Main Principles of Virology
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About the pagination of this eBook This eBook contains a multi-volume set. To navigate this eBook by page number, you will need to use the volume number and the page number, separated by a hyphen. For example, to go to page 5 of volume 1, type “1-5” in the Go box at the bottom of the screen and click "Go." To go to page 5 of volume 2, type “2-5”… and so forth. PRINCIPLES OF Virology 4th Edition ASM_POV4e_Vol1_FM.indd i 7/23/15 6:48 AM ASM_POV4e_Vol1_FM.indd ii 7/23/15 6:48 AM Molecular Biology VOLUME I PRINCIPLES OF Virology 4th Edition Jane Flint Vincent R. Racaniello Department of Molecular Biology Princeton University Princeton, New Jersey Department of Microbiology & Immunology College of Physicians and Surgeons Columbia University New York, New York Glenn F. Rall Anna Marie Skalka Fox Chase Cancer Center Philadelphia, Pennsylvania Fox Chase Cancer Center Philadelphia, Pennsylvania with Lynn W. Enquist Department of Molecular Biology Princeton University Princeton, New Jersey Washington, DC ASM_POV4e_Vol1_FM.indd iii 7/23/15 6:48 AM Copyright © 2015 American Society for Microbiology. All rights reserved. No part of this publication may be reproduced or transmitted in whole or in part or reused in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Disclaimer: To the best of the publisher’s knowledge, this publication provides information concerning the subject matter covered that is accurate as of the date of publication. The publisher is not providing legal, medical, or other professional services. Any reference herein to any specific commercial products, procedures, or services by trade name, trademark, manufacturer, or otherwise does not constitute or imply endorsement, recommendation, or favored status by the American Society for Microbiology (ASM). The views and opinions of the author(s) expressed in this publication do; not necessarily state or reflect those of ASM, and they shall not be used to advertise or endorse any product. Library of Congress Cataloging-in-Publication Data Flint, S. Jane, author. Principles of virology / Jane Flint, Department of Molecular Biology, Princeton University, Princeton, New Jersey; Vincent R. Racaniello, Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York; Glenn F. Rall, Fox Chase Cancer Center, Philadelphia, Pennsylvania; Anna Marie Skalka, Fox Chase Cancer Center, Philadelphia, Pennsylvania; with Lynn W. Enquist, Department of Molecular Biology, Princeton University, Princeton, New Jersey.—4th edition. pages cm Revision of: Principles of virology / S.J. Flint ... [et al.]. 3rd ed. Includes bibliographical references and index. ISBN 978-1-55581-933-0 (v. 1 pbk.)—ISBN 978-1-55581-934-7 (v. 2 pbk.)—ISBN 978-1-55581-9514 (set pbk.)—ISBN 978-1-55581-952-1 (set ebook) 1. Virology. I. Racaniello, V. R. (Vincent R.), author. II. Rall, Glenn F., author. III. Skalka, Anna M., author. IV. Enquist, L. W. (Lynn W.), author. V. Title. QR360.P697 2015 616.9’101--dc23 2015026213 doi:10.1128/9781555818951 (Volume I) doi:10.1128/9781555818968 (Volume II) doi:10.1128/9781555819521 (e-bundle) 10 9 8 7 6 5 4 3 2 1 All Rights Reserved Printed in the United States of America Address editorial correspondence to ASM Press, 1752 N St., N.W., Washington, DC 20036-2904, USA Send orders to ASM Press, P.O. Box 605, Herndon, VA 20172, USA Phone: 800-546-2416; 703-661-1593 Fax: 703-661-1501 E-mail: email@example.com Online: http://www.asmscience.org Illustrations and illustration concepting: Patrick Lane, ScEYEnce Studios Cover and interior design: Susan Brown Schmidler Cover image: Courtesy of Jason A. Roberts (Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Australia) Back cover photos: Peter Kurilla Photography ASM_POV4e_Vol1_FM.indd iv 7/25/15 12:15 AM We dedicate this book to the students, current and future scientists, physicians, and all those with an interest in the field of virology, for whom it was written. We kept them ever in mind. We also dedicate it to our families: Jonn, Gethyn, and Amy Leedham Doris, Aidan, Devin, and Nadia Eileen, Kelsey, and Abigail Rudy, Jeanne, and Chris And Kathy and Brian Oh, be wiser thou! Instructed that true knowledge leads to love. William Wordsworth Lines left upon a Seat in a Yew-tree ASM_POV4e_Vol1_FM.indd v 7/23/15 6:48 AM ASM_POV4e_Vol1_FM.indd vi 7/23/15 6:48 AM Contents Preface xvii Acknowledgments xxi About the Authors xxiii PART I The Science of Virology 1 Foundations 1 2 Luria’s Credo 3 Why We Study Viruses 3 Viruses Are Everywhere 3 Viruses Can Cause Human Disease 3 Viruses Infect All Living Things 3 Viruses Can Be Beneficial 4 Viruses Can Cross Species Boundaries 4 Viruses “R” Us 4 Viruses Are Unique Tools To Study Biology 5 Virus Prehistory 6 Viral Infections in Antiquity 6 The First Vaccines 7 Microorganisms as Pathogenic Agents 9 Discovery of Viruses 10 The Definitive Properties of Viruses 12 The Structural Simplicity of Virus Particles 12 The Intracellular Parasitism of Viruses 14 Viruses Defined 17 Cataloging Animal Viruses 17 The Classical System 17 Classification by Genome Type: the Baltimore System 20 vii ASM_POV4e_Vol1_FM.indd vii 7/23/15 6:48 AM viii Contents A Common Strategy for Viral Propagation Perspectives 21 References 23 2 The Infectious Cycle 21 24 Introduction 25 The Infectious Cycle 25 The Cell 25 The Architecture of Cell Surfaces 27 The Extracellular Matrix: Components and Biological Importance 27 Properties of the Plasma Membrane 29 Cell Membrane Proteins 30 Entering Cells 31 Making Viral RNA 31 Making Viral Proteins 31 Making Viral Genomes 31 Forming Progeny Virus Particles Viral Pathogenesis 32 Overcoming Host Defenses 32 Cultivation of Viruses 32 Cell Culture 32 Embryonated Eggs 35 Laboratory Animals 35 31 Assay of Viruses 36 Measurement of Infectious Units 36 Efficiency of Plating 39 Measurement of Virus Particles and Their Components 39 Viral Reproduction: the Burst Concept 46 The One-Step Growth Cycle 46 Initial Concept 46 One-Step Growth Analysis: a Valuable Tool for Studying Animal Viruses 49 Systems Biology 50 Perspectives 51 References 52 PART II Molecular Biology 3 53 Genomes and Genetics 54 Introduction 55 Genome Principles and the Baltimore System ASM_POV4e_Vol1_FM.indd viii 55 7/23/15 6:48 AM Contents Structure and Complexity of Viral Genomes DNA Genomes 56 RNA Genomes 58 ix 55 What Do Viral Genomes Look Like? 59 Coding Strategies 60 What Can Viral Sequences Tell Us? 60 The Origin of Viral Genomes 61 The “Big and Small” of Viral Genomes: Does Size Matter? Genetic Analysis of Viruses 65 Classical Genetic Methods 66 Engineering Mutations into Viral Genomes 67 Engineering Viral Genomes: Viral Vectors 73 65 Perspectives 78 References 79 4 Structure 80 Introduction 81 Functions of the Virion 81 Nomenclature 82 Methods for Studying Virus Structure 83 Building a Protective Coat 86 Helical Structures 86 Capsids with Icosahedral Symmetry 89 Other Capsid Architectures 102 Packaging the Nucleic Acid Genome 104 Direct Contact of the Genome with a Protein Shell 104 Packaging by Specialized Viral Proteins 105 Packaging by Cellular Proteins 105 Viruses with Envelopes 106 Viral Envelope Components 106 Simple Enveloped Viruses: Direct Contact of External Proteins with the Capsid or Nucleocapsid 109 Enveloped Viruses with an Additional Protein Layer 109 Large Viruses with Multiple Structural Elements Bacteriophage T4 111 Herpesviruses 112 Poxviruses 113 Giant Viruses 114 111 Other Components of Virions 116 Enzymes 116 Other Viral Proteins 116 Nongenomic Viral Nucleic Acid 117 Cellular Macromolecules 117 Perspectives 119 References 119 ASM_POV4e_Vol1_FM.indd ix 7/23/15 6:48 AM x Contents 5 Attachment and Entry 122 Introduction 123 Attachment of Virus Particles to Cells 123 General Principles 123 Identification of Receptors for Virus Particles 124 Virus-Receptor Interactions 126 Entry into Cells 132 Uncoating at the Plasma Membrane 132 Uncoating during Endocytosis 135 Membrane Fusion 137 Movement of Viral and Subviral Particles within Cells 147 Virus-Induced Signaling via Cell Receptors 148 Import of Viral Genomes into the Nucleus Nuclear Localization Signals 149 The Nuclear Pore Complex 149 The Nuclear Import Pathway 150 Import of Influenza Virus Ribonucleoprotein 151 Import of DNA Genomes 151 Import of Retroviral Genomes 151 148 Perspectives 153 References 154 6 Synthesis of RNA from RNA Templates 156 Introduction 157 The Nature of the RNA Template 157 Secondary Structures in Viral RNA 157 Naked or Nucleocapsid RNA 158 The RNA Synthesis Machinery 159 Identification of RNA-Dependent RNA Polymerases 159 Sequence Relationships among RNA Polymerases 161 Three-Dimensional Structure of RNA-Dependent RNA Polymerases 161 Mechanisms of RNA Synthesis Initiation 164 Capping 168 Elongation 168 Template Specificity 169 Unwinding the RNA Template 169 Role of Cellular Proteins 170 164 Paradigms for Viral RNA Synthesis 170 (⫹) Strand RNA 171 Synthesis of Nested Subgenomic mRNAs 172 (⫺) Strand RNA 173 Ambisense RNA 174 ASM_POV4e_Vol1_FM.indd x 7/23/15 6:48 AM Contents xi Double-Stranded RNA 175 Unique Mechanisms of mRNA and Genome Synthesis of Hepatitis Delta Satellite Virus 176 Why Are (⫺) and (⫹) Strands Made in Unequal Quantities? 177 Do Ribosomes and RNA Polymerases Collide? 179 Cellular Sites of Viral RNA Synthesis 179 Origins of Diversity in RNA Virus Genomes 182 Misincorporation of Nucleotides 182 Segment Reassortment and RNA Recombination 183 RNA Editing 185 Perspectives 185 References 185 7 Reverse Transcription and Integration 188 Retroviral Reverse Transcription 189 Discovery 189 Impact 189 The Process of Reverse Transcription 189 General Properties and Structure of Retroviral Reverse Transcriptases 198 Other Examples of Reverse Transcription 202 Retroviral DNA Integration Is a Unique Process 204 The Pathway of Integration: Integrase-Catalyzed Steps 205 Integrase Structure and Mechanism 210 Hepadnaviral Reverse Transcription 214 A DNA Virus with Reverse Transcriptase 214 The Process of Reverse Transcription 216 Perspectives 221 References 222 8 Synthesis of RNA from DNA Templates 224 Introduction 225 Properties of Cellular RNA Polymerases That Transcribe Viral DNA 225 Some Viral Genomes Must Be Converted to Templates Suitable for Transcription 226 Transcription by RNA Polymerase II 228 Regulation of RNA Polymerase II Transcription 228 Common Properties of Proteins That Regulate Transcription 234 The Cellular Machinery Alone Can Transcribe Viral DNA Templates 235 Viral Proteins That Govern Transcription of Viral DNA Templates 237 Patterns of Regulation 237 The Human Immunodeficiency Virus Type 1 Tat Protein Autoregulates Transcription 237 ASM_POV4e_Vol1_FM.indd xi 7/23/15 6:48 AM xii Contents The Transcriptional Cascades of DNA Viruses 245 Entry into One of Two Alternative Transcriptional Programs 254 Transcription of Viral Genes by RNA Polymerase III The VA-RNA I Promoter 257 Regulation of VA-RNA Gene Transcription 259 257 Inhibition of the Cellular Transcriptional Machinery 259 Unusual Functions of Cellular Transcription Components 260 A Viral DNA-Dependent RNA Polymerase 260 Perspectives 262 References 263 9 Replication of DNA Genomes 266 Introduction 267 DNA Synthesis by the Cellular Replication Machinery Eukaryotic Replicons 269 Cellular Replication Proteins 270 269 Mechanisms of Viral DNA Synthesis 271 Lessons from Simian Virus 40 271 Replication of Other Viral DNA Genomes 275 Properties of Viral Replication Origins 278 Recognition of Viral Replication Origins 280 Viral DNA Synthesis Machines 286 Resolution and Processing of Viral Replication Products 287 Exponential Accumulation of Viral Genomes 288 Viral Proteins Can Induce Synthesis of Cellular Replication Proteins 288 Synthesis of Viral Replication Machines and Accessory Enzymes 290 Viral DNA Replication Independent of Cellular Proteins 291 Delayed Synthesis of Structural Proteins Prevents Premature Packaging of DNA Templates 291 Inhibition of Cellular DNA Synthesis 291 Viral DNAs Are Synthesized in Specialized Intracellular Compartments 292 Limited Replication of Viral DNA Genomes 296 Integrated Parvoviral DNA Can Replicate as Part of the Cellular Genome 296 Different Viral Origins Regulate Replication of Epstein-Barr Virus 297 Limited and Amplifying Replication from a Single Origin: the Papillomaviruses 299 Origins of Genetic Diversity in DNA Viruses 301 Fidelity of Replication by Viral DNA Polymerases 301 Inhibition of Repair of Double-Strand Breaks in DNA 303 Recombination of Viral Genomes 304 Perspectives 307 References 307 ASM_POV4e_Vol1_FM.indd xii 7/23/15 6:48 AM Contents 10 Processing of Viral Pre-mRNA 310 Introduction 311 