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VOLUME I Molecular Biology

Virology
PRINCIPLES OF

Fifth Edition
 VOLUME I Molecular Biology

Virology
PRINCIPLES OF

Fifth Edition

Jane Flint Glenn F. Rall

Department of Molecular Biology Fox Chase Cancer Center
Princeton University Philadelphia, Pennsylvania
Princeton, New Jersey
Theodora Hatziioannou
Vincent R. Racaniello The Rockefeller University
Department of Microbiology & Immunology New York, New York
Vagelos College of Physicians and Surgeons
Columbia University Anna Marie Skalka
New York, New York Fox Chase Cancer Center
Philadelphia, Pennsylvania

Washington, DC
 Copyright © 2020 American Society for Microbiology. All rights reserved.

Copublication by the American Society for Microbiology and John Wiley & Sons, Inc.

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any
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The right of Jane Flint, Vincent R. Racaniello, Glenn F. Rall, Theodora Hatziioannou, and Anna Marie Skalka to be
identified as the author(s) of this work/the editorial material in this work has been asserted in accordance with law.

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While the publisher and author have used their best efforts in preparing this book, they make no repre­sen­ta­t ions or
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Library of Congress Cataloging-­in-­Publication Data

Names: Flint, S. Jane, author. | Racaniello, V. R. (Vincent R.), author. | Rall, Glenn F., author. | Hatziioannou,
Theodora, author. | Skalka, Anna Marie, author.
Title: Princi­ples of virology / Jane Flint, Department of Molecular Biology, Prince­ton University, Prince­ton,
New Jersey, Vincent R. Racaniello, Department of Microbiology & Immunology, Vagelos College of Physicians
and Surgeons, Columbia University, New York, New York, Glenn F. Rall, Fox Chase Cancer Center, Philadelphia,
Pennsylvania, Theodora Hatziioannou, The Rocke­fel­ler University, New York, New York, Anna Marie Skalka,
Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Description: Fifth edition. | Washington, DC : American Society for Microbiology [2020] ; Hoboken, NJ : Wiley,
[2020] | Includes bibliographical references and index. | Contents: volume 1. Molecular biology—­volume 2.
Pathogenesis and control.
Identifiers: LCCN 2020013722 (print) | LCCN 2020013723 (ebook) | ISBN 9781683670322 (set) | ISBN
9781683672845 (v. 1 ; paperback) | ISBN 9781683672852 (v. 2 ; paperback) | ISBN 9781683672821 (v. 1 ; adobe pdf) |
ISBN 9781683673606 (v. 1 ; epub) | ISBN 9781683672838 (v. 2 ; adobe pdf) | ISBN 9781683673590 (v. 2 ; epub) |
ISBN 9781683670339 (adobe pdf) | ISBN 9781683673583 (epub)
Subjects: LCSH: Virology.
Classification: LCC QR360 .P697 2020 (print) | LCC QR360 (ebook) | DDC 616.9/101—­dc23
LC rec­ord available at https://­lccn​.­loc​.­gov​/­2020013722
LC ebook rec­ord available at https://­lccn​.­loc​.­gov​/­2020013723

Illustrations and illustration concepting: Patrick Lane, ScEYEnce Studios

Cover image: Visual Science
Cover and interior design: Susan Brown Schmidler

Printed in the United States of Amer­i­ca

10 9 8 7 6 5 4 3 2 1
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
Paul, Stefan, and Eve
Rudy, Jeannie, and Chris

Oh, be wiser thou!

Instructed that true knowledge leads to love.
William Wordsworth
Lines left upon a Seat in a Yew-­tree
1888
 Trim Size: 216mm x 276mm Flint - 06/26/2020 11:50pm Page vi
k

About the Instructor Companion Website

This book is accompanied by a companion website for instructors:

www.wiley.com/go/flint/pov5

k The website includes: k

• PowerPoints of figures
• Author podcasts
• Study Questions and Answers

vi

k
Contents

Preface xvii
Acknowl­edgments xxi
About the Authors xxiii
Key of Repetitive Elements xxv

PA RT I
The Science of Virology 1
1 Foundations 2
Luria’s Credo 3
Viruses Defined 3
Why We Study Viruses 3
Viruses Are Everywhere 3
Viruses Infect All Living Th
­ ings 4
Viruses Can Cause ­Human Disease 5
Viruses Can Be Beneficial 5
Viruses “R” Us 6
Viruses Can Cross Species Bound­aries 6
Viruses Are Unique Tools To Study Biology 6
Virus Prehistory 7
Viral Infections in Antiquity 7
The First Vaccines 8
Microorganisms as Pathogenic Agents 9
Discovery of Viruses 11
The Defining Properties of Viruses 13
The Structural Simplicity of Virus Particles 13
The Intracellular Parasitism of Viruses 13
Cata­loging Animal Viruses 18
The Classical System 18
Classification by Genome Type: the Baltimore System 19
A Common Strategy for Viral Propagation 21
vii
viii Contents

Perspectives 21
References 24
Study Questions 24

2 The Infectious Cycle 26

Introduction 27
The Infectious Cycle 27
The Cell 27
Entering Cells 28
Viral RNA Synthesis 29
Viral Protein Synthesis 29
Viral Genome Replication 29
Assembly of Progeny Virus Particles 29
Viral Pathogenesis 29
Overcoming Host Defenses 30
Cultivation of Viruses 30
Cell Culture 30
Embryonated Eggs 35
Laboratory Animals 35
Assay of Viruses 35
Mea­sure­ment of Infectious Units 35
Efficiency of Plating 38
Mea­sure­ment of Virus Particles 40
Viral Reproduction: The Burst Concept 49
The One-­Step Growth Cycle 49
One-­Step Growth Analy­sis: a Valuable Tool for Studying
Animal Viruses 52
Global Analy­sis 53
DNA Microarrays 54
Mass Spectrometry 56
Protein-­Protein Interactions 56
Single-­Cell Virology 56
Perspectives 58
References 59
Study Questions 60

PA RT II
Molecular Biology 61
3 Genomes and Ge­ne­tics 62
Introduction 63
Genome Princi­ples and the Baltimore System 63
 Contents ix

Structure and Complexity of Viral Genomes 63

DNA Genomes 64
RNA Genomes 65
What Do Viral Genomes Look Like? 68
Coding Strategies 69
What Can Viral Sequences Tell Us? 69
The “Big and Small” of Viral Genomes: Does
Size ­Matter? 71
The Origin of Viral Genomes 73
Ge­ne­tic Analy­sis of Viruses 74
Classical Ge­ne­tic Methods 75
Engineering Mutations into Viral Genomes 77
Engineering Viral Genomes: Viral Vectors 83
Perspectives 87
References 87
Study Questions 88

4 Structure 90
Introduction 91
Functions of the Virion 91
Nomenclature 92
Methods for Studying Virus Structure 92
Building a Protective Coat 95
Helical Structures 96
Capsids with Icosahedral Symmetry 99
Other Capsid Architectures 111
Packaging the Nucleic Acid Genome 112
Direct Contact of the Genome with a Protein Shell 112
Packaging by Specialized Viral Proteins 113
Packaging by Cellular Proteins 113
Viruses with Envelopes 115
Viral Envelope Components 115
­Simple Enveloped Viruses: Direct Contact of External Proteins
with the Capsid or Nucleocapsid 117
Enveloped Viruses with an Additional Protein Layer 118
Large Viruses with Multiple Structure Elements 119
Particles with Helical or Icosahedral Parts 120
Alternative Architectures 123
Other Components of Virions 125
Enzymes 125
Other Viral Proteins 125
Cellular Macromolecules 126
Mechanical Properties of Virus Particles 126
Investigation of Mechanical Properties of Virus Particles 126
Stabilization and Destabilization of Virus Particles 128
x Contents

Perspectives 128
References 129
Study Questions 130

5 Attachment and Entry 132

Introduction 133
Attachment of Virus Particles to Cells 133
General Princi­ples 133
Identification of Receptors for Virus Particles 135
Virus-­Receptor Interactions 137
Entry into Cells 142
Virus-­induced Signaling via Cell Receptors 142
Routes of Entry 143
Membrane Fusion 145
Intracellular Trafficking and Uncoating 154
Movement of Viral and Subviral Particles within Cells 154
Uncoating of Enveloped Virus Particles 155
Uncoating of Nonenveloped Viruses 155
Import of Viral Genomes into the Nucleus 159
The Nuclear Pore Complex 159
Nuclear Localization Signals 159
Nuclear Import of RNA Genomes 161
Nuclear Import of DNA Genomes 162
Import of Retroviral Genomes 162
Perspectives 164
References 165
Study Questions 166

6 Synthesis of RNA from RNA Templates 168

Introduction 169
The Nature of the RNA Template 169
Secondary Structures in Viral RNA 169
Naked or Nucleocapsid RNA 170
The RNA Synthesis Machinery 171
Identification of RNA-­Dependent RNA Polymerases 171
Three-­Dimensional Structures of RNA-­Dependent RNA Polymerases 173
Mechanisms of RNA Synthesis 176
Initiation 176
Capping 179
Elongation 179
Functions of Additional Polymerase Domains 181
RNA Polymerase Oligomerization 181
Template Specificity 182
 Contents xi

Unwinding the RNA Template 182

Role of Cellular Proteins 183
Paradigms for Viral RNA Synthesis 183
(+) Strand RNA 184
Synthesis of Nested Subgenomic mRNAs 184
(−) Strand RNA 185
Ambisense RNA 189
Double-­Stranded RNA 189
Unique Mechanisms of mRNA and Genome Synthesis
of Hepatitis Delta Virus 190
Do Ribosomes and RNA Polymerases Collide? 192
Origins of Diversity in RNA Virus Genomes 193
Misincorporation of Nucleotides 193
Segment Reassortment and RNA Recombination 193
RNA Editing 194
Perspectives 195
References 196
Study Questions 197

7 Synthesis of RNA from DNA Templates 198

Introduction 199
Properties of Cellular RNA Polymerases That Transcribe Viral DNA 199
Some Viral Genomes Must Be Converted to Templates Suitable
for Transcription 200
Transcription by RNA Polymerase II 201
Regulation of RNA Polymerase II Transcription 203
Common Properties of Proteins That Regulate Transcription 206
Transcription of Viral DNA Templates by the Cellular
Machinery Alone 208
Viral Proteins That Govern Transcription of DNA
Templates 209
Patterns of Regulation 209
The H
­ uman Immunodeficiency Virus Type 1 Tat Protein Autoregulates
Transcription 211
The Transcriptional Cascades of DNA Viruses 217
Entry into One of Two Alternative Transcriptional Programs 226
Transcription of Viral Genes by RNA Polymerase III 230
The VA-­R NA I Promoter 231
Inhibition of the Cellular Transcriptional Machinery 232
Unusual Functions of Cellular Transcription Components in
Virus-­Infected Cells 233
Viral DNA-­Dependent RNA Polymerases 233
Perspectives 234
References 235
Study Questions 236
xii Contents

