformation,” 22, 27, 51, 67, 150; Lancet article (1915), 51–52; rivalry with d’Herelle over priorty, 25, 27, 28, 48; theories of transmissible lysis, 22; Twort-­d’Herelle phenomenon, 27–28, 52; ultramicroscopy, 67 Twort-­d’Herelle phenomenon, 27–28, 52 Tyler, Albert, 184 Typhoid Mary, 92 ultracentrifugation, 139–41 ultrafiltration of viruses, 137–41 ultramicroscopic viruses, 67 UV (ultraviolet) light and lycogenesis, 166 van Helvoort, Ton, 46, 70 Varley, Alan, 45–47 Varro, 68 viral theory of cancer, 30, 104–12, 114, 117, 118, 120 virology: early research, 58; golden age of, 99–103; origin of term, 58–60 The Virus: A History of the Concept (Hughes), 6 Virus as Organism (Burnet), 135 viruses: contrasted with bacteria, 67–72; crystallography, 141–43; cultivation of, 148–56; definition and history of term, 16–17, 25, 29, 57–61, 171; early concepts of/research on, 29–32, 57–61; effects of viral infections, 197–98; filterability, 13–15, 61–62, 65–67, 148; latent viruses, 131–32;

295

viruses (cont.): measurement of, 137–41; nature of, 5–6, 134–37; obligate parasitism, 148–50; overview of literature on, 6–8; ultramicroscopic, 67; virus theory of tumors, 30, 104–12, 114, 117, 118, 120 Vogt, Marguerite, 184 von Borries, Bodo, 145 Walter and Eliza Hall Institute, 99 Waterson, A. P., 6 Watson, James, 172, 173, 178, 184, 193 Weaver, Harry, 178 Weaver, Warren, 157 Weigle, Jean, 184–85 Welch, William H., 12, 30, 36 Weller, Thomas, 148, 156, 172

296

Whitehead, Alfred North, 143 Wilkinson, Lise, 6 Williams, Greer, 17 Witkowski, Jan, 154 Witz, Jean, 75 Wolbach, S. B., 29 Wollman, Elie, 98, 162 Wollman, Élisabeth, 52–53, 95–98, 161; photograph of, 96 Wollman, Eugène, 52–53, 95–99, 116, 161, 163–64; photograph of, 96 Wood, Francis Carter, 42–43, 110 Woodruff, Alice, 152 Woods, Albert, 72 Woolf, Virginia, xi–xii Yersin, Alexandre, 62

A Tale of Two Viruses