Covalent Modification during Viral Pre-mRNA Processing Capping the 5⬘ Ends of Viral mRNA 312 Synthesis of 3⬘ Poly(A) Segments of Viral mRNA 315 Splicing of Viral Pre-mRNA 317 Alternative Processing of Viral Pre-mRNA 322 Editing of Viral mRNAs 325 Export of RNAs from the Nucleus The Cellular Export Machinery 327 Export of Viral mRNA 327 xiii 312 327 Posttranscriptional Regulation of Viral or Cellular Gene Expression by Viral Proteins 330 Temporal Control of Viral Gene Expression 330 Viral Proteins Can Inhibit Cellular mRNA Production 333 Regulation of Turnover of Viral and Cellular mRNAs in the Cytoplasm 335 Regulation of mRNA Stability by Viral Proteins 336 mRNA Stabilization Can Facilitate Transformation 338 Production and Function of Small RNAs That Inhibit Gene Expression 338 Small Interfering RNAs, Micro-RNAs, and Their Synthesis 338 Viral Micro-RNAs 342 Viral Gene Products That Block RNA Interference 345 Perspectives 345 References 346 11 Protein Synthesis 348 Introduction 349 Mechanisms of Eukaryotic Protein Synthesis General Structure of Eukaryotic mRNA 349 The Translation Machinery 350 Initiation 351 Elongation and Termination 360 The Diversity of Viral Translation Strategies Polyprotein Synthesis 363 Leaky Scanning 365 Reinitiation 366 Suppression of Termination 366 Ribosomal Frameshifting 368 Bicistronic mRNAs 368 349 362 Regulation of Translation during Viral Infection 368 Inhibition of Translation Initiation after Viral Infection 369 Regulation of eIF4F 372 ASM_POV4e_Vol1_FM.indd xiii 7/23/15 6:48 AM xiv Contents Regulation of Poly (A)-Binding Protein Activity 376 Regulation of eIF3 376 Interfering with RNA 376 Stress-Associated RNA Granules 377 Perspectives 377 References 379 12 Intracellular Trafficking 380 Introduction 381 Assembly within the Nucleus 382 Import of Viral Proteins for Assembly 383 Assembly at the Plasma Membrane 384 Transport of Viral Membrane Proteins to the Plasma Membrane 386 Sorting of Viral Proteins in Polarized Cells 401 Disruption of the Secretory Pathway in Virus-Infected Cells 404 Signal Sequence-Independent Transport of Viral Proteins to the Plasma Membrane 406 Interactions with Internal Cellular Membranes 409 Localization of Viral Proteins to Compartments of the Secretory Pathway 410 Localization of Viral Proteins to the Nuclear Membrane 411 Transport of Viral Genomes to Assembly Sites 411 Transport of Genomic and Pregenomic RNA from the Nucleus to the Cytoplasm 411 Transport of Genomes from the Cytoplasm to the Plasma Membrane 411 Perspectives 413 References 414 13 Assembly, Exit, and Maturation 416 Introduction 417 Methods of Studying Virus Assembly and Egress 418 Structural Studies of Virus Particles 418 Visualization of Assembly and Exit by Microscopy 418 Biochemical and Genetic Analyses of Assembly Intermediates 418 Methods Based on Recombinant DNA Technology 421 Assembly of Protein Shells 421 Formation of Structural Units 421 Capsid and Nucleocapsid Assembly 423 Self-Assembly and Assisted Assembly Reactions 425 Selective Packaging of the Viral Genome and Other Components of Virus Particles 430 Concerted or Sequential Assembly 430 Recognition and Packaging of the Nucleic Acid Genome 431 Incorporation of Enzymes and Other Nonstructural Proteins 438 ASM_POV4e_Vol1_FM.indd xiv 7/23/15 6:48 AM Contents xv Acquisition of an Envelope 439 Sequential Assembly of Internal Components and Budding from a Cellular Membrane 439 Coordination of the Assembly of Internal Structures with Acquisition of the Envelope 440 Release of Virus Particles 441 Assembly and Budding at the Plasma Membrane 441 Assembly at Internal Membranes: the Problem of Exocytosis 444 Release of Nonenveloped Viruses 450 Maturation of Progeny Virus Particles 450 Proteolytic Processing of Structural Proteins 450 Other Maturation Reactions 456 Cell-to-Cell Spread Perspectives 460 References 460 457 14 The Infected Cell 464 Introduction 465 Signal Transduction 465 Signaling Pathways 465 Signaling in Virus-Infected Cells 466 Gene Expression 470 Inhibition of Cellular Gene Expression 470 Differential Regulation of Cellular Gene Expression 474 Metabolism 477 Methods To Study Metabolism 477 Glucose Metabolism 479 The Citric Acid Cycle 483 Electron Transport and Oxidative Phosphorylation 484 Lipid Metabolism 486 Remodeling of Cellular Organelles The Nucleus 491 The Cytoplasm 495 491 Perspectives 498 References 500 APPENDIX Structure, Genome Organization, and Infectious Cycles Glossary 537 Index 501 543 ASM_POV4e_Vol1_FM.indd xv 7/23/15 6:48 AM ASM_POV4e_Vol1_FM.indd xvi 7/23/15 6:48 AM Preface The enduring goal of scientific endeavor, as of all human enterprise, I imagine, is to achieve an intelligible view of the universe. One of the great discoveries of modern science is that its goal cannot be achieved piecemeal, certainly not by the accumulation of facts. To understand a phenomenon is to understand a category of phenomena or it is nothing. Understanding is reached through creative acts. A. D. HERSHEY Carnegie Institution Yearbook 65 All four editions of this textbook have been written according to the authors’ philosophy that the best approach to teaching introductory virology is by emphasizing shared principles. Studying the phases of the viral reproductive cycle, illustrated with a set of representative viruses, provides an overview of the steps required to maintain these infectious agents in nature. Such knowledge cannot be acquired by learning a collection of facts about individual viruses. Consequently, the major goal of this book is to deﬁne and illustrate the basic principles of animal virus biology. In this information-rich age, the quantity of data describing any given virus can be overwhelming, if not indigestible, for student and expert alike. The urge to write more and more about less and less is the curse of reductionist science and the bane of those who write textbooks meant to be used by students. In the fourth edition, we continue to distill information with the intent of extracting essential principles, while providing descriptions of how the information was acquired. Boxes are used to emphasize major principles and to provide supplementary material of relevance, from explanations of terminology to descriptions of trail-blazing experiments. Our goal is to illuminate process and strategy as opposed to listing facts and ﬁgures. In an eﬀort to make the book readable, rather than comprehensive, we are selective in our choice of viruses and examples. The encyclopedic Fields Virology (2013) is recommended as a resource for detailed reviews of speciﬁc virus families. What’s New This edition is marked by a change in the author team. Our new member, Glenn Rall, has brought expertise in viral immunology and pathogenesis, pedagogical clarity, and down-toearth humor to our work. Although no longer a coauthor, our colleague Lynn Enquist has continued to provide insight, advice, and comments on the chapters. Each of the two volumes of the fourth edition has a unique appendix and a general glossary. Links to Internet resources such as websites, podcasts, blog posts, and movies are provided; the digital edition provides one-click access to these materials. xvii ASM_POV4e_Vol1_FM.indd xvii 7/23/15 6:48 AM xviii Preface A major new feature of the fourth edition is the incorporation of in-depth video interviews with scientists who have made a major contribution to the subject of each chapter. Students will be interested in these conversations, which also explore the factors that motivated the scientists’ interest in the ﬁeld and the personal stories associated with their contributions. Volume I covers the molecular biology of viral reproduction, and Volume II focuses on viral pathogenesis, control of virus infections, and virus evolution. The organization into two volumes follows a natural break in pedagogy and provides considerable ﬂexibility and utility for students and teachers alike. The volumes can be used for two courses, or as two parts of a one-semester course. The two volumes diﬀer in content but are integrated in style and presentation. In addition to updating the chapters and Appendices for both volumes, we have organized the material more eﬃciently and new chapters have been added. As in our previous editions, we have tested ideas for inclusion in the text in our own classes. We have also received constructive comments and suggestions from other virology instructors and their students. Feedback from students was particularly useful in ﬁnding typographical errors, clarifying confusing or complicated illustrations, and pointing out inconsistencies in content. For purposes of readability, references are generally omitted from the text, but each chapter ends with an updated list of relevant books, review articles, and selected research papers for readers who wish to pursue speciﬁc topics. In general, if an experiment is featured in a chapter, one or more references are listed to provide more detailed information. Principles Taught in Two Distinct, but Integrated Volumes These two volumes outline and illustrate the strategies by which all viruses reproduce, how infections spread within a host, and how they are maintained in populations. The principles of viral reproduction established in Volume I are essential for understanding the topics of viral disease, its control, and the evolution of viruses that are covered in Volume II. Volume I The Science of Virology and the Molecular Biology of Viruses This volume examines the molecular processes that take place in an infected host cell. It begins with a general introduction and historical perspectives, and includes descriptions of the unique properties of viruses (Chapter 1). The unifying principles that are the foundations of virology, including the concept of a common strategy for viral propagation, are then described. An introduction to cell biology, the principles of the infectious cycle, descriptions of the basic techniques for cultivating and assaying viruses, and the concept of the single-step growth cycle are presented in Chapter 2. The fundamentals of viral genomes and genetics, and an overview of the surprisingly limited repertoire of viral strategies for genome replication and mRNA synthesis, are topics of Chapter 3. The architecture of extracellular virus particles in the context of providing both protection and delivery of the viral genome in a single vehicle are considered in Chapter 4. Chapters 5 through 13 address the broad spectrum of molecular processes that characterize the common steps of the reproductive cycle of viruses in a single cell, from decoding genetic information to genome replication and production of progeny virions. We describe how these common steps are accomplished in cells infected by diverse but representative viruses, while emphasizing common principles. Volume I concludes with a new chapter, “The Infected Cell,” which presents an integrated description of cellular responses to illustrate the marked, and generally, irreversible, impact of virus infection on the host cell. The appendix in Volume I provides concise illustrations of viral life cycles for members of the main virus families discussed in the text; ﬁve new families have been added in the fourth edition. It is intended to be a reference resource when reading individual chapters and a convenient visual means by which speciﬁc topics may be related to the overall infectious cycles of the selected viruses. ASM_POV4e_Vol1_FM.indd xviii 7/23/15 6:48 AM Preface xix Volume II Pathogenesis, Control, and Evolution This volume addresses the interplay between viruses and their host organisms. The first five chapters have been reorganized and rewritten to reflect our growing appreciation of the host immune response and how viruses cause disease. In Chapter 1 we introduce the discipline of epidemiology, provide historical examples of epidemics in history, and consider basic aspects that govern how the susceptibility of a population is controlled and measured. With an understanding of how viruses affect human populations, subsequent chapters focus on the impact of viral infections on hosts, tissues and individual cells. Physiological barriers to virus infections, and how viruses spread in a host, invade organs, and spread to other hosts are the topics of Chapter 2. The early host response to infection, comprising cell autonomous (intrinsic) and innate immune responses, are the topics of Chapter 3, while the next chapter considers adaptive immune defenses, that are tailored to the pathogen, and immune memory. Chapter 5 focuses on the classic patterns of virus infection within cells and hosts, the myriad ways that viruses cause illness, and the value of animal models in uncovering new principles of viral pathogenesis. In Chapter 6, we discuss virus infections that transform cells in culture and promote oncogenesis (the formation of tumors) in animals. Chapter 7 is devoted entirely to the AIDS virus, not only because it is the causative agent of the most serious current worldwide epidemic, but also because of its unique and informative interactions with the human immune defenses. Next, we consider the principles involved in treatment and control of infection. Chapter 8 focuses on vaccines, and Chapter 9 discusses the approaches and challenges of antiviral drug discovery. The topics of viral evolution and emergence have now been divided into two chapters. The origin of viruses, the drivers of viral evolution, and host-virus conﬂicts are the subjects of Chapter 10. The principles of emerging virus infections, and humankind’s experiences with epidemic and pandemic viral infections, are considered in Chapter 11. Volume II ends with a new chapter on unusual infectious agents, viroids, satellites, and prions. The Appendix of Volume II provides snapshots of the pathogenesis of common human viruses. This information is presented in four illustrated panels that summarize the viruses and diseases, epidemiology, disease mechanisms, and human infections. Reference Knipe DM, Howley PM (ed). 