8 Pro­cessing 238
Introduction 239
Covalent Modification during Viral Pre-­mRNA Pro­cessing 240
Capping the 5′ Ends of Viral mRNA 240
Synthesis of 3′ Poly(A) Segments of Viral mRNA 243
Internal Methylation of Adenosine Residues 245
Splicing of Viral Pre-­mRNA 246
Regulated Pro­cessing of Viral Pre-­mRNA 249
Editing of Viral mRNAs 255
Export of RNAs from the Nucleus 257
The Cellular Export Machinery 257
Export of Viral mRNA 258
Posttranscriptional Regulation of Viral or Cellular
Gene Expression by Viral Proteins 262
Temporal Control of Viral Gene Expression 262
Viral Proteins Can Inhibit Cellular mRNA Production 264
Regulation of Turnover of Viral and Cellular mRNAs
in the Cytoplasm 266
Intrinsic Turnover 266
Regulation of mRNA Stability by Viral Proteins 267
mRNA Stabilization Can Facilitate Transformation 267
Nonsense-­Mediated mRNA Decay 267
Noncoding RNAs 271
Small Interfering RNAs and Micro-­R NAs 271
Long Noncoding RNAs 276
Circular RNAs 278
Perspectives 278
References 279
Study Questions 281

9 Replication of DNA Genomes 282

Introduction 283
DNA Synthesis by the Cellular Replication Machinery 284
Eukaryotic Replicons 284
Cellular Replication Proteins 287
Mechanisms of Viral DNA Synthesis 287
Lessons from Simian Virus 40 288
Replication of Other Viral DNA Genomes 290
Properties of Viral Replication Origins 294
Recognition of Viral Replication Origins 296
Viral DNA Synthesis Machines 301
Resolution and Pro­cessing of Viral Replication Products 301
Exponential Accumulation of Viral Genomes 302
Viral Proteins Can Induce Synthesis of Cellular Replication Proteins 303
 Contents xiii

Synthesis of Viral Replication Machines and Accessory Enzymes 304

Viral DNA Replication In­de­pen­dent of Cellular Proteins 304
Delayed Synthesis of Structural Proteins Prevents Premature
Packaging of DNA Templates 305
Inhibition of Cellular DNA Synthesis 305
Synthesis of Viral DNA in Specialized Intracellular Compartments 305
­Limited Replication of Viral DNA Genomes 308
Integrated Parvoviral DNA Can Be Replicated as Part of the
Cellular Genome 308
Dif­fer­ent Viral Origins Regulate Replication of Epstein-­Barr
Virus 310
­Limited and Amplifying Replication from a Single Origin:
the Papillomaviruses 313
Origins of Ge­ne­tic Diversity in DNA Viruses 315
Fidelity of Replication by Viral DNA Polymerases 315
Modulation of the DNA Damage Response 316
Recombination of Viral Genomes 318
Perspectives 321
References 321
Study Questions 323

10 Reverse Transcription and Integration 324

Retroviral Reverse Transcription 325
Discovery 325
Impact 325
The Pro­cess of Reverse Transcription 326
General Properties and Structure of Retroviral Reverse
Transcriptases 334
Other Examples of Reverse Transcription 337
Retroviral DNA Integration 340
The Pathway of Integration: Integrase-­Catalyzed Steps 341
Integrase Structure and Mechanism 347
Hepadnaviral Reverse Transcription 350
A DNA Virus with Reverse Transcriptase 350
The Pro­cess of Hepadnaviral Reverse Transcription 352
Perspectives 358
References 359
Study Questions 360

11 Protein Synthesis 362

Introduction 363
Mechanisms of Eukaryotic Protein Synthesis 363
General Structure of Eukaryotic mRNA 363
The Translation Machinery 364
xiv Contents

Initiation 365
Elongation and Termination 375
The Diversity of Viral Translation Strategies 378
Polyprotein Synthesis 378
Leaky Scanning 378
Reinitiation 381
StopGo Translation 382
Suppression of Termination 382
Ribosomal Frameshifting 383
Bicistronic mRNAs 384
Regulation of Translation during Viral Infection 385
Inhibition of Translation Initiation ­after Viral Infection 385
Regulation of eIF4F 389
Regulation of Poly(A)-­Binding Protein Activity 392
Regulation of eIF3 392
Interfering with RNA 392
Stress-­Associated RNA Granules 393
Perspectives 395
References 396
Study Questions 397

12 Intracellular Trafficking 398

Introduction 399
Assembly within the Nucleus 400
Import of Viral Proteins for Assembly 401
Assembly at the Plasma Membrane 403
Transport of Viral Membrane Proteins to the Plasma
Membrane 404
Sorting of Viral Proteins in Polarized Cells 419
Disruption of the Secretory Pathway in Virus-­Infected Cells 421
Signal Sequence-­Independent Transport of Viral Proteins
to the Plasma Membrane 422
Interactions with Internal Cellular Membranes 426
Localization of Viral Proteins to Compartments of the
Secretory Pathway 426
Localization of Viral Proteins to the Nuclear Membrane 426
Transport of Viral Genomes to Assembly Sites 427
Transport of Genomic and Pregenomic RNA from the
Nucleus to the Cytoplasm 427
Transport of Genomes from the Cytoplasm to the Plasma
Membrane 429
Perspectives 430
References 431
Study Questions 432
 Contents xv

13 Assembly, Release, and Maturation 434

Introduction 435
Methods of Studying Virus Assembly and Egress 435
Structural Studies of Virus Particles 436
Visualization of Assembly and Exit by Microscopy 436
Biochemical and Ge­ne­tic Analyses of Assembly Intermediates 436
Methods Based on Recombinant DNA Technology 439
Assembly of Protein Shells 439
Formation of Structural Units 439
Capsid and Nucleocapsid Assembly 441
Self-­Assembly and Assisted Assembly Reactions 445
Selective Packaging of the Viral Genome and Other
Components of Virus Particles 447
Concerted or Sequential Assembly 447
Recognition and Packaging of the Nucleic Acid Genome 448
Incorporation of Enzymes and Other Nonstructural Proteins 458
Acquisition of an Envelope 459
Sequential Assembly of Internal Components and Budding
from a Cellular Membrane 459
Coordination of the Assembly of Internal Structures with
Acquisition of the Envelope 460
Release of Virus Particles 460
Assembly and Budding at the Plasma Membrane 461
Assembly at Internal Membranes: the Prob­lem of Exocytosis 464
Release of Nonenveloped Virus Particles 470
Maturation of Progeny Virus Particles 470
Proteolytic Pro­cessing of Structural Proteins 470
Other Maturation Reactions 474
Cell-­to-­Cell Spread 475
Perspectives 479
References 479
Study Questions 481

14 The Infected Cell 482

Introduction 483
Signal Transduction 483
Signaling Pathways 483
Signaling in Virus-­Infected Cells 485
Gene Expression 489
Inhibition of Cellular Gene Expression 489
Differential Regulation of Cellular Gene Expression 492
Metabolism 496
Methods To Study Metabolism 496
xvi Contents

Glucose Metabolism 497

The Citric Acid Cycle 501
Electron Transport and Oxidative Phosphorylation 502
Lipid Metabolism 504
Remodeling of Cellular Organelles 507
The Nucleus 509
The Cytoplasm 511
Perspectives 516
References 518
Study Questions 519

A P P E N D I X Structure, Genome Organ­ization, and Infectious Cycles

of Viruses Featured in This Book 521
Glossary 557
Index 563
Preface

The enduring goal of scientific endeavor, as of all h

­ uman 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 five 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 common steps of the viral reproductive cycle, illustrated with a set of represen-
tative viruses, and considering mechanisms by which ­these viruses can cause disease pro-
vides an integrated overview of the biology of ­t hese infectious agents. 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 virus biology.
In this information-­rich age, the quantity of data describing any given virus can be over-
whelming, 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 t­ hose who write text-
books meant to be used by students. In the fifth edition, we continue to distill information
with the intent of extracting essential princi­ples, while providing descriptions of how the in-
formation was acquired and tools to encourage our readers’ exploration of the primary lit­er­a­
ture. Boxes are used to emphasize major princi­ples and to provide supplementary material of
relevance, from explanations of terminology to descriptions of trailblazing experiments. Our
goal is to illuminate pro­cess and strategy as opposed to listing facts and figures. In an effort to
make the book readable, we have been selective in our choice of viruses that are used as ex-
amples. The encyclopedic Fields’ Virology [Knipe DM, Howley PM (ed). 2020. Fields Virology,
7th ed. Lippincott Williams & Wilkins, Philadelphia, PA] is recommended as a resource for
detailed reviews of specific virus families.

What’s New
This edition is marked by a welcome addition to the author team. Our new member, Theo-
dora Hatziioannou, brings expertise in retrovirology, entry, and intrinsic immunity, as well
as authority regarding ancient Greek my­thol­ogy and philosophy that the attentive reader
­w ill see is generously sprinkled throughout the text.

xvii
xviii Preface

We have added an impor­tant new chapter in Volume II, “Therapeutic Viruses.” While the
majority of the chapters define how viruses reproduce and cause mayhem to both cell and
host, this new chapter turns the ­tables to discuss how viruses can be beneficial to eliminate
tumor cells, deliver therapeutic genes to specific cells, and expand our arsenal of vaccines for
prevention of virus-­mediated diseases.
The authors continually strive to make this text accessible and relevant to our readers,
many of whom are undergraduates, gradu­ate students, and postdoctoral fellows. Conse-
quently, for this edition, we enlisted the aid of more than twenty of t­ hese trainees to provide
guidance and commentary on our chapters and ensure that concepts are clearly explained
and that the text is compelling to read. This unique group of editors has been invaluable in
the design of all of our fully reworked and up-­to-­date chapters and appendices, and we ex-
tend a par­tic­u ­lar thank-­you to them for sharing their perspectives.
A new feature is the inclusion of a set of study questions and/or, in some cases, puzzles, as
aids to ensure that the key princi­ples are evident within each chapter. This section comple-
ments the Princi­ples that begin each chapter, focusing on unifying core concepts.
Fi­nally, although the SARS-­CoV-2 pandemic began as we ­were preparing to go to press,
we have included additions to relevant chapters on the epidemiology, emergence, and repli-
cation of this global scourge, as well as some hopeful information concerning vaccine devel-
opment. What is apparent is that, now more than ever, an appreciation of how viruses impact
their hosts is not just an academic pursuit, but rather literally a ­matter of life and death. We
extend our gratitude to all ­t hose who serve in patient care settings.

Princi­ples Taught in Two Distinct, but Integrated Volumes

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 vol-
umes follows a natu­ral break in pedagogy and provides considerable flexibility and utility for
students and teachers alike. 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 as noted above, added a new chapter that we be-
lieve reflects an exciting direction for the field. Links to Internet resources such as websites,
podcasts, blog posts, and movies are provided within each chapter; the digital edition pro-
vides one-­click access to ­t hese materials.
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 our readers was particularly useful in finding
typographical errors, clarifying confusing or complicated illustrations, and pointing out in-
consistencies in content.
For purposes of readability, references are not included within the text; each chapter ends
with an updated list of relevant books, review articles, and selected research papers for read-
ers who wish to pursue specific topics. New to this edition are short descriptions of the key
messages from each of the cited papers of special interest. Fi­nally, each volume has a general
glossary of essential terms.
­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. We have
focused primarily on animal viruses, but have drawn insights from studies of viruses that
reproduce in plants, bacteria, and archaea.

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. Chap-
ter 1 provides a general introduction and historical perspective, and includes descriptions of
the unique properties of viruses. The unifying princi­ples that are the foundations of virology,
 Preface xix

including the concept of a common strategy for viral propagation, are then described. The
princi­ples of the infectious cycle, descriptions of the basic techniques for cultivating and as-
saying 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 is considered in Chapter 4.
Chapters 5 to 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 in-
formation to genome replication and production of progeny virions. We describe how ­t hese
common steps are accomplished in cells infected by diverse but representative viruses, while
emphasizing common princi­ples. Volume I concludes with a chapter that pre­sents an inte-
grated 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 reproductive cycles for
members of the main virus families discussed in the text. It is intended to be a reference re-
source 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.

Volume II: Pathogenesis, Control, and Evolution

This volume addresses the interplay between viruses and their host organisms. In Chapter 1,
we introduce the discipline of epidemiology, and consider basic aspects that govern how the
susceptibility of a population is controlled and mea­sured. Physiological barriers to virus in-
fections, and how viruses spread in a host, and 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, which are tailored to the pathogen, and immune memory. Chapter 5 focuses on the
classical patterns of virus infection within cells and hosts, and the myriad ways that viruses
cause illness. In Chapter 6, we discuss virus infections that transform cells in culture and
promote oncogenesis (the formation of tumors) in animals. Next, we consider the princi­ples
under­lying treatment and control of infection. Chapter 7 focuses on vaccines, and Chapter 8
discusses the approaches and challenges of antiviral drug discovery. In Chapter 9, the new
chapter in this edition, we describe the rapidly expanding applications of viruses as thera-
peutic agents. The origin of viruses, the d
­ rivers of viral evolution, and host-­v irus 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.
Chapter 12 is devoted entirely to the “AIDS virus,” ­human immunodeficiency virus type 1,
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 h ­ uman immune defenses.
Volume II ends with a chapter on unusual infectious agents, viroids, satellites, and prions.
The Appendix of Volume II affords snapshots of the pathogenesis of common h ­ uman vi-
ruses. This appendix has been completely re-­envisioned in this edition, and now includes
panels that define pathogenesis, vaccine and antiviral options, and the course of the infec-
tion through the h ­ uman body. This consistent format should allow students to find informa-
tion more easily, and compare properties of the selected viruses.

For some behind-­t he-­scenes information about how the authors created the previous edi-
tion of Princi­ples of Virology, see: http://­bit​.­ly​/­Virology​_ ­MakingOf.
Acknowl­edgments

­ ese two volumes of Princi­ples could not have been composed and revised without help and
Th
contributions from many individuals. We are most grateful for the continuing encourage-
ment from our colleagues in virology and the students who use the text. Our sincere thanks
also go to colleagues 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 w­ ere invaluable and are inextricably
woven into the final form of the book.
We also are grateful to ­t hose who gave so generously of their time to serve as expert re-
viewers of individual chapters or specific topics in ­t hese two volumes: Siddharth Balachan-
dran (Fox Chase Cancer Center), Paul Bieniasz (Rocke­fel­ler University), Christoph Seeger
(Fox Chase Cancer Center), and Laura Steel (Drexel University College of Medicine). Their
rapid responses to our requests for details and checks on accuracy, as well as their assistance
in simplifying complex concepts, ­were invaluable.
As noted in “What’s New,” we benefited from the efforts of the students and postdoctoral
fellows who provided critiques on our chapters and helped to guide our revisions: Pradeep
Morris Ambrose, Ruchita Balasubramanian, Mariana Nogueira Batista, Pierre Michel Jean
Beltran, Marni S. Crow, Qiang Ding, Florian Douam, Jenna M. Gaska, Laura J. Halsey, Eliana
Jacobson, Orkide O. Koyuncu, Robert LeDesma, Rebecca Markham, Alexa McIntyre, Kate-
lynn A. Milora, Laura A. M. Nerger, Morgan Pantuck, Chen Peng, Katrien Poelaert, Daniel
Poston, Anagha Prasanna, Pavithran T. Ravindran, Inna Ricardo-­Lax, Fabian Schmidt, An-
dreas Solomos, Nikhila Shree Tanneti, Sharon M. Washio, Riley M. Williams, and Kai Wu.
Since the inception of this work, our belief has been that the illustrations must comple-
ment and enrich the text. 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 ScEY-
Ence Studios. A key to common figure ele­ments is provided following the “About the Authors”
section. 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 in this latest edition: Jônatas Abrahão (Universidade Federal de Minas Gerais), Mark
Andrake (Fox Chase Cancer Center), Irina Arkhipova (Marine Biological Laboratory, Woods
Hole), Brian Baker (University of Notre Dame), Ben Beaden (Australia Zoo, Queensland),
Paul Bieniasz (Rocke­fel­ler University), Kartik Chandran (Albert Einstein College of Medi-
cine), Elliot Lefkowitz (University of Alabama), Joseph Pogliano (University of California,
xxi
xxii Acknowl­edgments

San Diego), B.V. Venkatar Prasad and Liya Hu (Baylor College of Medicine), Bonnie Quigley
(University of the Sunshine Coast, Australia), Jason Roberts (Victorian Infectious Diseases
Reference Laboratory, Doherty Institute, Melbourne, Australia), Michael Rout (Rocke­fel­ler
University), and Nuria Verdaguer (Molecular Biology Institute of Barcelona, CSIC).
The collaborative work undertaken to prepare the fifth edition was facilitated greatly by
several authors’ retreats. ASM Press generously provided financial support for ­t hese as well
as for our many other meetings over the three years that this edition has been in preparation.
We thank all ­t hose who guided and assisted in its production: Christine Charlip (Director,
ASM Press) for her enduring support of our efforts; Megan Angelini (Managing Develop-
mental Editor, ASM Press) for steering us through the complexities inherent in a team effort,
and for keeping us on track during production; Susan Schmidler for her elegant and creative
designs for the layout and cover; and Lindsay Williams (Editorial Rights Coordinator, ASM
Press) for obtaining permissions for images and figures.
­There is ­little doubt that in undertaking such a massive effort typographical errors and/or
confusing statements still remain; we hope that the readership of this edition w ­ ill help to
remedy any ­mistakes. Even so, the three authors who have been part of this endeavor since it
was first published in 1995, and the two who joined along the way, feel that with each new
edition we get closer to our idealized vision of what this book would be. We aspire to convey
more than information: we hope to educate, excite, and encourage ­f uture generations of sci-
ence consumers. As Antoine de Saint-­Exupéry, author of The L ­ ittle Prince, once said: “If you
want to build a ship, d ­ on’t drum up the workers to gather wood, divide the l­abor, and give
­orders. Instead, teach them to yearn for the vast and endless sea.”
This often-­consuming enterprise was made pos­si­ble by the emotional, intellectual, and
logistical support of our families, to whom the two volumes are dedicated.
About the Authors

L to R: Jane Flint, Vincent Racaniello, Theodora Hatziioannou, Ann Skalka, Glenn Rall

Jane Flint is a Professor Emerita of Molecular Biology at dergraduate virology lectures have been viewed by thousands
Prince­ton University. Dr. Flint’s research focused on investi- at iTunes University, Coursera, and on YouTube. Vincent
gation of the molecular mechanisms by which viral gene blogs about viruses at virology.ws and is host of the popu­lar
products modulate host cell pathways and antiviral defenses science program This Week in Virology, which, together with
to allow efficient reproduction in normal ­human cells of ade- six other science podcasts, can be found at microbe.tv.
noviruses, viruses that are widely used in such therapeutic
applications as gene transfer and cancer treatment. Her ser­ Glenn F. Rall is a Professor and the Chief Academic Offi-
vice to the scientific community includes membership on cer at the Fox Chase Cancer Center in Philadelphia. He is an
vari­ous editorial boards, several NIH study sections, and the Adjunct Professor in the Microbiology and Immunology
NIH Recombinant DNA Advisory Committee. departments at the University of Pennsylvania and Thomas
Jefferson, Drexel, and ­Temple Universities. Dr. Rall’s labo-
Vincent R. Racaniello is Higgins Professor of Micro- ratory studies viral infections of the brain and the immune
biology & Immunology at Columbia University Vagelos College responses to t­ hose infections, with the goal of defining how
of Physicians & Surgeons. Dr. Racaniello has been studying viruses contribute to disease in ­humans. His ser­v ice to the
viruses for over 40 years, including poliovirus, rhinovirus, en- scientific community includes former membership on the
teroviruses, hepatitis C virus, and Zika virus. He teaches Autism Speaks Scientific Advisory Board, Editor of PLoS
virology to undergraduate, gradu­ate, medical, dental, and Pathogens, ­Career Development Chair and Program Chair of
nursing students and uses social media to communicate the the American Society for Virology, and membership on mul-
subject outside of the classroom. His Columbia University un- tiple NIH grant review panels.