2013. Fields Virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA. For some behind-the-scenes information about how the authors created the fourth edition of Principles of Virology, see: http://bit.ly/Virology_MakingOf ASM_POV4e_Vol1_FM.indd xix 7/23/15 6:48 AM ASM_POV4e_Vol1_FM.indd xx 7/23/15 6:48 AM Acknowledgments These two volumes of Principles could not have been composed and revised without help and contributions from many individuals. We are most grateful for the continuing encouragement from our colleagues in virology and the students who use the text. Our sincere thanks also go to colleagues (listed in the Acknowledgments for the third edition) who have taken considerable time and effort to review the text in its evolving manifestations. Their expert knowledge and advice on issues ranging from teaching virology to organization of individual chapters and style were invaluable, and are inextricably woven into the final form of the book. We also are grateful to those who gave so generously of their time to serve as expert reviewers of individual chapters or speciﬁc topics in these two volumes: Siddharth Balachandran (Fox Chase Cancer Center), Patrick Moore (University of Pittsburgh), Duane Grandgenett (St. Louis University), Frederick Hughson (Princeton University), Bernard Moss (Laboratory of Viral Diseases, National Institutes of Health), Christoph Seeger (Fox Chase Cancer Center), and Thomas Shenk (Princeton University). Their rapid responses to our requests for details and checks on accuracy, as well as their assistance in simplifying complex concepts, were invaluable. All remaining errors or inconsistencies are entirely ours. Since the inception of this work, our belief has been that the illustrations must complement and enrich the text. Execution of this plan would not have been possible without the support of Christine Charlip (Director, ASM Press), and the technical expertise and craft of our illustrator. The illustrations are an integral part of the text, and credit for their execution goes to the knowledge, insight, and artistic talent of Patrick Lane of ScEYEnce Studios. We also are indebted to Jason Roberts (Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Australia) for the computational expertise and time he devoted to producing the beautiful renditions of poliovirus particles on our new covers. As noted in the ﬁgure legends, many could not have been completed without the help and generosity of numerous colleagues who provided original images. Special thanks go to those who crafted ﬁgures or videos tailored speciﬁcally to our needs, or provided multiple pieces: Chantal Abergel (CNRS, Aix-Marseille Université, France), Mark Andrake (Fox Chase Cancer Center), Timothy Baker (University of California), Bruce Banﬁeld (The University of Colorado), Christopher Basler and Peter Palese (Mount Sinai School of Medicine), Ralf Bartenschlager (University of Heidelberg, Germany), Eileen Bridge (Miami University, Ohio), Richard Compans (Emory University), Kartik Chandran (Albert Einstein College of Medicine), Paul Duprex (Boston University School of Medicine), Ramón González (Universidad Autónoma del Estado xxi ASM_POV4e_Vol1_FM.indd xxi 7/23/15 6:48 AM xxii Acknowledgments de Morelos), Urs Greber (University of Zurich), Reuben Harris (University of Minnesota), Hidesaburo Hanafusa (deceased), Ari Helenius (University of Zurich), David Knipe (Harvard Medical School), J. Krijnse-Locker (University of Heidelberg, Germany), Petr G. Leiman (École Polytechnique Fédérale de Lausanne), Stuart Le Grice (National Cancer Institute, Frederick MD), Hongrong Liu (Hunan Normal University), David McDonald (Ohio State University), Thomas Mettenleiter (Federal Institute for Animal Diseases, Insel Reims, Germany), Bernard Moss (Laboratory of Viral Diseases, National Institutes of Health), Norm Olson (University of California), B. V. Venkataram Prasad (Baylor College of Medicine), Andrew Rambaut (University of Edinburgh), Jason Roberts (Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Australia), Felix Rey (Institut Pasteur, Paris, France), Michael Rossmann (Purdue University), Anne Simon (University of Maryland), Erik Snijder (Leiden University Medical Center), Alasdair Steven (National Institutes of Health), Paul Spearman (Emory University), Wesley Sundquist (University of Utah), Livia Varstag (Castleton State College, Vermont), Jiri Vondrasek (Institute of organic Chemistry and Biochemistry, Czech Republic), Matthew Weitzman (University of Pennsylvania), Sandra Weller (University of Connecticut Health Sciences Center, Connecticut), Tim Yen (Fox Chase Cancer Center), and Z. Hong Zhou (University of California, Los Angeles). The collaborative work undertaken to prepare the fourth edition was facilitated greatly by several authors’ retreats. ASM Press generously provided ﬁnancial support for these retreats as well as for our many other meetings. We thank all those who guided and assisted in the preparation and production of the book: Christine Charlip (Director, ASM Press) for steering us through the complexities inherent in a team eﬀort, Megan Angelini and John Bell (Production Managers, ASM Press) for keeping us on track during production, and Susan Schmidler for her elegant and creative designs for the layout and cover. We are also grateful for the expert secretarial and administrative support from Ellen Brindle-Clark (Princeton University) that facilitated preparation of this text. Special thanks go to Ellen for obtaining many of the permissions required for the ﬁgures. There is little doubt in undertaking such a massive eﬀort that inaccuracies still remain, despite our best eﬀorts to resolve or prevent them. We hope that the readership of this edition will draw our attention to them, so that these errors can be eliminated from future editions of this text. This often-consuming enterprise was made possible by the emotional, intellectual, and logistical support of our families, to whom the two volumes are dedicated. ASM_POV4e_Vol1_FM.indd xxii 7/23/15 6:48 AM About the Authors Jane Flint is a Professor of Molecular Biology at Princeton University. Dr. Flint’s research focuses on investigation of the molecular mechanisms by which viral gene products modulate host cell pathways and antiviral defenses to allow efficient reproduction in normal human cells of adenoviruses, viruses that are widely used in such therapeutic applications as gene transfer and cancer treatment. Her service to the scientific community includes membership of various editorial boards and several NIH study sections and other review panels. Dr. Flint is currently a member of the Biosafety Working Group of the NIH Recombinant DNA Advisory Committee. Vincent Racaniello is Higgins Professor of Microbiology & Immunology at Columbia University Medical Center. Dr. Racaniello has been studying viruses for over 35 years, including poliovirus, rhinovirus, enteroviruses, and hepatitis C virus. He teaches virology to graduate, medical, dental, and nursing students and uses social media to communicate the subject outside of the classroom. His Columbia University undergraduate virology lectures have been viewed by thousands at iTunes University, Coursera, and on YouTube. Vincent blogs about viruses at virology.ws and is host of the popular science program This Week in Virology. Glenn Rall is a Professor and the Co-Program Leader of the Blood Cell Development and Function Program at the Fox Chase Cancer Center in Philadelphia. At Fox Chase, Dr. Rall is also the Associate Chief Academic Officer and Director of the Postdoctoral Program. He is an Adjunct Professor in the Microbiology and Immunology departments at the University of Pennsylvania, Thomas Jefferson, Drexel, and Temple Universities. Dr. Rall’s laboratory studies viral infections of the brain and the immune responses to those infections, with the goal of defining how viruses contribute to disease in humans. His service to the scientific community includes membership on the Autism Speaks Scientific Advisory Board, Opinions Editor of PLoS Pathogens, chairing the Education and Career Development Committee of the American Society for Virology, and membership on multiple NIH grant review panels. Anna Marie Skalka is a Professor and the W.W. Smith Chair in Cancer Research at Fox Chase Cancer Center in Philadelphia and an Adjunct Professor at the University of Pennsylvania. Dr. Skalka’s major research interests are the molecular aspects of the replication of retroviruses. Dr. Skalka is internationally recognized for her contributions to the understanding of the biochemical mechanisms by which such viruses (including the AIDS virus) replicate and insert their genetic material into the host genome. Both an administrator and researcher, she has been deeply involved in state, national, and international advisory groups concerned with the broader, societal implications of scientific research, including the NJ Commission on Cancer Research and the U.S. Defense Science Board. Dr. Skalka has served on the editorial boards of peerreviewed scientific journals and has been a member of scientific advisory boards including the National Cancer Institute Board of Scientific Counselors, the General Motors Cancer Research Foundation Awards Assembly, the Board of Governors of the American Academy of Microbiology, and the National Advisory Committee for the Pew Biomedical Scholars Program. xxiii ASM_POV4e_Vol1_FM.indd xxiii 7/23/15 6:48 AM ASM_POV4e_Vol1_FM.indd xxiv 7/23/15 6:48 AM PART I The Science of Virology 1 2 ASM_POV4e_Vol1_Ch01.indd 1 Foundations The Infectious Cycle 7/22/15 12:27 PM 1 Foundations Luria’s Credo Why We Study Viruses Viruses Are Everywhere Viruses Can Cause Human Disease Viruses Infect All Living Things Viruses Can Be Beneficial Viruses Can Cross Species Boundaries Viruses “R” Us Viruses Are Unique Tools To Study Biology Virus Prehistory Viral Infections in Antiquity The First Vaccines Microorganisms as Pathogenic Agents The Definitive Properties of Viruses The Structural Simplicity of Virus Particles The Intracellular Parasitism of Viruses Viruses Defined Cataloging Animal Viruses The Classical System Classification by Genome Type: the Baltimore System A Common Strategy for Viral Propagation Perspectives References Discovery of Viruses LINKS FOR CHAPTER 1 Video: Interview with Dr. Donald Henderson http://bit.ly/Virology_Henderson This Week in Virology (TWIV): A weekly podcast about viruses featuring informal yet informative discussions and interviews with guests about the latest topics in the field. http://www.twiv.tv Marine viruses and insect defense http://bit.ly/Virology_Twiv301 Giants among viruses http://bit.ly/Virology_Twiv261 Latest update of virus classification from the ICTV. http://www.ictvonline.org/virusTaxonomy. asp?bhcp=1 The abundant and diverse viruses of the seas. http://bit.ly/Virology_3-20-09 How many viruses on Earth? http://bit.ly/Virology_9-6-13 ASM_POV4e_Vol1_Ch01.indd 2 7/22/15 12:27 PM Luria’s Credo “There is an intrinsic simplicity of nature and the ultimate contribution of science resides in the discovery of unifying and simplifying generalizations, rather than in the description of isolated situations—in the visualization of simple, overall patterns rather than in the analysis