xxiii
xxiv About the Authors

Theodora Hatziioannou is a Research Associate Profes- is internationally recognized for her contributions to the un-
sor at Rocke­fel­ler University in New York. Throughout her derstanding of the biochemical mechanisms by which such
­career, Dr. Hatziioannou has worked on multiple viruses, viruses (including the AIDS virus) replicate and insert their
with a par­tic­u­lar focus on retroviruses and the molecular ge­ne­tic material into the host genome. Both an administrator
mechanisms that govern virus tropism and on the improve- and researcher, Dr. Skalka has been deeply involved in state,
ment of animal models for ­human disease. She is actively in- national, and international advisory groups concerned with
volved in teaching programs at the Rocke­fel­ler University the broader, societal implications of scientific research. She
and the Albert Einstein College of Medicine, is an editor of has also served on the editorial boards of peer-­reviewed sci-
Journal of General Virology, and serves as a reviewer for mul- entific journals and has been a member of scientific advisory
tiple scientific journals and NIH grant review panels. boards including the National Cancer Institute Board of Sci-
entific Counselors, the General Motors Cancer Research
Anna Marie Skalka is a Professor Emerita and former Foundation Awards Assembly, the Board of Governors of the
Se­nior Vice President for Basic Research at the Fox Chase Can- American Acad­emy of Microbiology, and the National Advi-
cer Center in Philadelphia. Dr. Skalka’s major research inter- sory Committee for the Pew Biomedical Scholars.
ests are the molecular aspects of retrovirus biology. Dr. Skalka
 Key of Repetitive Elements

Viral DNA Transcription start site

Inhibition Enlargement arrow

Newly synthesized viral DNA Activation

Lipid Membrane
Cellular DNA

DNA (bar style)

5' AT CG 3'
3' T AG C 5'
Protein bar style

Viral RNA

U U U U U P
+ RNA – RNA ± RNA
Ubiquitin Phosphorylation
mRNA
5' c
AnAOH3’

mRNA (bar style)

5' c ACUG AnAOH3’

5' c Exon 1 Intron Exon 2 AnAOH3’

miRNA
5'
3'

Neutrophils Macrophages CD4+ T cells CD8+ T cells NK cells

B cells Endothelial cells Epithelial cells

Dendritic cells

xxv
PART I
The Science
of Virology
1 Foundations
2 The Infectious Cycle
1 Foundations

Luria’s Credo Discovery of Viruses

Viruses Defined The Defining Properties of
Viruses
Why We Study Viruses
The Structural Simplicity of Virus
Viruses Are Everywhere
Particles
Viruses Infect All Living Things
The Intracellular Parasitism of Viruses
Viruses Can Cause Human Disease
Viruses Can Be Beneficial Cataloging Animal Viruses
Viruses “R” Us The Classical System
Viruses Can Cross Species Boundaries Classification by Genome Type:
the ­Baltimore System
Viruses Are Unique Tools To Study
Biology A Common Strategy for Viral
Propagation
Virus Prehistory
Viral Infections in Antiquity Perspectives
The First Vaccines References
Microorganisms as Pathogenic Agents Study Questions

LINKS FOR CHAPTER 1

Video: Interview with Dr. Donald Hen­der­son Whiter reefs, fresh breath
http://​bit.​ly/​Virology_​Henderson http://​w ww.​microbe.​t v/​t wiv/​t wiv-​391/​
This Week in Virology (TWIV): A weekly Latest up­date of vi­rus clas­si­fi­ca­tion from
pod­cast about vi­ruses fea­tur­ing in­for­mal yet the ICTV
in­for­ma­tive dis­cus­sions and in­ter­views with https://​talk.​ictvonline.​org/​taxonomy/​
guests about the lat­est top­ics in the field The abun­dant and di­verse vi­ruses of
http://​w ww.​microbe.​t v/​t wiv the seas
Marine vi­ruses and in­sect de­fense http:// ​bit.​ly/​Virology_ ​3-​20- ​09
http://​bit.​ly/​Virology_​Twiv301 How many vi­ruses on Earth?
Giants among vi­rus­es http:// ​bit.​ly/​Virology_​9- ​6 -​13
http:// ​bit.​ly/​Virology_​Twiv261
 tious par ticles called virions, which contain genomes com-
prising RNA or DNA surrounded by a protective protein
coat. Upon par ticle entry and disassociation in a host cell, the
viral genome directs synthesis of viral components by cellu-
lar systems. Progeny virus particles are formed in the infected
cell by de novo self-assembly from the newly synthesized com-
Luria’s Credo ponents.
“There is an intrinsic simplicity of nature and the ultimate As will be discussed in the follow ing chapters, advances in
contribution of science resides in the discovery of unify ing knowledge of the structure of virus par ticles and the mecha-
and simplify ing generalizations, rather than in the descrip- nisms by which they are produced in their host cells have been
tion of isolated situations—in the visualization of simple, accompa nied by increasingly accurate definitions of these
overall pat terns rather than in the analysis of patchworks.” unique agents. The earliest pathogenic viruses, distinguished
More than half a century has passed since Salvador Luria by their small size and dependence on a host organism for re-
wrote this credo in the introduction to the classic textbook production, emphasized the impor tance of viruses as agents
General Virology. of disease. But there are many other impor tant reasons to
Despite an explosion of information in biology since Lu- study viruses.
ria wrote these words, his vision of unity in diversity is as
relevant now as it was then. That such unify ing principles Why We Study Viruses
ex ist may not be obvious considering the bewildering array
Viruses Are Everywhere
of viruses, genes, and proteins recognized in modern virol-
Viruses are all around us, comprising an enormous propor-
ogy. Indeed, new viruses are being described reg u larly, and
tion of our environment, in both number and total mass (Box
viral diseases such as acquired immunodeficiency syndrome
1.1). All liv ing things encounter billions of virus par ticles ev-
(AIDS), hepatitis, and influenza continue to chal lenge our
ery day. For example, they enter our lungs in the 6 liters of air
eforts to control them. Yet Luria’s credo still stands: even as
each of us inhales every minute; they enter our digestive sys-
our knowledge of viruses continues to increase, it is clear
tems with the food we eat; and they are transferred to our
that their reproduction and sur vival depend on simi lar
eyes, mouths, and other points of entry from the surfaces we
pathways. This insight has been hard-won over many years
touch and the people with whom we interact. Viral nucleic ac-
of obser vation, research, and debate; the history of virology
ids (the virome) can be found in the respiratory, gastrointes-
is rich and instructive.
tinal, and urogenital tracts even of normal, healthy individuals
(Fig. 1.1). Our bloodstreams harbor up to 100,000 virus par ti-
Viruses Defined cles per milliliter. In addition to viruses that can infect us, our
Viruses are microscopic infectious agents that can reproduce intestinal tracts are loaded with myriad plant and insect vi-
only inside a cell that they infect: they are obligate parasites ruses, as well as many hundreds of bacterial species that har-
of their host cells. Viruses spread from cell to cell via infec- bor their own constellations of viruses.

P R I N C I P L E S Foundations
Viruses are obligate intracellular parasites and depend on While Koch’s postulates were essential for defining many
their host cell for all aspects of their reproduction. agents of disease, not all pathogenic viruses can be shown to
fulfill these criteria.
The field of virology encompasses viral discovery; the study
of virus structure and reproduction; and the importance of Viruses can be described based on their appearance, the
viruses in biology, ecology, and disease. hosts they infect, or the nature of their nucleic acid genome.
This text focuses primarily on viruses that infect vertebrates, All viruses must produce mRNA that can be translated by
especially humans, but it is important to keep in mind that cellular ribosomes. The Baltimore classification allows rela-
viruses infect all living things including insects, plants, and tionships among viruses with RNA or DNA genomes to be
bacteria. determined based on the pathway required for mRNA pro-
duction.
Viruses are not solely pathogenic nuisances; they can be ben-
eficial. Viruses contribute to ecological homeostasis, keep A common program underlies the propagation of all vi-
our immune responses activated and alert, and can be used ruses. This textbook describes that strategy and the similar-
as molecular flashlights to illuminate cellular processes. ities and diferences in the manner in which diferent viruses
are reproduced, spread, and cause disease.
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 ani-
mals (zoonoses).

3
4 Chapter 1

Viruses Infect All Living Things DNA viruses

While most of this text­book fo­cuses on vi­ral in­fec­tions of
RNA viruses
hu­mans, it is im­por­tant to bear in mind that vi­ruses also in­ Nervous system
fect pets, do­mes­tic and wild an­i­mals, plants, and in­sects (>3)
through­out­the world. They in­fect mi­crobes such as al­gae,

Skin, hair, nails

B OX 1.1 (>13)
B A C K G R O U N D Respiratory tract
Some as­tound­ing num­bers (>17)

• Viruses are the most abun­dant en­ti­ties in the bio­sphere. The

bio­mass on our planet of bac­te­rial vi­ruses alone ex­ceeds that
of all­of Earth’s el­e­phants by more than 1,000-fold. There are Digestive tract
more than 1030 particles of bacterial viruses in the world’s (>19) Urogenital tract
oceans, enough to extend out into space for 200 million light-​ (>6)
years if arranged head to tail (http://​w ww.​v irology.​ws/​2009/​
03/​20/​t he-​abundant-​and-​diverse-​v iruses-​of-​t he-​seas/​; http://​
www.​phagehunter.​org/​2 008/​0 9/​how-​f ar-​do-​t hose-​phages-​
stretch.​html).
• W hales are com­monly in­fected with a mem­ber of the vi­rus
fam­i ly Caliciviridae that causes rashes, blis­ ters, in­ tes­
ti­
nal Blood
prob­lems, and di­ar­rhea, and that can also in­fect hu­mans. In- (>19)
fected whales ex­crete more than 1013 calicivirus par­ti­cles dai­ly.
• The av­er­age hu­man body con­tains ap­prox­i­ma­tely 1013 cells,
but al­most an equal num­ber of bac­te­ria, and as many as 100-
fold more vi­rus par­ti­cles.
• With about 1016 hu­man im­mu­no­de­fi­ciency vi­rus type 1 (HIV-1)
ge­nomes on the planet to­day, it is highly prob­a­ble that some­ Figure 1.1 The hu­man virome. Our knowl­edge of the di­ver­sity of
where there ex­ist HIV-1 ge­nomes that are re­sis­tant to ev­ery one vi­ruses that can be pres­ent in or on a nor­mal hu­man (in­clud­ing some
po­ten­t ial path­o­gens) has in­creased greatly with the de­vel­op­ment of
of the an­ti­v i­ral drugs that we have now or are likely to have in
high-throughput se­quenc­i ng tech­niques and new bioinformatic tools.
the fu­ture.
Current es­ti­ma­tes of the num­bers of dis­tinct vi­ral fam­i­lies with DNA
or RNA ge­nomes in var­i­ous sites are in pa­ren­t he­ses; the > sym­bol sig­ni­
fies the pres­ence of ad­di­tional vi­ruses not yet as­signed to known fam­i­
lies. The num­bers may in­crease as di­ag­nos­tic tools im­prove and new
vi­ral fam­i ­lies are iden­ti­fied. Data from Popgeorgiev N et al. 2013. Inter-
virology 56:395-412; see also http://​w ww.​v irology.​w s/​2 017/​03/​23/​t he​
-​v iruses-​in-​your-​blood/​.

fungi, and bac­te­ria, and some even in­ter­fere with the re­pro­
duc­tion of other vi­r uses. Viral in­fec­tion of ag­ri­cul­tural
plants and an­i­mals can have enor­mous eco­nomic and so­ci­e­
tal im­pact. Outbreaks of in­fec­tion by foot-and-mouth dis­
ease and avian in­flu­enza vi­r uses have led to the de­struc­tion
(cull­ing) of mil­li­ons of cat­t le, sheep, and poul­try, in­clud­ing
healthy an­i­mals, to pre­vent fur­ther spread. Losses in the
United Kingdom dur­ ing the 2001 out­­ break of foot-and-
mouth dis­ease ran into bil­li­ons of dol­lars, and caused havoc
for both farm­ers and the gov­ern­ment (Box 1.2). More re­cent
out­­
breaks of the avian in­ flu­enza vi­r us H5N1 and other
Viruses re­side in Earth’s vast oceans and
strains in Asia have re­sulted in sim­i­lar dis­r up­tion and eco­
ev­ery­where else on our plan­et. Courtesy of NASA’s
Earth Observatory, Suomi NPP sat­el­lite im­age nomic loss. Viruses that in­fect crops such as po­ta­toes and
cour­tesy of NASA/GSFC. fruit trees are com­mon, and can lead to se­ri­ous food short­
ages as well as fi­nan­cial dev­as­ta­tion.
 Foundations 5

B OX 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 do­mes­ mals. The as­so­ci­ated eco­nomic, so­ci­e­tal, and pigs, roughly 12% of its pop­u ­la­tion, to curb a
tic cat­t le, pigs, and sheep, as well as many spe­ po­lit­i­cal costs jolted the Brit­ish gov­ern­ment. more se­ri­ous out­­break spread of the vi­rus.
cies of wild an­i­mals. Although mor­tal­ity is Images of mass gra­ves and hor­rific pyres con­
Hunt J.3 Jan­u­a ry 2013. Foot-and-mouth is knock­ing
low, mor­bid­ity (ill­ness) is high and in­fected sum­ing the corpses of dead an­i­mals (see fig­ on Eu­rope’s door. Farmers Weekly. http://​w ww.​f wi.​
farm an­ i­mals lose their com­ mer­ cial value. ure) sen­ si­
tized the pub­ lic as never be­ fore. co.​u k/​a rticles/​03/​01/​2 013/​136943/​foot-​a nd-​mouth-​
The vi­rus is highly con­ta­gious, and the most Minor out­­breaks that oc­curred later in the is-​k nocking-​on-​europe39s-​door.​htm.
com­mon and ef­fec­tive method of con­trol is by United Kingdom and parts of Asia were also Mur­phy FA, Gibbs EPJ, Horzinek MC, Studdert MJ.
the slaugh­ter of en­tire herds in af­fected ar­eas. con­trolled by cull­ing. But in 2011, South Ko­ 1999. Veterinary Virology, 3rd ed. Academic Press,
Inc, San Di­ego, CA.
Outbreaks of foot-and-mouth dis­ ease rea was re­ported to have de­stroyed 1.5 mil­lion
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 en­try
into the Chi­cago stock­yards, the vi­rus spread
to more than 3,500 herds in 22 states. This ca­
lam­ity ac­cel­er­ated ep­i­de­mi­o­log­i­cal and dis­
ease con­trol pro­grams, even­tu­a lly lead­ing to
the field- and lab­o­ra­to­r y-based sys­tems main­
tained by the U.S. Department of Agriculture
to pro­tect do­mes­tic live­stock from for­eign an­
i­mal and plant dis­eases. Similar con­trol sys­
tems have been es­tab­lished in other Western
coun­tries, but this vi­rus still pres­ents a for­mi­
da­ble chal­lenge through­out­the 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 ep­ i­
demic out­­ break in the
United Kingdom spread to other coun­tries in Mass burn­ ing of cat­ tle car­
casses dur­ing the 2001 foot-and-
Eu­rope and led to the slaugh­ter of more than mouth dis­ease out­­break in the United Kingdom. Courtesy of Dr.
6 mil­lion in­fected and un­in­fected farm an­i­ Pamela Hullinger, Cal­i­for­nia Department of Food and Agriculture.

Viruses Can Cause Human Disease and gas­tro­in­tes­ti­nal tracts kill mil­li­ons of chil­dren in the de­
With such con­stant ex­po­sure, it is noth­ing short of amaz­ vel­op­ing world each year. As sum­ma­rized in Volume II, Ap-
ing that the vast ma­jor­ity of vi­r uses that in­fect us have lit­t le pendix, there is no ques­tion about the bio­med­i­cal im­por­tance
or no im­pact on our health or well-be­ing. As de­scribed in of these agents.
Volume II, we owe such rel­a­t ive safety to our elab­o­rate im­
mune de­fense sys­tems, which have evolved un­der the se­lec­ Viruses Can Be Beneficial
tive pres­sure im­posed by mi­cro­bial in­fec­tion. When these Despite the ap­pall­ing sta­tis­tics from hu­man and ag­ri­cul­tural
de­fenses are com­pro­mised, even the most com­mon in­fec­tion ep­i­dem­ics, it is im­por­tant to re­a l­i ze that vi­r uses can also be
can be le­thal. Despite such de­fenses, some of the most dev­as­ ben­e­fi­cial. Such ben­e­fit can be seen most clearly in the ma­
tat­ing hu­man dis­eases have been or still are caused by vi­rus­es; rine eco­sys­tem, where vi­rus par­ti­cles are the most abun­dant
these dis­eases in­clude small­pox, yel­low fe­ver, po­l io­my­eli­t is, bi­o­log­i­c al en­t i­t ies (Box 1.1). Indeed, they com­prise 94% of
in­flu­enza, mea­sles, and AIDS. Viral in­fec­t ions can lead to all­nu­cleic ac­id-containing par­t i­cles in the oceans and are
life-threatening dis­eases that im­pact vir ­tu­a lly all­ or­gans, 15 times more abun­dant than Bacteria and Archaea. Viral
in­clud­ing the lungs, liver, cen­tral ner­vous sys­tem, and in­tes­ in­fec­tions in the ocean kill 20 to 40% of ma­rine mi­crobes daily,
tines. Viruses are re­spon­si­ble for ap­prox­i­ma­tely 15% of the con­vert­ing these liv­ing or­gan­isms into par­tic­u­late mat­ter. In so
hu­man can­cer bur­den, and vi­ral in­fec­tions of the re­spi­ra­tory do­ing they re­lease es­sen­tial nu­tri­ents that sup­ply phy­to­plank­ton
6 Chapter 1