of patchworks.” More than half a century has passed since Salvador Luria wrote this credo in the introduction to the classic textbook General Virology. Despite an explosion of information in biology since Luria wrote these words, his vision of unity in diversity is as relevant now as it was then. That such unifying principles exist may not be obvious considering the bewildering array of viruses, genes, and proteins recognized in modern virology. Indeed, new viruses are being described regularly, and viral diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis, and inﬂuenza continue to challenge our eﬀorts to control them. Yet Luria’s credo still stands: even as our knowledge continues to increase, it is clear that all viruses follow the same simple strategy to ensure their survival. This insight has been hard-won over many years of observation, research, and debate; the history of virology is rich and instructive. Why We Study Viruses Viruses Are Everywhere Viruses are all around us, comprising an enormous proportion of our environment, in both number and total mass (Box 1.1). All living things encounter billions of virus particles every day. For example, they enter our lungs in the 6 liters of P R I N C I P L E S air each of us inhales every minute; they enter our digestive systems with the food we eat; and they are transferred to our eyes, mouths, and other points of entry from the surfaces we touch and the people with whom we interact. Our bodies are reservoirs for viruses that reside in our respiratory, gastrointestinal, and urogenital tracts. In addition to viruses that can infect us, our intestinal tracts are loaded with myriad plant and insect viruses, as well as hundreds of bacterial species that harbor their own constellations of viruses. Viruses Can Cause Human Disease With such constant exposure, it is nothing short of amazing that the vast majority of viruses that infect us have little or no impact on our health or well-being. As described in Volume II, we owe such relative safety to our elaborate immune defense systems, which have evolved to fight microbial infection. When these defenses are compromised, even the most common infection can be lethal. Despite such defenses, some of the most devastating human diseases have been or still are caused by viruses; these diseases include smallpox, yellow fever, poliomyelitis, influenza, measles, and AIDS. Viral infections can lead to life-threatening diseases that impact virtually all organs, including the lungs, liver, central nervous system, and intestines. Viruses are responsible for approximately 20% of the human cancer burden, and viral infections of the respiratory and gastrointestinal tracts kill millions of children in the developing world each year. As summarized in Volume II, Appendix, there is no question about the biomedical importance of these agents. Viruses Infect All Living Things While most of this textbook focuses on viral infections of humans, it is important to bear in mind that viruses also infect pets, food animals, plants, insects, and wildlife throughout Foundations The field of virology encompasses viral discovery, the study of virus structure and reproduction, and the importance of viruses in biology and disease. While this text focuses primarily on viruses that infect vertebrates, especially humans, it is important to keep in mind that viruses infect all living things including insects, plants, bacteria, and even other viruses. Viruses are not solely pathogenic nuisances; they can be beneficial. Viruses contribute to ecological homeostasis, keep our immune responses activated and alert, and can be used as molecular flashlights to illuminate cellular processes. Viruses have been part of all of human history: they were present long before Homo sapiens evolved, and the majority of human infections were likely acquired from other animals (zoonoses). As viruses continue to be discovered, our understanding of how human health and well-being are affected by these agents remains incomplete. Viruses are obligate intracellular parasites and depend on their host cell for all aspects of the viral life cycle. While Koch’s postulates were essential for defining many agents of disease, not all pathogenic viruses fulfilled these criteria. Viruses can be cataloged based on their appearance, the hosts they infect, or the nature of their nucleic acid genome. The Baltimore classification allows relationships among various viral genomes and the pathway to mRNA to be determined. A common strategy underlies the propagation of all viruses. This textbook describes that strategy and the similarities and differences in the manner in which it is accomplished by different viruses. 3 ASM_POV4e_Vol1_Ch01.indd 3 7/22/15 12:27 PM 4 Chapter 1 BOX 1.1 B A C K G R O U N D Some astounding numbers • Viruses are the most abundant entities in the biosphere. The biomass on our planet of bacterial viruses alone exceeds that of all of Earth’s elephants by more than 1,000-fold. There are more than 1030 bacteriophage particles in the world’s oceans, enough to extend out into space for 200 million light-years if arranged head to tail (http://www.virology.ws/2009/03/20/the-abundantand-diverse-viruses-of-the-seas/). • Whales are commonly infected with a member of the virus family Caliciviridae that causes rashes, blisters, intestinal problems, and diarrhea and can also infect humans. Infected whales excrete more than 1013 calicivirus particles daily. • The average human body contains approximately 1013 cells, but these are outnumbered 10-fold by bacteria and as much as 100-fold by virus particles. • With about 1016 human immunodeficiency virus (HIV) genomes on the planet today, it is highly probable that somewhere there exist HIV genomes that are resistant to every one of the antiviral drugs that we have now or are likely to have in the future. Earth and its oceans. Courtesy: NASA/Goddard Space Flight Center. Viruses Can Be Beneficial Despite the appalling statistics from human and agricultural epidemics, it is important to realize that viruses can also be beneficial. Such benefit can be seen most clearly in marine ecology, where virus particles are the most abundant biological entities (Box 1.1). Indeed, they comprise 94% of all nucleic acid-containing particles in the oceans and are 15 times more abundant than the Bacteria and Archaea. Viral infections in the ocean kill 20 to 40% of marine microbes daily, converting these living organisms into particulate matter, and in so doing release essential nutrients that supply phytoplankton at the bottom of the ocean’s food chain, as well as carbon dioxide and other gases that affect the climate of the earth. Pathogens can also influence one another: infection by one virus can have an ameliorating effect on the pathogenesis of a second virus or even bacteria. For example, human immunodeficiency virus-infected AIDS patients show a substantial decrease in their disease progression if they are persistently infected with hepatitis G virus, and mice latently infected with some murine herpesviruses are resistant to infection with the bacterial pathogens Listeria monocytogenes and Yersinia pestis. The idea that viruses are solely agents of disease is giving way to the notion that they can exert positive, even necessary, effects. Viruses Can Cross Species Boundaries the world. They infect microbes such as algae, fungi, and bacteria, and some even interfere with the reproduction of other viruses. Viral infection of agricultural plants and animals can have enormous economic and societal impact. Outbreaks of infection by foot-and-mouth disease and avian influenza viruses have led to the destruction (culling) of millions of cattle, sheep, and poultry to prevent further spread. Losses in the United Kingdom during the 2001 outbreak of foot-andmouth disease ran into billions of dollars and caused havoc for both farmers and the government (Box 1.2). More recent outbreaks of the avian influenza virus H5N1 in Asia have resulted in similar disruption and economic loss. Viruses that infect crops such as potatoes and fruit trees are common and can lead to serious food shortages as well as financial devastation. ASM_POV4e_Vol1_Ch01.indd 4 Although viruses generally have a limited host range, they can and do spread across species barriers. As the world’s human population continues to expand and impinge on the wilderness, cross-species (zoonotic) infections of humans are occurring with increasing frequency. In addition to the AIDS pandemic, the highly fatal Ebola hemorrhagic fever and the severe acute respiratory syndrome (SARS) are recent examples of viral diseases to emerge from zoonotic infections. The current pandemic of influenza virus H5N1 in avian species has much of the world riveted by the frightening possibility that a new, highly pathogenic strain might emerge following transmission from birds to human hosts. Indeed, given the eons over which viruses have had the opportunity to interact with various species, today’s “natural” host may simply be a way station in viral evolution. Viruses “R” Us Every cell in our body contains viral DNA. Human endogenous retroviruses, and elements thereof, make up about 5 to 8% of our DNA. Most are inactive, fossil remnants from infections of germ cells that have occurred over millions of years during our evolution. Some of them are suspected to be associated with specific diseases, but the protein products of other endogenous retroviruses are essential for placental development. Recent genomic studies have revealed that our viral “heritage” is not limited to retroviruses. Human and other 7/22/15 12:27 PM Foundations BOX 5 1.2 D I S C U S S I O N The first animal virus discovered remains a scourge today Foot-and-mouth disease virus infects domestic cattle, pigs, and sheep, as well as many species of wild animals. Although mortality is low, morbidity is high and infected farm animals lose their commercial value. The virus is highly contagious, and the most common and effective method of control is by the slaughter of entire herds in affected areas. Outbreaks of foot-and-mouth disease were widely reported in Europe, Asia, Africa, and South and North America in the 1800s. The largest epidemic ever recorded in the United States occurred in 1914. After gaining entry into the Chicago stockyards, the virus spread to more than 3,500 herds in 22 states. This calamity accelerated epidemiological and disease control programs, eventually leading to the field- and laboratory-based systems maintained by the U.S. Department of Agriculture to protect domestic livestock from foreign animal and plant diseases. Similar control systems have been established in other Western countries, but this virus still presents a formidable challenge throughout the world. A 1997 outbreak of foot-and-mouth disease among pigs in Taiwan resulted in economic losses of greater than $10 billion. In 2001, an epidemic outbreak in the United Kingdom spread to other countries in Europe and led to the slaughter of more than 3 million infected and uninfected farm animals. The associated economic, societal, and political costs threatened to bring down the British government. Images of mass graves and horrific pyres consuming the corpses of dead animals (see figure) sensitized the public as never before. Recent outbreaks and societal unrest in Turkey and regions of North Africa, including Libya and Egypt, make the threat of further spread a serious concern for other countries. Mass burning of cattle carcasses during the 2001 foot-and-mouth disease outbreak in the United Kingdom. vertebrate genomes harbor sequences derived from several DNA and RNA viruses that, in contrast to the retroviruses, lack mechanisms to invade host DNA. As many of these insertions are estimated to have occurred some 40 million to 90 million years ago, this knowledge has provided unique insight into the ages and evolution of some currently circulating viruses. Furthermore, the conservation of some of the viral sequences in vertebrate genomes suggests that they may have been selected for beneﬁcial properties over evolutionary time. Viruses Are Unique Tools To Study Biology Because viruses are dependent on their hosts for propagation, studies that focus on viral reprogramming of cellular mechanisms have provided unique insights into cellular biology and functioning of host defenses. Groundbreaking studies of viruses that infect bacteria, the bacteriophages, laid the ASM_POV4e_Vol1_Ch01.indd 5 Hunt J. 3 January 2013. Foot-and-mouth is knocking on Europe’s door. Farmers Weekly. http://www.fwi. co.uk/articles/03/01/2013/136943/foot-and-mouth-isknocking-on-europe39s-door.htm. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ. 1999. Veterinary Virology, 3rd ed. Academic Press, Inc, San Diego, CA. foundations of modern molecular biology (Table 1.1), and crystallization of the plant virus tobacco mosaic virus was a landmark in structural biology. Studies of animal viruses established many fundamental principles of cellular function, including the presence of intervening sequences in eukaryotic genes. The study of cancer (transforming) viruses revealed the genetic basis of this disease. It seems clear that studies of viruses will continue to open up such paths of discovery in the future. With the development of recombinant DNA technology and our increased understanding of some viral systems, it has become possible to use viral genomes as vehicles for the delivery of genes to cells and organisms for both scientiﬁc and therapeutic purposes. The use of viral vectors to introduce genes into various cells and organisms to study their function has become a standard method in biology. Viral vectors are 7/22/15 12:27 PM 6 Chapter 1 Table 1.1 Bacteriophages: landmarks in molecular biologya Year Discovery (discoverer[s]) 1939 1946 1947 1952 1952 1952 1955 1958 One-step growth of viruses (Ellis and Delbrück) Mixed phage infection leads to genetic recombination (Delbrück) Mutation and DNA repair (multiplicity reactivation) (Luria) Transduction of genetic information (Zinder and Lederberg) DNA, not protein, found to be the genetic material (Hershey and Chase) Restriction and modification of DNA (Luria) Definition of a gene (cis-trans test) (Benzer) Mechanisms of control of gene expression by repressors and activators established (Pardee, Jacob, and Monod) Definition of the episome (Jacob and Wollman) Discovery of mRNA (Brenner, Jacob, and Meselson) Elucidation of the triplet code by genetic analysis (Crick, Barnett, Brenner, and Watts-Tobin) Genetic definition of nonsense codons as stop signals for translation (Campbell, Epstein, and Bernstein) Colinearity of the gene with the polypeptide chain (Sarabhai, Stretton, and Brenner) Pathways of macromolecular assembly (Edgar and Wood) Vectors for recombinant DNA technology (Murray and Murray, Thomas, Cameron, and Davis) 1958 1961 1961 1961 1964 1966 1974 a Sources: T. D. Brock, The Emergence of Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990); K. Denniston and L. Enquist, Recombinant DNA. Benchmark Papers in Microbiology, vol. 15 (Dowden, Hutchinson and Ross, Inc., Stroudsburg, PA, 1981); and C. K. Mathews, E. Kutter, G. Mosig, and P. Berget, Bacteriophage T4 (American Society for Microbiology, Washington, DC, 1983). also being used to treat human disease via “gene therapy,” in which functional genes delivered by viral vectors compensate for faulty genes in the host cells. Virus Prehistory Although viruses have been known as distinct biological entities for little more than 100 years, evidence of viral infection can be found among the earliest recordings of human activity, and methods for combating viral disease were practiced long before the first virus was recognized. Consequently, efforts to understand and control these important agents of disease are phenomena of the 20th century. Viral Infections in Antiquity Reconstruction of the prehistoric past to provide a plausible account of when or how viruses established themselves in human populations is a challenging task. However, extrapolating from current knowledge, we can deduce that some modern viruses were undoubtedly associated with the earliest precursors of mammals and coevolved with humans. Other viruses entered human populations only recently. The last 10,000 years of history was a time of radical change for humans and our viruses: animals were domesticated, the human population increased dramatically, large population centers appeared, and commerce drove worldwide travel and interactions among unprecedented numbers of people. ASM_POV4e_Vol1_Ch01.indd 6 Viruses that established themselves in human populations were undoubtedly transmitted from animals, much as still happens today. Early human groups that domesticated and lived with their animals were almost certainly exposed to different viruses than were nomadic hunter societies. Similarly, as many diﬀerent viruses are endemic in the tropics, human societies in that environment must have been exposed to a greater variety of viruses than societies established in temperate climates. When nomadic groups met others with domesticated animals, human-to-human contact could have provided new avenues for virus spread. Even so, it seems unlikely that viruses such as those that cause measles or smallpox could have entered a permanent relationship with small groups of early humans. Such highly virulent viruses, as we now know them to be, either kill their hosts or induce lifelong immunity. Consequently, they can survive only when large, interacting host populations oﬀer a suﬃcient number of naive and permissive hosts for their continued propagation. Such viruses could not have been established in human populations until large, settled communities appeared. Less virulent viruses that enter into a long-term relationship with their hosts were therefore more likely to be the ﬁrst to become adapted to reproduction in the earliest human populations. These viruses include the modern retroviruses, herpesviruses, and papillomaviruses. Evidence of several viral diseases can be found in ancient records. The Greek poet Homer characterizes Hector as 7/22/15 12:27 PM Foundations A 7 B Here this firebrand, rabid Hector, leads the charge. Homer, The Iliad, translated by Robert Fagels (Viking Penguin) Figure 1.1 References to viral diseases abound in the ancient literature. (A) An image of Hector from an ancient Greek vase. Courtesy of the University of Pennsylvania Museum (object 30-44-4). (B) An Egyptian stele, or stone tablet, from the 18th dynasty (1580–1350 b.c.) depicting a man with a withered leg and the “drop foot” syndrome characteristic of polio. Panel B is reprinted from W. Biddle, A Field Guide to Germs (Henry Holt and Co., LLC, New York, NY, 1995; © 1995 by Wayne Biddle), with permission from the publisher. “rabid” in The Iliad (Fig. 1.1A), and Mesopotamian laws that outline the responsibilities of the owners of rabid dogs date from before 1000 b.c. Their existence indicates that the communicable nature of this viral disease was already well-known by that time. Egyptian hieroglyphs that illustrate what appear to be the consequences of poliovirus infection (a withered leg typical of poliomyelitis [Fig. 1.1B]) or pustular lesions characteristic of smallpox also date from that period. The smallpox virus, which was probably endemic in the Ganges River basin by the ﬁfth century b.c. and subsequently spread to other parts of Asia and Europe, has played an important part in human history. Its introduction into the previously unexposed native populations of Central and South America by colonists in the 16th century led to lethal epidemics, which are considered an important factor in the conquests achieved by a small number of European soldiers. Other viral diseases known in ancient times include mumps and, perhaps, inﬂuenza. Yellow fever has been described since the discovery ASM_POV4e_Vol1_Ch01.indd 7 of Africa by Europeans, and it has been suggested that this scourge of the tropical trade was the basis for legends about ghost ships, such as the Flying Dutchman, in which an entire ship’s crew perished mysteriously. Humans have not only been subject to viral disease throughout much of their history but have also manipulated these agents, albeit unknowingly, for much longer than might be imagined. One classic example is the cultivation of marvelously patterned tulips, which were of enormous value in 17th-century Holland. Such eﬀorts included deliberate spread of a virus (tulip breaking virus or tulip mosaic virus) that we now know causes the striping of tulip petals so highly prized at that time (Fig. 1.2). Attempts to control viral disease have an even more venerable history. The First Vaccines Measures to control one viral disease have been used with some success for the last millennium. The disease is smallpox 7/22/15 12:27 PM 8 Chapter 1 Figure 1.3 Characteristic smallpox lesions in a young victim. Illustrations like these were used as examples to track down individuals infected with the smallpox virus (variola virus) during the World Health Organization campaign to eradicate the disease. Photo courtesy of the Immunization Action Coalition (original source: Centers for Disease Control and Prevention). (See also the interview with Dr. Donald Henderson: http://bit.ly/Virology_Henderson) Figure 1.2 Three Broken Tulips. A painting by Nicolas Robert (1624–1685), now in the collection of the Fitzwilliam Museum, Cambridge, United Kingdom. Striping patterns (color breaking) in tulips were described in 1576 in western Europe and were caused by a viral infection. This beautiful image depicts the remarkable consequences of infection with the tulip mosaic virus. Courtesy of the Fitzwilliam Museum, University of Cambridge. (Fig. 1.3), and the practice is called variolation, inoculation of healthy individuals with material from a smallpox pustule into a scratch made on the arm. Variolation, widespread in China and India by the 11th century, was based on the recognition that smallpox survivors were protected against subsequent bouts of the disease. Variolation later spread to Asia Minor, where its value was recognized by Lady Mary Wortley Montagu, wife of the British ambassador to the Ottoman Empire. She introduced this practice into England in 1721, where it became quite widespread following the successful inoculation of children of the royal family. George Washington is said to have introduced variolation among Continental Army soldiers in 1776. However, the consequences of variolation were unpredictable and never pleasant: serious skin lesions invariably developed at the site of inoculation and were often accompanied by more generalized rash and disease, with a fatality rate of 1 to 2%. From the comfortable viewpoint of an affluent country in the 21st century, such a death rate seems unacceptably high. However, in the 18th century, variolation ASM_POV4e_Vol1_Ch01.indd 8 was perceived as a much better alternative than contracting natural smallpox, a disease with a fatality rate of 25% in the whole population and 40% in babies and young children. In the 1790s, Edward Jenner, an English country physician, recognized the principle on which modern methods of viral immunization are based, even though viruses themselves were not to be identiﬁed for another 100 years. Jenner himself was variolated as a boy and also practiced this procedure. He was undoubtedly familiar with its eﬀects and risks. Perhaps this experience spurred his great insight upon observing that milkmaids were protected against smallpox if they previously contracted cowpox (a mild disease in humans). Jenner followed up this astute observation with direct experiments. In 1794 to 1796, he demonstrated that inoculation with extracts from cowpox lesions induced only mild symptoms but protected against the far more dangerous disease. It is from these experiments with cowpox that we derive the term vaccination (vacca ⫽ “cow” in Latin); Louis Pasteur coined this term in 1881 to honor Jenner’s accomplishments. Initially, the only way to propagate and maintain the cowpox vaccine was by serial infection of human subjects. This method was eventually banned, as it was often associated with transmission of other diseases such as syphilis and hepatitis. By 1860, the vaccine had been passaged in cows; later, sheep and water buﬀaloes were also used. While Jenner’s original vaccine was based on the virus that causes cowpox, sometime during the human-to-human or cow-to-cow transfers, the poxvirus now called vaccinia virus replaced the cowpox virus. Vaccinia virus is the basis for the modern smallpox vaccine, but its origins remain a mystery: it exhibits limited genetic similarity to the viruses that cause cowpox or smallpox, or to many of the 7/22/15 12:27 PM Foundations BOX 9 1.3 D I S C U S S I O N Origin of vaccinia virus Over the years, at least three hypotheses have been advanced to explain the curious substitution of cowpox virus by vaccinia virus: Needle is held perpendicular to the arm 1. Recombination of cowpox virus with smallpox virus after variolation of humans 2. Recombination between cowpox virus and animal poxviruses during passage in various animals 3. Genetic drift of cowpox virus after repeated passage in humans and