at the bot­tom of the ocean’s food chain, as well as car­bon birds in ar­eas of the Middle East and Asia. The vi­rus is
di­ox­ide and other gases that af­fect the cli­mate of the earth. deadly to hu­mans who catch it from in­fected birds. The
Pathogens can also in­flu­ence one an­oth­er: in­fec­tion by one fright­en­ing pos­si­bil­ity that it could gain the abil­ity to spread
vi­rus can have an ame­lio­rat­ing ef­fect on the path­o­gen­e­sis of a among hu­mans is a ma­jor in­cen­tive for mon­i­tor­ing for per­
sec­ond vi­rus or even bac­te­ria. For ex­am­ple, mice la­tently in­ son-to-person trans­mis­sion in case of in­fec­tion by this and
fected with some mu­rine her­pes­v i­ruses are re­sis­tant to in­ other path­o­genic avian in­flu­enza vi­ruses. Given the eons
fec­tion with the bac­te­rial path­o­gens Listeria monocytogenes over which vi­r uses have had the op­por­tu­nity to in­ter­act with
and Yersinia pestis. The idea that vi­ruses are solely agents of var­i­ous spe­cies, to­day’s “nat­u­ral” host may sim­ply be a way
dis­ease is giv­ing way to an ap­pre­ci­a­tion of their pos­i­tive, even sta­tion in vi­ral evo­lu­tion.
nec­es­sary, ef­fects, and a re­a l­i­za­tion that their unique prop­er­
ties can ac­tu­a lly be har­nessed for hu­man ben­e­fit (Volume II, Viruses Are Unique Tools To Study
Chapter 9). Biology
Because vi­ruses are de­pen­dent on their hosts for prop­a­ga­
Viruses “R” Us tion, stud­ies that fo­cus on vi­ral re­pro­gram­ming of cel­lu­lar
Every cell in our body con­tains vi­ral DNA. Human en­dog­e­ mech­a­nisms have pro­v ided unique in­sights into ge­net­ics,
nous ret­ro­v i­ruses, and el­e­ments thereof, make up about 8% of cel­lu­lar bi­­ol­ogy, and func­tion­ing of host de­fenses. Ground-
our ge­nome. Most are in­ac­tive, fos­sil rem­nants from in­fec­ breaking stud­ies of vi­r uses that in­fect bac­te­ria (called bac­te­
tions of germ cells that oc­curred over mil­li­ons of years dur­ rio­phag­es) in the mid-20th cen­tury es­tab­lished the mo­lec­u ­lar
ing our evo­lu­tion. Some of them are sus­pected to be as­so­ci­ated ba­sis of ge­netic in­her­i­tance. Through de­vel­op­ment and use
with spe­cific dis­eases, but the reg­u ­la­tory se­quences and pro­ of strin­gent, quan­ti­ta­tive meth­ods with these rel­a­tively sim­
tein prod­ucts of other en­dog­e­nous ret­ro­v i­ruses have been ple bi­o­log­i­cal en­ti­ties, this re­search con­firmed that DNA en­
coopted dur­ing our evo­lu­tion for their unique func­tions. For codes genes and genes en­code pro­teins. Ge­ne­ral mech­a ­nisms
ex­am­ple, ret­ro­v i­ral gene prod­ucts may play a role in the reg­ of ge­netic re­com­bi­na­tion, re­pair, and con­trol of gene ex­pres­
u­la­tion of pluripotency in germ cells, in trans­mis­sion of sig­ sion were also elu­ci­dated, lay­ing the foun­da­tions of mod­ern
nals at neu­ro­nal syn­apses, and clearly in the way that we give mo­lec­u­lar bi­­ol­ogy and re­com­bi­nant DNA tech­nol­ogy. Sub-
birth. The de­vel­op­ment of the hu­man pla­centa de­pends on sequent stud­ies of an­i­mal vi­ruses es­tab­lished many fun­da­
cell fu­sion pro­moted by a ret­ro­v i­ral pro­tein. If not for these men­tal prin­ci­ples of cel­lu­lar func­tion, in­clud­ing the pres­ence
en­dog­e­nous ret­ro­v i­ruses, we might be pro­duc­ing our young of in­ter­ven­ing se­quences in eu­kary­otic genes. The study of
in eggs, like birds and rep­tiles. can­cer (trans­form­ing) vi­ruses es­tab­lished the ge­netic ba­sis
Recent ge­no­mic stud­ies have re­vealed that our vi­ral “her­i­ of this dis­ease.
tage” is not lim­ited to ret­ro­vi­ruses. Human and other ver­te­ With the de­vel­op­ment of re­com­bi­nant DNA tech­nol­ogy
brate ge­nomes har­bor se­quences de­rived from sev­eral other and our in­creased un­der­stand­ing of vi­ral sys­tems, it has be­
RNA and DNA vi­ruses. As many of these in­ser­tions are es­ti­ come pos­si­ble to use vi­ral ge­nomes as ve­hi­cles for the de­liv­
mated to have oc­curred some 40 mil­lion to 90 mil­lion years ery of genes to cells and or­gan­isms for both sci­en­tific and
ago, this knowl­edge has pro­vided unique in­sight into the ages ther­a­peu­tic pur­poses. The use of vi­ral vec­tors to in­tro­duce
and evo­lu­tion of their cur­rently cir­cu­lat­ing rel­a­tives. The con­ genes into var­i­ous cells and or­gan­isms to study their func­
ser­va­tion of some of these vi­ral se­quences in ver­te­brate ge­ tion has be­come a stan­dard method in bi­­ol­ogy. Viral vec­tors
nomes sug­gests that they may have been se­lected for ben­e­fi­cial are also be­i ng used to treat hu­man dis­ease, for ex­a m­ple, via
prop­er­ties over evo­lu­tion­ary time. “gene ther­a­py,” in which func­tional genes de­liv­ered by vi­ral
vec­tors com­pen­sate for faulty genes in the host cells (Volume II,
Viruses Can Cross Species Boundaries Chapter 9).
Although vi­r uses gen­er­a lly have a lim­ited host range, they The study of vi­ruses has con­trib­uted in a unique way to the
can and do spread across spe­cies bar­ri­ers. As the world’s hu­ field of an­thro­pol­ogy. As an­cient hu­mans moved from one
man pop­u ­la­tion con­tin­ues to ex­pand and im­pinge on the geo­graphic area to an­other, the vi­ral strains unique to their
wil­der­ness, cross-species (zoo­not­ic) in­fec­tions of hu­mans orig­i­nal lo­ca­tions came along with them. The pres­ence of such
are oc­cur­ring with in­creas­ing fre­quency. In ad­di­tion to the strains can be de­tected by anal­y­sis of vi­ral nu­cleic ac­ids, pro­
AIDS pan­demic, the highly fa­tal Ebola hem­or­rhagic fe­ver, teins, and an­ti­bod­ies from an­cient hu­man spec­i­mens and in
se­vere acute re­spi­ra­tory syn­drome (SARS), and Middle East mod­ern pop­u ­la­tions. Together with archeological in­for­ma­
re­spi­ra­tory syn­drome (MERS) are re­cent ex­a m­ples of vi­ral tion, iden­ti­fi­ca­tion of these vi­ro­log­i­cal mark­ers has been used
dis­eases to emerge from zoo­notic in­fec­tions. The in­flu­enza to trace the path­ways by which hu­mans came to in­habit var­i­
vi­rus H5N1 con­tin­ues to spread among poul­try and wild ous re­gions of our planet (Fig. 1.2).
 Foundations 7

fer­ent vi­ruses than were no­madic hunt­er/gatherer so­ci­e­ties.

Similarly, as many dif­fer­ent vi­ruses are en­dem­ic in the trop­
ics, hu­man so­ci­e­ties in that en­v i­ron­ment must have been ex­
posed 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
pro­v ided new av­e­nues for vi­rus spread. Even so, it seems un­
likely that vi­ruses such as those that cause mea­sles or small­
pox could have en­tered a per­ma­nent re­la­tion­ship with small
groups of early hu­mans. Such highly vir­u ­lent vi­ruses, as we
now know them to be, ei­t her kill their hosts or in­duce life­long
im­mu­nity. Consequently, they can sur­v ive only when large,
Figure 1.2 Tracking an­cient hu­man mi­gra­tions by the vi­ruses in­ter­act­ing host pop­u ­la­tions of­fer a suf
­fi­cient num­ber of na­
they car­ried. The poly­oma­v i­r us known as JC vi­r us is trans­m it­ted ive and per­mis­sive hosts for their con­tin­ued prop­a­ga­tion.
among fam­i ­lies and pop­u ­la­tions and has co­evolved with hu­mans since Such vi­ruses could not have been es­tab­lished in hu­man pop­
the time of their or­i­gin in Af­rica. This vi­rus pro­duces no dis­ease in nor­ u­la­tions un­til large, set­t led com­mu­ni­ties ap­peared. Less vir­
mal, healthy peo­ple. Most in­di­v id­u­a ls are in­fected in child­hood, af­ter
which the vi­rus es­tab­lishes a per­sis­tent in­fec­tion in the gas­tro­in­tes­ti­nal u­lent vi­r uses that en­ter into a long-term re­la­tion­ship with
tract and is shed in urine. Analysis of the ge­nomes of JC vi­rus in hu­man their hosts were there­fore more likely to be the first to be­come
pop­u ­la­t ions from dif­fer­ent geo­g raphic lo­ca­t ions has sug­gested an ex­ adapted to re­pro­duc­tion in the ear­li­est hu­man pop­u ­la­tions.
pan­sion of an­cient hu­mans from Af­rica via two dis­tinct mi­gra­tions,
each car­r y­ing a dif­fer­ent lin­e­age of the vi­rus. Results from these stud­ies
These vi­ruses in­clude the mod­ern ret­ro­vi­ruses, her­pes­vi­ruses,
are con­sis­tent with an­a ­ly­ses of hu­man DNAs (shown by the solid line). and pap­il­lo­ma­v i­rus­es.
They also sug­gest an ad­di­tional route that was un­de­tect­able in the hu­ Evidence for knowl­edge of sev­eral dis­eases that we now
man DNA an­a ­ly­ses (in­di­cated by the dashed line). Data from Pavesi A. know to be caused by vi­ruses can be found in an­cient re­cords.
2005. J Gen Virol 86:1315–1326.
The Greek poet Homer char­ac­ter­izes Hector as “ra­bid” in The
Il­i­ad (Fig. 1.3A), and Mes­o­po­ta­mian laws that out­­line the re­
Virus Prehistory spon­si­bil­i­ties of the own­ers of ra­bid dogs date from be­fore
Although vi­ruses have been known as dis­tinct bi­o­log­i­cal en­ 1000 b.c.e. Their ex­is­tence in­di­cates that the com­mu­ni­ca­ble
ti­ties for only about 120 years, ev­i­dence of vi­ral in­fec­tion can na­ture of this vi­ral dis­ease was al­ready well-known by that
be found among the ear­li­est re­cord­ings of hu­man ac­tiv­ity, and time. Egyp­tian hi­ero­glyphs il­lus­trate what ap­pear to be the
meth­ods for com­bat­ing vi­ral dis­ease were prac­ticed long be­ con­se­quences of po­lio­vi­rus in­fec­tion (a with­ered leg typ­i­cal of
fore the first vi­r us was rec­og­nized. Consequently, ef­forts to po­lio­my­eli­tis [Fig. 1.3B]). Pustular le­sions char­ac­ter­is­tic of
un­der­stand and con­trol these im­por­tant agents of dis­ease be­ small­pox have also been found on Egyp­tian mum­mies. The
gan only in the last cen­tu­r y. small­pox vi­rus was prob­a­bly en­demic in the Gan­ges River ba­
sin by the fifth cen­tury b.c.e. and sub­se­quently spread to
Viral Infections in Antiquity other parts of Asia and Eu­rope. This vi­ral path­o­gen has played
Reconstruction of the pre­his­toric past to pro­vide a plau­si­ble an im­por­tant part in hu­man his­tory. Its in­tro­duc­tion into the
ac­count of when or how vi­ruses es­tab­lished them­selves in hu­ pre­v i­ously un­ex­posed na­tive pop­u ­la­tions of Central and
man pop­u­la­tions is chal­leng­ing. However, ex­trap­o­lat­ing from South Amer­ica by col­o­nists in the 16th cen­tury led to le­t hal
cur­rent knowl­edge, we can de­duce that some mod­ern vi­ruses ep­i­dem­ics, which are con­sid­ered an im­por­tant fac­tor in the
were un­doubt­edly as­so­ci­ated with the ear­li­est pre­cur­sors of con­quests achieved by a small num­ber of Eu­ro­pean sol­diers.
mam­mals and co­evolved with hu­mans. Other vi­ruses en­tered Other vi­ral dis­eases known in an­cient times in­clude mumps
hu­man pop­u­la­tions only re­cently. The last 10,000 years of his­ and, per­haps, in­flu­enza. Eu­ro­pe­ans have de­scribed yel­low fe­
tory was a time of rad­i­cal change for hu­mans and our vi­rus­es: ver since they dis­cov­ered Af­rica, and it has been sug­gested
an­i­mals were do­mes­ti­cated, the hu­man pop­u­la­tion in­creased that this scourge of the trop­i­cal trade was the ba­sis for leg­ends
dra­mat­i­cally, large pop­u­la­tion cen­ters ap­peared, and com­ about ghost ships, such as the Flying Dutchman, in which an
merce and tech­nol­ogy drove world­wide travel and in­ter­ac­tions en­tire ship’s crew per­ished mys­te­ri­ous­ly.
among un­prec­e­dented num­bers of peo­ple. Humans have not only been sub­ ject to vi­ ral dis­ ease
Viruses that es­tab­lished them­selves in hu­man pop­u ­la­tions through­out­much of their his­tory but have also ma­nip­u ­lated
were un­doubt­edly trans­mit­ted from an­i­mals, much as still these agents, al­beit un­k now­ingly, for much lon­ger than might
hap­pens to­day. Early hu­man groups that do­mes­ti­cated and be imag­ined. One clas­sic ex­am­ple is the cul­ti­va­tion of mar­
lived with their an­i­mals were al­most cer­tainly ex­posed to dif­ vel­ously pat­terned tu­lips, which were of enor­mous value in
8 Chapter 1

Here this firebrand, rabid Hector,

leads the charge.
Homer, The Iliad,
translated by Robert Fagels
(Viking Penguin)

Figure 1.3 References to vi­ral dis­eases from the an­cient lit­er­

a­ture. (A) An im­age of Hector from an an­cient Greek vase. Courtesy
of the Penn Museum, ob­ject 30-44-4. (B) An Egyp­t ian stele, or stone
tab­let, from the 18th dy­nasty (1580–1350 b.c.e.) de­pict­ing a man with a
with­ered leg and the “drop foot” syn­drome char­ac­ter­is­tic of po­lio­my­
eli­t is. Image cour­tesy of SPL/Science Source.

Figure 1.4 Three Broken Tulips. A paint­ing by Ni­co­las Rob­ert

(1624–1685), now in the col­lec­t ion of the Fitzwilliam Museum, Cam­
17th-century Holland. Such ef­forts in­cluded de­lib­er­ate spread bridge, United Kingdom. Striping pat­terns (color break­ing) in tu­lips
of a vi­rus (tu­lip break­ing vi­rus or tu­lip mo­saic vi­rus) that we were de­scribed in 1576 in west­ern Eu­rope and were caused by a vi­ral
now know causes the strip­ing of tu­lip pet­a ls so highly prized in­fec­t ion. This beau­t i­f ul im­age de­picts the re­mark­able con­se­quences
at that time (Fig. 1.4). Attempts to con­trol vi­ral dis­ease have of in­fec­tion with the tu­lip mo­saic vi­rus. © Fitzwilliam Museum,
Cam­bridge.
an even more ven­er­a­ble his­to­r y.

The First Vaccines

Measures to con­trol one vi­ral dis­ease have been used for the
last mil­len­nium. The dis­ease is small­pox (Fig. 1.5), and the
prac­tice is called variolation. The pro­cess en­tails tak­ing ma­
te­rial di­rectly from the small­pox le­sions of an in­fected in­di­
vid­ual and scratch­ing it onto the skin of healthy in­di­v id­u­a ls
with a lan­cet. Widespread in China and In­dia by the 11th
cen­tury, variolation was based on the rec­og­ni­tion that small­
pox sur­v i­vors 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 in­tro­
duced this prac­tice into En­gland in 1721, where it be­came
quite wide­spread fol­low ­ing the suc­cess­f ul in­oc­u­la­tion of
chil­dren of the royal fam­ily. George Wash­ing­ton is said to
have in­tro­duced the prac­tice among Continental Army sol­
diers in 1776. However, the con­se­quences of variolation were Figure 1.5 Characteristic small­pox le­sions in a young vic­tim.
un­pre­dict­able and never pleas­a nt: se­ri­ous skin le­sions in­ Illustrations like these were used as ex­a m­ples to track down in­di­v id­u­a ls
vari­ably de­vel­oped at the site of in­oc­u ­la­tion and were of­ten in­fected with the small­pox vi­rus (va­ri­ola vi­rus) dur­ing the World Health
Organization cam­paign to erad­i­cate the dis­ease. Courtesy of CDC/Dr.
ac­com­pa­nied by more gen­er­a l­ized rash and dis­ease, with a Rob­in­son (CDC PHIL ID#10398). See also the in­ter­v iew with Dr. Don-
fa­tal­ity rate of 1 to 2%. From the com­fort­able view­point of ald Hen­der­son: http://​bit.​ly/​Virology_​Henderson.
 Foundations 9

an af ­fl u­ent coun­try in the 21st cen­tury, such a death rate Today, vi­ral vac­cine strains se­lected for re­duced vir­u­lence
seems un­ac­cept­ably high. However, in the 18th cen­tury, var- are called at­ten­u­at­ed, a term de­rived from the Latin pre­fi x ad,
iolation was per­ceived as a much bet­ter al­ter­na­tive than nat­ mean­ing “to,” and ten­u­is, mean­ing “weak.” Safer and more ef­
u­rally con­tract­ing nat­u­ral small­pox, a dis­ease with a fa­tal­ity fi­cient meth­ods for the pro­duc­tion of larger quan­ti­ties of these
rate of 25% in the whole pop­u ­la­tion and 40% in ba­bies and first vac­cines awaited the rec­og­ni­tion of vi­ruses as dis­tinc­tive
young chil­dren. bi­o­log­i­cal en­ti­ties and par­a­sites of cells in their hosts. Indeed,
In the 1790s, Ed­ward Jen­ner, an En­glish coun­try phy­si­ it took al­most 50 years to dis­cover the next an­ti­vi­ral vac­cines:
cian, es­tab­lished the prin­ci­ple on which mod­ern meth­ods of a vac­cine for yel­low fe­ver vi­rus was de­vel­oped in 1935, and an
vi­ral im­mu­ni­za­tion are based, even though vi­ruses them­ in­flu­enza vac­cine was avail­­able in 1936. These ad­vances be­
selves were not to be iden­ti­fied for an­other 100 years. Jen­ner came pos­si­ble only with rad­i­cal changes in our knowl­edge of
him­self was variolated with small­pox as a young boy and was liv­ing or­gan­isms and of the causes of dis­ease.
un­doubt­edly fa­mil­iar with its ef­fects and risks. Perhaps this
ex­pe­ri­ence spurred his abid­ing in­ter­est in this method. Al- Microorganisms as Pathogenic Agents
though it is com­monly as­serted that Jen­ner’s de­vel­op­ment of The 19th cen­tury was a pe­riod of rev­o­lu­tion in sci­en­tific
the small­pox vac­cine was in­spired by his ob­ser­va­tions of thought, par­tic­u ­larly in ideas about the or­i­gins of liv­ing
milk­maids, the re­a l­ity is more pro­saic. As a phy­si­cian’s ap­ things. The pub­li­ca­tion of Charles Dar­win’s The Origin of Spe-
pren­tice at age 13, Jen­ner learned about a cu­ri­ous ob­ser­va­tion cies in 1859 crys­ tal­
lized star­ t ling (and, to many peo­ ple,
of lo­cal prac­ti­tion­ers who had been variolating farm­ers with shock­ing) new ideas about the or­i­gin of di­ver­sity in plants and
small­pox. No ex­pected skin rash or dis­ease ap­peared in farm­ an­i­mals, un­til then gen­er­a lly at­trib­uted di­rectly to the hand
ers who had pre­v i­ously suf­fered a bout with cow­pox. This of God. These in­sights per­ma­nently un­der­mined the per­cep­
lack of re­sponse was typ­i­cal of in­di­v id­u­a ls who had sur­v ived tion that hu­mans were some­how set apart from all­other
ear­lier in­fec­tion with small­pox and were known to be im­ mem­bers of the an­i­mal king­dom. From the point of view of
mune to the dis­ease. It was sup­posed there­fore that, like the sci­ence of vi­rol­ogy, the most im­por­tant changes were in
small­ pox sur­ v i­
vors, these nonresponding farm­ ers must ideas about the causes of dis­ease.
some­how be im­mune to small­pox. Although the phe­nom­e­ The di­ver­sity of mac­ro­scopic or­gan­isms has been ap­pre­
non was first ob­served and later re­ported by oth­ers, Jen­ner ci­ated and cata­loged since the dawn of re­corded hu­man his­
was the first to ap­pre­ci­ate its sig­nif­i­cance fully and to fol­low tory. However, a vast new world of or­gan­isms too small to
up with di­rect ex­per­i­ments. From 1794 to 1796, he dem­on­ be vis­i­ble to the na­ked eye was re­vealed through the mi­cro­
strated that in­oc­u ­la­tion with ma­te­rial from cow­pox le­sions scopes of An­tony van Leeu­wen­hoek (1632–1723). Van Leeu­
in­duced only mild symp­toms in the re­cip­i­ent but pro­tected wen­hoek’s vivid and ex­cit­i ng de­scrip­t ions of liv ­i ng
against the far more dan­ger­ous dis­ease. It is from these ex­ mi­cro­or­gan­isms, the “wee an­i­mal­cules” pres­ent in such or­
per­i­ments that we de­rive the term vac­ci­na­tion (vacca = “cow” di­nary ma­te­ri­a ls as rain or sea­wa­ter, in­cluded ex­a m­ples of
in Lat­in); Louis Pas­teur coined this term in 1881 to honor pro­to­zoa, al­gae, and bac­te­r ia. By the early 19th cen­t ury, the
Jen­ner’s ac­com­plish­ments. sci­en­t ific com­mu­nity had ac­cepted the ex­is­tence of mi­cro­
Initially, the only way to prop­a­gate and main­tain the cow­ or­gan­isms and turned to the ques­t ion of their or­i­g in, a topic
pox-derived vac­cine was by se­rial in­fec­tion of hu­man sub­ of fierce de­bate. Some be­lieved that mi­cro­or­gan­isms arose
jects. This method was even­tu­a lly banned, as it was of­ten spon­ta­ne­ously, for ex­a m­ple, in de­com­pos­i ng mat­ter, where
as­so­ci­ated with trans­mis­sion of other dis­eases such as syph­i­ they were es­pe­cially abun­dant. Others held the view that all­
lis and hep­a­ti­tis. By 1860, the vac­cine had been pas­saged in were gen­er­ated by their re­pro­duc­t ion, as are mac­ro­scopic
cows; later, horses, sheep, and wa­ter buf­fa­loes were also used. or­gan­isms. The death knell of the spon­ta­ne­ous-generation
The or­i­gin of the cur­rent vac­cine vi­rus, vac­cinia vi­rus, is now hy ­poth­e­sis was sounded with the fa­mous ex­per­i­ments of
thought to be horsepox vi­rus (Box 1.3). Pas­teur. He dem­on­strated that boiled (i.e., ster­il­ized) me­
The first ra­bies vac­cine was made by Louis Pas­teur, al­ dium re­mained free of mi­cro­or­gan­isms as long as it was
though he had no idea at the time that the rel­e­vant agent main­tained in spe­cial flasks with curved, nar­row necks de­
was a vi­r us. In 1885, he in­oc­u ­lated rab­bits with ma­te­r ial signed to pre­vent en­t ry of air­borne mi­crobes (Fig. 1.6). Pas­
from the brain of a cow suf­fer­ing from ra­bies and then used teur also es­tab­lished that dis­t inct mi­cro­or­gan­isms were
aque­ous sus­pen­sions of dried spi­nal cords from these an­i­ as­so­ci­ated with spe­cific pro­cesses, such as fer­men­ta­t ion, an
mals to in­fect other rab­bits. After sev­eral such pas­sages, the idea that was cru­cial in the de­vel­op­ment of mod­ern ex­pla­
re­sult­ing prep­a­ra­t ions were ad­min­is­tered to hu­man sub­ na­t ions for the causes of dis­ease.
jects, where they pro­duced mild dis­ease but ef­fec­t ive im­mu­ From the ear­li­est times, poi­son­ous air (mi­asma) was
nity against ra­bies. gen­er­a lly in­voked to ac­count for ep­i­dem­ics of con­ta­g ious
10 Chapter 1