animals None of these hypotheses has been proven conclusively, and all fail to account fully for the origins of the sequences in the vaccinia virus genome. Evans DH. 2 June 2013. Episode 235, This Week in Virology. http://www.twiv.tv/2013/06/02/twiv-235live-in-edmonton-eh/ Qin L, Upton C, Hazes B, Evans DH. 2011. Genomic analysis of the vaccinia virus strain variants found in Dryvax vaccine. J Virol 24:13049–13060. Wrist of vaccinator rests on the arm Drop of vaccine is held in the fork of the needle Smallpox vaccine is delivered via multiple punctures with a special two-pronged needle (inset) that has been dipped in the vaccine (Adapted from WHO, with permission). other known members of the poxvirus family. Scientists have recovered the smallpox vaccine used in New York in 1876 and have veriﬁed that it contains vaccinia virus and not cowpox virus. Speculation about when and how the switch occurred has produced some possible scenarios (Box 1.3). The ﬁrst deliberately attenuated viral vaccine was made by Louis Pasteur, although he had no idea at the time that the relevant agent was a virus. In 1885, he inoculated rabbits with material from the brain of a cow suﬀering from rabies and then used aqueous suspensions of dried spinal cords from these animals to infect other rabbits. After several such passages, the resulting preparations caused mild disease (i.e., were attenuated) yet produced effective immunity against rabies. Safer and more eﬃcient methods for the production of larger quantities of these ﬁrst vaccines awaited the recognition of viruses as distinctive biological entities and parasites of cells in their hosts. Indeed, it took almost 50 years to discover the next antiviral vaccines: a vaccine for yellow fever virus was developed in 1935, and an inﬂuenza vaccine was available in 1936. These advances became possible only with radical changes in our knowledge of living organisms and of the causes of disease. Microorganisms as Pathogenic Agents The 19th century was a period of revolution in scientific thought, particularly in ideas about the origins of living things. ASM_POV4e_Vol1_Ch01.indd 9 The publication of Charles Darwin’s The Origin of Species in 1859 crystallized startling (and, to many people, shocking) new ideas about the origin of diversity in plants and animals, until then generally attributed directly to the hand of God. These insights permanently undermined the perception that humans were somehow set apart from all other members of the animal kingdom. From the point of view of the science of virology, the most important changes were in ideas about the causes of disease. The diversity of macroscopic organisms has been appreciated and cataloged since the dawn of recorded human history. A vast new world of organisms too small to be visible to the naked eye was revealed through the microscopes of Antony van Leeuwenhoek (1632–1723). Van Leeuwenhoek’s vivid and exciting descriptions of living microorganisms, the “wee animalcules” present in such ordinary materials as rain or seawater, included examples of protozoa, algae, and bacteria. By the early 19th century, the scientiﬁc community had accepted the existence of microorganisms and turned to the question of their origin, a topic of ﬁerce debate. Some believed that microorganisms arose spontaneously, for example in decomposing matter, where they were especially abundant. Others held the view that all were generated by the reproduction of like microorganisms, as were macroscopic organisms. The death knell of the spontaneous-generation hypothesis was sounded with the famous experiments of Pasteur. He demonstrated 7/22/15 12:27 PM 10 Chapter 1 articulated in an 1890 presentation in Berlin. These criteria, Koch’s postulates, can be summarized as follows. • The organism must be regularly associated with the disease and its characteristic lesions. • The organism must be isolated from the diseased host and grown in culture. • The disease must be reproduced when a pure culture of the organism is introduced into a healthy, susceptible host. • The same organism must be reisolated from the experimentally infected host (Box 1.4). Broth Figure 1.4 Pasteur’s famous swan-neck flasks provided passive exclusion of microbes from the sterilized broth. Although the flask was freely open to the air at the end of the long curved stem, the broth remained sterile as long as the microbe-bearing dust that collected in the neck of the stem did not reach the liquid. that boiled (i.e., sterilized) medium remained free of microorganisms as long as it was maintained in special ﬂasks with curved, narrow necks designed to prevent entry of airborne microbes (Fig. 1.4). Pasteur also established that particular microorganisms were associated with speciﬁc processes, such as fermentation, an idea that was crucial in the development of modern explanations for the causes of disease. From the earliest times, poisonous air (miasma) was generally invoked to account for epidemics of contagious diseases, and there was little recognition of the diﬀerences among causative agents. The association of particular microorganisms, initially bacteria, with specific diseases can be attributed to the ideas of the German physician Robert Koch. He developed and applied a set of criteria for identiﬁcation of the agent responsible for a speciﬁc disease (a pathogen), BOX By applying his criteria, Koch demonstrated that anthrax, a common disease of cattle, was caused by a speciﬁc bacterium (designated Bacillus anthracis) and that a second, distinct bacterial species caused tuberculosis in humans. Guided by these postulates and the methods for the sterile culture and isolation of pure preparations of bacteria developed by Pasteur, Joseph Lister, and Koch, many pathogenic bacteria (as well as yeasts and fungi) were identiﬁed and classiﬁed during the last part of the 19th century (Fig. 1.5). From these beginnings, investigation into the causes of infectious disease was placed on a secure scientiﬁc foundation, the ﬁrst step toward rational treatment and ultimately control. Furthermore, during the last decade of the 19th century, failures of the paradigm that bacterial or fungal agents are responsible for all diseases led to the identiﬁcation of a new class of infectious agents— submicroscopic pathogens that came to be called viruses. Discovery of Viruses The first report of a pathogenic agent smaller than any known bacterium appeared in 1892. The Russian scientist Dimitrii Ivanovsky observed that the causative agent of tobacco mosaic disease was not retained by the unglazed filters used at 1.4 D I S C U S S I O N New methods extend Koch’s principles While it is clear that a microbe that fulfills Koch’s postulates is almost certainly the cause of the disease in question, we now know that microbes that do not fulfill such criteria may still represent the etiological agents of disease. In the latter part of the 20th century, new methods were developed to associate particular viruses with disease based on immunological evidence of infection, for example, the presence of antibodies in blood. The availability of these methods led to the proposal of modified “molecular Koch’s postulates” based on the application of molecular techniques to monitor the role played by virulence genes in bacteria. ASM_POV4e_Vol1_Ch01.indd 10 The most revolutionary advances in our ability to link particular viruses with disease (or benefit) come from the more recent development of high-throughput nucleic acid sequencing methods and bioinformatics tools that allow detection of viral genetic material directly in environmental or biological samples, an approach called viral metagenomics. Based on these developments, alternative “metagenomic Koch’s postulates” have been proposed in which (i) the definitive traits are molecular markers such as genes or full genomes that can uniquely distinguish samples obtained from diseased subjects from those obtained from matched, healthy control subjects and (ii) inoculating a healthy individual with a sample from a diseased subject results in transmission of the disease as well as the molecular markers. Falkow S. 1988. Molecular Koch’s postulates applied to microbial pathogenicity. Rev Infect Dis 10(Suppl 2): S274–S276. Fredericks DN, Relman DA. 1996. Sequence-based identification of microbial pathogens: a reconsideration of Koch’s postulates. Clin Microbiol Rev 9:18–33. Mokili JL, Rohwer F, Dutilh BE. 2012. Metagenomics and future perspectives in virus discovery. Curr Opin Virol 2:63–77. Racaniello V. 22 January 2010. Koch’s postulates in the 21st century. Virology Blog. http://www.virology. ws/2010/01/22/kochs-postulates-in-the-21st-century/ 7/22/15 12:27 PM Foundations 11 60 Cumulative number of discoveries 50 Fungi (17) Bacteria (50) Protozoa (11) Filterable viruses (19) 40 30 20 Koch's Koch'sintroduction introductionof of efﬁcient efﬁcientbacteriological bacteriological methods methods Discovery of TMV 10 0 1835 1845 1855 1865 1875 1885 Year 1895 1905 1915 1925 1935 Figure 1.5 The pace of discovery of new infectious agents in the 19th and 20th centuries. Koch’s introduction of efficient bacteriological techniques spawned an explosion of new discoveries of bacterial agents in the early 1880s. Similarly, the discovery of filterable agents launched the field of virology in the early 1900s. Despite an early surge of virus discovery, only 19 distinct human viruses had been reported by 1935. TMV, tobacco mosaic virus. Adapted from K. L. Burdon, Medical Microbiology (Macmillan Co., New York, NY, 1939), with permission. that time to remove bacteria from extracts and culture media (Fig. 1.6A). Six years later in Holland, Martinus Beijerinck independently made the same observation. More importantly, Beijerinck made the conceptual leap that this must be a distinctive agent, because it was so small that it could pass through filters that trapped all known bacteria. However, Beijerinck thought that the agent was an infectious liquid. It was two former students and assistants of Koch, Friedrich Loeffler and Paul Frosch, who in the same year (1898) deduced that such infectious filterable agents comprised small particles: they observed that while the causative agent of footand-mouth disease (Box 1.2) passed through filters that held back bacteria, it could be retained by a finer filter. Not only were the tobacco mosaic and foot-and-mouth disease pathogens much smaller than any previously recognized microorganism, but also they were replicated only in their host organisms. For example, extracts of an infected tobacco plant diluted into sterile solution produced no additional infectious agents until introduced into leaves of healthy plants, which subsequently developed tobacco mosaic disease. The serial transmission of infection by diluted extracts established that these diseases were not caused by a bacterial toxin present in the original preparations derived from infected tobacco plants or cattle. The failure of both pathogens to multiply in solutions that readily supported the growth of bacteria, as well as their dependence on host ASM_POV4e_Vol1_Ch01.indd 11 organisms for reproduction, further distinguished these new agents from pathogenic bacteria. Beijerinck termed the submicroscopic agent responsible for tobacco mosaic disease contagium vivum fluidum to emphasize its infectious nature and distinctive reproductive and physical properties. Agents passing through ﬁlters that retain bacteria came to be called ultrafilterable viruses, appropriating the term “virus” from the Latin for “poison.” This term eventually was simpliﬁed to “virus.” The discovery of the ﬁrst virus, tobacco mosaic virus, is often attributed to the work of Ivanovsky in 1892. However, he did not identify the tobacco mosaic disease pathogen as a distinctive agent, nor was he convinced that its passage through bacterial ﬁlters was not the result of some technical failure. It may be more appropriate to attribute the founding of the ﬁeld of virology to the astute insights of Beijerinck, Loeﬄer, and Frosch, who recognized the distinctive nature of the plant and animal pathogens they were studying more than 100 years ago. The pioneering work on tobacco mosaic and foot-andmouth disease viruses was followed by the identification of viruses associated with specific diseases in many other organisms. Important landmarks from this early period include the identiﬁcation of viruses that cause leukemias or solid tumors in chickens by Vilhelm Ellerman and Olaf Bang in 1908 and Peyton Rous in 1911, respectively. The study of viruses associated with cancers in chickens, particularly Rous 7/22/15 12:27 PM 12 Chapter 1 A B Bacteria + virus Berkefeld ﬁlters have three grades of porosities, two of which hold back all bacteria. These ﬁlters are made of diatomaceous earth. Virus Figure 1.6 Filter systems used to characterize/purify virus particles. (A) The earliest, the Berkefeld filter, was invented in Germany in 1891. It was a “candle”-style filter comprising diatomaceous earth, or Kieselguhr, pressed into the shape of a hollow candle. The white candle is in the upper chamber of the apparatus, which is open at the top to receive the liquid to be filtered into the suction flask. The smallest pore size retained bacteria and allowed virus particles to pass through. Such filters were probably used by Ivanovsky, Loeffler, and Frosch to isolate the first plant and animal viruses. (B) A typical Millipore membrane filter apparatus. Such modern-day filter systems are disposable plastic laboratory items in which the upper and lower chambers are separated by a biologically inert membrane, available in a variety of pore sizes. Such filtration approaches may have limited our detection of giant viruses. Image provided courtesy of EMD Millipore Corporation. sarcoma virus, eventually led to an understanding of the molecular basis of cancer (Volume II, Chapter 7). The fact that bacteria could also be hosts to viruses was ﬁrst recognized by Frederick Twort in 1915 and Félix d’Hérelle in 1917. d’Hérelle named such viruses bacteriophages because of their ability to lyse bacteria on the surface of agar plates (“phage” is derived from the Greek for “eating”). In an interesting twist of serendipity, Twort made his discovery of bacterial viruses while testing the smallpox vaccine virus to see if it would grow on simple media. He found bacterial contaminants, some of them appearing more transparent, which proved to be the result of lysis by a bacteriophage. Investigation of bacteriophages established the foundations for the ﬁeld of molecular biology, as well as fundamental insights into how viruses interact with their host cells. The Definitive Properties of Viruses Throughout the early period of virology when many viruses of plants, animals, and bacteria were cataloged, ideas about the origin and nature of these distinctive infectious agents were quite controversial. Arguments centered on whether viruses originated from parts of a cell or were built from unique components. Little progress was made toward resolving these issues and establishing the definitive properties of viruses until the development of new techniques that allowed their visualization or propagation in cultured cells. ASM_POV4e_Vol1_Ch01.indd 12 The Structural Simplicity of Virus Particles Dramatic confirmation of the structural simplicity of virus particles came in 1935, when Wendell Stanley obtained crystals of tobacco mosaic virus. At that time, nothing was known of the structural organization of any biologically important macromolecules, such as proteins and DNA. Indeed, the crucial role of DNA as genetic material had not even been recognized. The ability to obtain an infectious agent in crystalline form, a state that is more generally associated with inorganic material, created much wonder and speculation about whether a virus is truly a life form. In retrospect, it is obvious that the relative ease with which tobacco mosaic virus could be crystallized was a direct result of both its structural simplicity and the ability of many particles to associate in regular arrays. The 1930s saw the introduction of the instrument that rapidly revolutionized virology: the electron microscope. The great magnifying power of this instrument (eventually more than 100,000-fold) allowed direct visualization of virus particles for the ﬁrst time. It has always been an exciting experience for investigators to obtain images of viruses, especially as they appear to be remarkably elegant (Fig. 1.7). Images of many diﬀerent virus particles conﬁrmed that these agents are very small (Fig. 1.8) and that most are far simpler in structure than any cellular organism. Many appeared 7/22/15 12:27 PM Foundations A C B 13 D Figure 1.7 Electron micrographs of virus particles following negative staining. (A) The complex, nonenveloped virus bacteriophage T4. Note the intricate tail and tail fibers. Courtesy of R. L. Duda, University of Pittsburgh, Pittsburgh, PA. (B) The helical, nonenveloped particle of tobacco mosaic virus. Reprinted from the Universal Virus Database of the International Committee on Taxonomy of Viruses (http://ictvonline.org/), with permission. (C) Enveloped particles of the rhabdovirus vesicular stomatitis virus. Courtesy of F. P. Williams, University of California, Davis. (D) Nonenveloped, icosahedral human rotavirus particles. Courtesy of F. P. Williams, U.S. Environmental Protection Agency, Washington, DC. Figure 1.8 Size matters. (A) Sizes of animal and plant cells, bacteria, viruses, proteins, molecules, and atoms are indicated. The resolving powers of various techniques used in virology, including light microscopy, electron microscopy, X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy, are indicated. Viruses span a broad range from that equal to some small bacteria to just under ribosome size. The units commonly used in descriptions of virus particles or their components are the nanometer (nm [10⫺9 m]) and the angstrom (Å [10⫺10 m]). Adapted from A. J. Levine, Viruses (Scientific American Library, New York, NY, 1991); used with permission of Henry Holt and Company, LLC. (B) Illustration of the size differences among two viruses and a typical host cell. A Plant cells Animal cells Bacteria Ribosomes Viruses Proteins Small molecules Atoms Meters 10–2 (1 cm) 10–3 (1 mm) 10–4 10–5 10–6 (1 μm) 10–7 10–8 10–9 (1 nm) 10–10 (1 Å) Light microscope Electron microscope X ray NMR B Ribosomes (20 nm) Herpesvirus (200 nm) Poliovirus (30 nm) ASM_POV4e_Vol1_Ch01.indd 13 7/22/15 12:27 PM 14 Chapter 1 as regular helical or spherical particles. The description of the morphology of virus particles made possible by electron microscopy also opened the way for the ﬁrst rational classiﬁcation of viruses. The Intracellular Parasitism of Viruses Organisms as Hosts The defining characteristic of viruses is their absolute dependence on a living host for reproduction: they are obligate parasites. Transmission of plant viruses such as tobacco mosaic virus can be achieved readily, for example, by applying extracts of an infected plant to a scratch made on the leaf of a healthy plant. Furthermore, as a single infectious particle of many plant viruses is suﬃcient to induce the characteristic lesion (Fig. 1.9), the concentration of the infectious agent could be measured. Plant viruses were therefore the ﬁrst to be studied in detail. Some viruses of humans and other species could also be propagated in laboratory animals, and methods were developed to quantify them by determining the lethal dose. The transmission of yellow fever virus to mice by Max Theiler in 1930 was an achievement that led to the isolation of an attenuated strain, still considered one of the safest and most eﬀective ever produced for the vaccination of humans. After speciﬁc viruses and host organisms were identiﬁed, it became possible to produce suﬃcient quantities of virus particles for study of their physical and chemical properties and the consequences of infection for the host. Features such as the incubation period, symptoms of infection, and eﬀects on speciﬁc tissues and organs were investigated. Laboratory Figure 1.9 Lesions induced by tobacco mosaic virus on an infected tobacco leaf. In 1886, Adolph Mayer first described the characteristic patterns of light and dark green areas on the leaves of tobacco plants infected with tobacco mosaic virus. He demonstrated that the mosaic lesions could be transmitted from an infected plant to a healthy plant by aqueous extracts derived from infected plants. The number of local necrotic lesions that result is directly proportional to the number of infectious particles in the preparation. Courtesy J. P. Krausz; Reproduced, by permission of APS, from Scholthof, K.-B. G. 2000. Tobacco mosaic virus. The Plant Health Instructor. doi:10.1094/PHI-I-2000-1010-01. ASM_POV4e_Vol1_Ch01.indd 14 animals remain an essential tool in investigations of the pathogenesis of viruses that cause disease. However, real progress toward understanding the mechanisms of virus reproduction was made only with the development of cell culture systems. Among the simplest, but crucial to both virology and molecular biology, were cultures of bacterial cells. Lessons from Bacteriophages In the late 1930s and early 1940s, bacteriophages, or “phages,” received increased attention as a result of controversy centering on how they were formed. John Northrup, a biochemist at the Rockefeller Institute in Princeton, NJ, championed the theory that a phage was a metabolic product of a bacterium. On the other hand, Max Delbrück, in his work with Emory Ellis and later with Luria, regarded phages as autonomous, stable, self-replicating entities characterized by heritable traits. According to this paradigm, phages were seen as ideal tools with which to investigate the nature of genes and heredity. Probably the most critical early contribution of Delbrück and Ellis was the perfection of the one-step growth method for synchronization of the reproduction of phages, an achievement that allowed analysis of a single cycle of phage reproduction in a population of bacteria. This approach introduced highly quantitative methods to virology, as well as an unprecedented rigor of analysis. The ﬁrst experiments showed that phages indeed multiplied in the bacterial host and were liberated in a “burst” by lysis of the cell. Delbrück was a zealot for phage research and recruited talented scientists to pursue the fundamental issues of what is now known as the ﬁeld of molecular biology. This group of scientists, working together in what came to be called the “phage school,” focused their attention on speciﬁc phages of the bacterium Escherichia coli. Progress was rapid, primarily because of the simplicity of the phage infectious cycle. Phages reproduce in bacterial hosts, which can be obtained in large numbers by overnight culture. By the mid-1950s, it was evident that viruses from bacteria, animals, and plants share many fundamental properties. However, the phages provided a far more tractable experimental system. Consequently, their study had a profound impact on the ﬁeld of virology. One critical lesson came from a deﬁnitive experiment that established that viral nucleic acid carries genetic information. It was known from studies of the “transforming principle” of pneumococcus by Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) that nucleic acid was both necessary and suﬃcient for the transfer of genetic traits of bacteria. However, in the early 1950s, protein was still suspected to be an important component of viral heredity. In a brilliantly simple experiment that included the use of a common kitchen food blender, Alfred Hershey and Martha Chase showed that this hypothesis was incorrect (Box 1.5). 7/22/15 12:27 PM Foundations BOX 15 1.5 E X P E R I M E N T S The Hershey-Chase experiment By differentially labeling the nucleic acid and protein components of virus particles with radioactive phosphorus ( 32P) and radioactive sulfur ( 35S), respectively, Alfred Hershey and Martha Chase showed that the Infection protein coat of the infecting virus could be removed soon after infection by agitating the bacteria for a few minutes in a blender. In contrast, 32P-labeled phage DNA entered and remained associated with the bacterial cells Blending/separation under these conditions. Because such blended cells produced a normal burst of new virus particles, it was clear that the DNA contained all of the information necessary to produce progeny phages. Centrifugation/detection Viral protein labeled with radioactive sulfur Radioactivity predominantly in the supernatant fraction No radioactivity detected in next generation of phage Radioactivity predominantly in the cell pellet Radioactive DNA is detected in progeny phage Viral DNA labeled with radioactive phosphorus Bacteriophages were originally thought to be lethal agents, killing their host cells after infection. In the early 1920s, a previously unknown interaction was discovered, in which the host cell not only survived the infection but also stably inherited the genetic information of the virus. It was also observed that certain bacterial strains could lyse spontaneously and produce bacteriophages after a period of growth in culture. Such strains were called lysogenic, and the phenomenon, lysogeny. Studies of lysogeny uncovered many previously unrecognized features of virus-host cell interactions (Box 1.6). Recognition of this phenomenon came from the work of many scientists, but it began with the elegant experiments of André Lwoﬀ and colleagues at the Institut Pasteur in Paris. Lwoﬀ showed that a viral genome exists in lysogenic cells in the form of a silent genetic element called the prophage. This element determined the ability of lysogenic bacteria to produce infectious bacteriophages. ASM_POV4e_Vol1_Ch01.indd 15 Subsequent studies of the E. coli phage lambda established a paradigm for one mechanism of lysogeny, the integration of a phage genome into a speciﬁc site on the bacterial chromosome. Bacteriophages became inextricably associated with the new ﬁeld of molecular biology (Table 1.1). Their study established many fundamental principles: for example, control of the decision to enter a lysogenic or a lytic pathway is encoded in the genome of the virus. The ﬁrst mechanisms discovered for the control of gene expression, exempliﬁed by the elegant operon theory of Nobel laureates François Jacob and Jacques Monod, were deduced in part from studies of lysogeny by phage lambda. The biology of phage lambda provided a fertile ground for work on gene regulation, but study of virulent T phages (T1 to T7, where T stands for “type”) of E. coli paved the way for many other important advances (Table 1.1). As we shall see, these systems also 7/22/15 12:27 PM 16 BOX Chapter 1 1.6 B A C K G R O U N D Properties of lysogeny shared with animal viruses Lytic versus Lysogenic Response to Infection Some bacterial viruses can enter into either destructive (lytic) or relatively benign (lysogenic) relationships with their host cells. Such bacteriophages were called temperate. In a lysogenic bacterial cell, viral genetic information persists but viral gene expression is repressed. Such cells are called lysogens, and the quiescent viral genome, a prophage. By analogy with the prophage, an integrated DNA copy of a retroviral genome in an animal genome is termed a provirus. Propagation as a Prophage For some bacteriophages like lambda and Mu (Mu stands for “mutator”), prophage DNA is integrated into the host genome of lysogens and passively replicated by the host. Virally encoded enzymes, known as integrase (lambda) and transposase (Mu), mediate the covalent insertion of viral DNA into the chromosome of the host bacterium, establishing it as a prophage. The prophage DNA of other bacteriophages, such as P1, exists as a plasmid, a self-replicating, autonomous chromosome in a lysogen. Both forms of propagation have been identified in certain animal viruses. Gene Repression and Induction Prophage gene expression in lysogens is turned off by the action of viral proteins called repressors. Expression can be turned on when repressors are inactivated (a process called induction). Elucidation of the mechanisms of these processes set the stage for later investigation of the control of gene expression in experiments with other viruses and their host cells. Transduction of Host Genes Bacteriophage genomes can pick up cellular genes and deliver them to new cells (a process known as transduction). The process can be generalized, with the acquisition by the virus of any segment from the host chromosome, or specialized, as is the case for viruses that integrate into specific sites in the host chromosome. For example, occasional mistakes in excision of the lambda prophage after induction result in production of unusual progeny phage that have lost some of their own DNA but have acquired the bacterial DNA adjacent to the prophage. As described in Volume II, Chapter 7, the acute transforming retroviruses also arise via capture of genes in the vicinity of their integration as proviruses. These cancer-inducing cellular genes are then transduced along with viral genes during subsequent infection. Pioneers in the study of lysogeny: Nobel laureates François Jacob, Jacques Monod, and André Lwoff. Insertional Mutagenesis Bacteriophage Mu inserts its genome into many random locations on the host chromosome, causing numerous mutations. This process is called insertional mutagenesis and is a phenomenon observed with retroviruses. provided an extensive preview of mechanisms of animal virus reproduction (Box 1.7). Animal Cells as Hosts The culture of animal cells in the laboratory was initially more of an art than a science, restricted to cells that grew out of organs or tissues maintained in nutrient solutions under sterile conditions. The ﬁnite life span of such primary cells; their dependence for growth on natural components in their media such as lymph, plasma, or chicken embryo extracts; and the technical demands of sterile culture prior to the discovery of antibiotics made reproducible experimentation very difficult. However, by 1955, the work of many investigators had led to a series of important methodological advances. These included the development of deﬁned media ASM_POV4e_Vol1_Ch01.indd 16 optimal for growth of mammalian cells, incorporation of antibiotics into cell culture media, and development of immortal cell lines such as the mouse L and human HeLa cells that are still in widespread use. These advances allowed growth of animal cells in culture to become a routine, reproducible exercise. The availability of well-characterized cell cultures had several important consequences for virology. It allowed the discovery of new human viruses, such as adenovirus, measles virus, and rubella virus, for which animal hosts were not available. In 1949, John Enders and colleagues used cell cultures to propagate poliovirus, a feat that led to the development of polio vaccines a few years later. Cell culture technology revolutionized the ability to investigate the reproduction of viruses. Viral infectious cycles could be studied 7/22/15 12:27 PM Foundations B OX The definitive properties of viruses are summarized as follows: 1.7 T E R M I N O L O G Y The episome In 1958, François Jacob and Elie Wollman realized that lambda prophage and the E. coli F sex factor had many common properties. This remarkable insight led to the definition of the episome. An episome is an exogenous genetic element that is not necessary for cell survival. Its defining characteristic is the ability to reproduce in two alternative states: while integrated in the host chromosome or autonomously. However, this term is often applied to genomes that can be maintained in cells by autonomous replication and never integrate, for example, the DNA genomes of certain animal viruses. F 17 Integrated F • A virus is an infectious, obligate intracellular parasite. • The viral genome comprises DNA or RNA. • The viral genome directs the synthesis of viral components by cellular systems within an appropriate host cell. • Infectious progeny virus particles, called virions, are formed by de novo self-assembly from newly synthesized components. • A progeny virion assembled during the infectious cycle is the vehicle for transmission of the viral genome to the next host cell or organism, where its disassembly initiates the next infectious cycle. While viruses lack the complex energy-generating and biosynthetic systems necessary for independent existence (Box 1.8), they are not the simplest biologically active agents: viroids, which are infectious agents of a variety of economically important plants, comprise a single small molecule of noncoding RNA, whereas other agents, termed prions, are thought to be single protein molecules (Volume II, Chapter 12). Cataloging Animal Viruses under precisely controlled conditions by employing the analog of the one-step growth cycle of bacteriophages and simple methods for quantiﬁcation of infectious particles described in Chapter 2. Our current understanding of the molecular basis of viral parasitism, the focus of this volume, is based almost entirely on analyses of one-step growth cycles in cultured cells. Such studies established that viruses are molecular parasites: for example, their reproduction depends absolutely on their host cell’s biosynthetic machinery for synthesis of the components from which they are built. In contrast to cells, viruses are not reproduced by growth and division. Rather, the infecting genome contains the information necessary to redirect cellular systems to the production of many copies of all the components needed for the de novo assembly of new virus particles. Viruses Defined Advances in knowledge of the structure of virus particles and the mechanisms by which they are produced in their host cells have been accompanied by increasingly accurate definitions of these unique agents. The earliest pathogenic agents, distinguished by their small size and dependence on a host organism for reproduction, emphasized the importance of viruses as agents of disease. We can now provide a much more precise definition, elaborating their relationship with the host cell and the important features of virus particles. ASM_POV4e_Vol1_Ch01.indd 17 Virus classification was at one time a subject of colorful and quite heated controversy (Box 1.9). As new viruses were being discovered and studied by electron microscopy, the virus world was seen to be a veritable zoo of particles with different sizes, shapes, and compositions (see, for example, Fig. 1.10). Very strong opinions were advanced concerning classification and nomenclature. One camp pointed to the inability to infer, from the known properties of viruses, anything about their evolutionary origin or their relationships to one another—the major goal of classical taxonomy. The other camp maintained that despite such limitations, there were significant practical advantages in grouping isolates with similar properties. A major sticking point, however, was finding agreement on which properties should be considered most important in constructing a scheme for virus classification. The Classical System Lwoff, Robert Horne, and Paul Tournier, in 1962, advanced a comprehensive scheme for the classification of all viruses (bacterial, plant, and animal) under the classical Linnaean hierarchical system consisting of phylum, class, order, family, genus, and species. Although a subsequently formed international committee on the nomenclature of viruses did not adopt this system in toto, its designation of families, genera, and species was used for the classification of animal viruses. One of the most important principles embodied in the system advanced by Lwoﬀ and his colleagues was that viruses 7/22/15 12:27 PM 18 BOX Chapter 1 1.8 D I S C U S S I O N Are viruses living entities? What can/can’t they do? Viruses can be viewed as microbes that exist in two phases: an inanimate phase, the virion; and a multiplying phase in an infected cell. Some researchers have promoted the idea that viruses are organisms and that the inanimate virions may be viewed as “spores” that come “alive” in cells, or in factories within cells. This has long been a topic of intense discussion, stimulated most recently by the discovery of giant viruses such as the mimiviruses and pandoraviruses. Check out what the contemporary general public feels about this topic (http://www.virology.ws/are-viruses-alive/). Apart from attributing “life” to viruses, many scientists have succumbed to the temptation of ascribing various actions and motives when discussing them. While remarkably effective in enlivening a lecture or an article, anthropomorphic characterizations are inaccurate and also quite misleading. Infected cells and hosts respond in many ways after infection, but viruses are passive agents, totally at the mercy of their environments. Therefore viruses cannot employ, ensure, synthesize, exhibit, display, destroy, deploy, depend, reprogram, avoid, retain, evade, exploit, generate, etc. As virologists can be very passionate about their subject, it is exceedingly difficult to purge such anthropomorphic terms from virology communications. Indeed, hours were spent doing so in the preparation of this textbook, Bândea Cl. 1983. A new theory on the origin and the nature of viruses. J Theor Biol 105:591–602. Claverie JM, Abergel C. 2013. Open questions about giant viruses. Adv Virus Res 85:25-56. or should be grouped according to their shared properties rather than the properties of the cells or organisms they infect. A second principle was a focus on the nucleic acid genome as the primary criterion for classiﬁcation. The importance of the genome had become clear when it was inferred from the Hershey-Chase experiment that viral nucleic acid alone can be infectious (Box 1.5). Four cha