B OX 1.3
D I S C U S S I O N
Origin of vac­cinia vi­rus
Over the years, many hy­poth­e­ses have been
ad­vanced to ex­plain the cu­ri­ous or­i­gin of vac­
cinia vi­r us. However, re­cent in­ves­ti­ga­tions
into this mys­ tery by col­ lab­
o­ tors in the
ra­
United States, Ger­many, and Bra­zil in­d i­cate
that horsepox, not cow­pox, was the likely
pre­cur­sor of vac­cine strains of vac­cinia vi­rus.
The pro­ver­bial smok­ing gun was an orig­i­
nal wooden and glass con­tainer that held capil­
lar­ies with the small­pox vac­cine pro­duced in
1902 by H.K. Mulford in Phil­a­del­phia (a com­
pany that merged with Sharpe and Dohme in
1929). Sequence anal­y­sis of the DNA showed The orig­i­nal wooden (top) and glass (bot­tom) con­tain­ers that held
capil­lar­ies con­tain­ing the Mulford 1902 small­pox vac­cine. Photo kindly
that the core ge­nome of the vi­rus in that vial
pro­v ided by Dr. Jose Esparza, Institute of Human Virology, University of
had the high­est de­gree of sim­i­lar­ity (99.7%) to Mary­land School of Medicine, Bal­t i­more. ©Merck Sharp & Dohme Corp.,
horsepox vi­rus. A re­view of the his­tor­i­cal re­ Merck & Co., Inc.
cord shows that dur­ing the 19th cen­tury, pus­
tu­lar ma­te­rial de­rived from both cow­pox and
horsepox le­sions was used to im­mu­nize against sis is that the an­ces­tor of the cur­rent vac­cine • Cows from which pus­tu­lar ma­te­rial was
small­pox. The lat­ter tech­nique was called equi­ strain was a nat­u­rally oc­cur­ring vac­cinia vi­ ob­tained for vac­ci­na­t ion were most of­
nation. Although the dis­ease is now rare in rus pres­ent in the widely dis­trib­uted French ten in­fected with horsepox, trans­mit­ted
horses and was never re­ported in the Amer­i­cas, prep­a­ra­t ion. Alternatively, the vac­cine strain by their han­d lers or by ro­dents.
it was prev­a­lent in Eu­rope, where most vac­cine may have evolved from horsepox vi­rus dur­
sam­ples were ob­tained at the time. ing an­i­mal pas­sage. The stu­dent is in­v ited to con­jure up other
Most small­pox vac­cines used in the United It is im­por­tant to con­sider that de­vel­op­ plau­si­ble ex­pla­na­t ions.
States, Bra­zil, and many Eu­ro­pean coun­tries ment of the small­pox vac­cine took place more
Damaso CR. 2018. Revisiting Jen­ner’s mys­ter­ies, the
were pro­duced in the United States from calves than a cen­tury be­fore mod­ern con­cepts of vi­ role of the Beaugency lymph in the evo­lu­tion­ary
in­oc­u­lated with ma­te­rial col­lected in 1866 rol­ogy were es­tab­lished. One can think of other path of an­cient small­pox vac­cines. Lancet Infect Dis
from spon­ta­ne­ous cases of cow­pox in France. sce­nar­ios to ex­plain why the vac­cine strain of 18:e55–e63.
Genetic anal­y­sis of ex­ist­ing sam­ples of these vac­cinia vi­rus is closely re­lated to horsepox and Esparza J, Schrick L, Damaso CR, Nitsche A. 2017.
Equination (in­oc­u ­la­t ion of horsepox): an early al­ter­
early vac­cines in­di­cates that they con­tained a not cow­pox, as orig­i­nally sup­posed. na­t ive to vac­ci­na­t ion (in­oc­u ­la­t ion of cow­pox) and
vi­rus more sim­i­lar to horsepox and vac­cinia the po­ten­t ial role of horsepox vi­r us in the or­i­g in of
vi­ruses than to cow­pox vi­rus. While nat­u­rally • The milk­maid with le­sions that were the the small­pox vac­cine. Vaccine 35:7222–7230.
oc­c ur­r ing vac­cinia vi­r uses are found to­d ay source of Jen­ner’s orig­i­nal in­oc­u ­lum in Schrick L, Tausch SH, Dabrowski PW, Damaso CR,
only in In­dia (in buf­fa­los) and Bra­zil (in cows), 1796 was in­fected with horsepox, not Esparza J, Nitsche A. 2017. An early Amer­ i­
can
small­pox vac­cine based on horsepox. N Engl J Med
they can in­fect horses and peo­ple, pro­duc­ing cow­pox. Horsepox can be trans­mit­ted 377:1491–1492.
pus­tu­lar le­sions sim­i­lar to those caused by to cows, and both an­i­mals are com­mon TWIV 478: A pox on your horse. http://​w ww.​microbe.​t v​
horsepox and cow­pox vi­ruses. One hy­poth­e­ on farms. /​twiv/​twiv-​478/​.

dis­eases, and there was lit­t le rec­og­n i­t ion of the dif­fer­ences • The or­gan­ism must be iso­lated from the dis­eased host
among caus­a­t ive agents. The as­so­ci­a­t ion of par­t ic­u ­lar mi­ and grown in cul­ture.
cro­or­gan­isms, ini­t ially bac­te­r ia, with spe­cific dis­eases can • The dis­ease must be re­pro­duced when a pure cul­ture of
be at­t rib­uted to the ideas of the Ger­man phy­si­cian Rob­ert the or­gan­ism is in­tro­duced into a healthy, sus­cep­ti­ble
Koch. He de­vel­oped and ap­plied a set of cri­te­r ia for iden­t i­ host.
fi­c a­tion of the agent re­spon­si­ble for a spe­cific dis­ease (a • The same or­gan­ism must be reisolated from the ex­per­i­
path­o­gen), ar ­t ic­u­lated in an 1890 pre­sen­ta­t ion in Ber­l in. men­tally in­fected host.
These cri­te­r ia, Koch’s pos­tu­lates, can be sum­ma­rized as
fol­lows. Modern tech­nol­ogy has al­lowed some of Koch’s prin­ci­ples
to be amended by the ap­pli­ca­tion of other types of ev­i­dence
• The or­gan­ism must be reg­u­larly as­so­ci­ated with the dis­ (Box 1.4). However, by ap­ply­ing his cri­te­ria, Koch dem­on­
ease and its char­ac­ter­is­tic le­sions. strated that an­t hrax, a com­mon dis­ease of cat­t le, was caused
 Foundations 11

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 bac­te­r ium ap­peared in 1892. The Rus­sian sci­en­tist
Dimitrii Ivanovsky ob­served that the caus­a­t ive agent of to­
bacco mo­saic dis­ease was not re­tained by the un­g lazed fil­
ters used at that time to re­move bac­te­r ia from ex­t racts and
cul­ture me­d ium (Fig. 1.8A). Six years later in Holland, Mar-
Broth tinus Beijerinck in­de­pen­dently made the same ob­ser ­va­t ion.
More im­por­tantly, Beijerinck made the con­cep­t ual leap that
Figure 1.6 Pas­teur’s fa­mous swan-neck flasks pro­vided pas­
this must be a dis­t inc­t ive agent, be­cause it was so small that
sive ex­clu­sion of mi­crobes from the ster­il­ized broth. Although it could pass through fil­ters that trapped all­known bac­te­ria.
the flask was freely open to the air at the end of the long, curved stem, However, Beijerinck thought that the agent was an in­fec­
the broth re­mained ster­ile, pro­v ided that mi­crobe-bearing dust that col­ tious liq­u id. It was two for­mer stu­dents and as­sis­tants of
lected in the neck of the stem did not reach the liq­uid.
Koch, Frie­d rich Loeffler and Paul Frosch, who in the same
year (1898) de­duced that such in­fec­tious fil­ter­able agents
by a spe­cific bac­te­rium (des­ig­nated Bacillus anthracis) and com­prised small par­ti­cles: they ob­served that while the
that a sec­ond, dis­tinct bac­te­rial spe­cies caused tu­ber­cu­lo­sis in caus­a­t ive agent of foot-and-mouth dis­ease (Box 1.2) passed
hu­mans. Guided by these pos­tu­lates and the meth­ods for the through fil­ters that held back bac­te­r ia, it could be re­tained
ster­ile cul­ture and iso­la­tion of pure prep­a­ra­tions of bac­te­ria by a finer fil­ter.
de­vel­oped by Pas­teur, Jo­seph Lis­ter, and Koch, many path­o­ Not only were the to­bacco mo­saic and foot-and-mouth
genic bac­te­ria (as well as yeasts and fungi) were iden­ti­fied and dis­ease path­o­gens much smaller than any pre­v i­ously rec­og­
clas­si­fied dur­ing the last part of the 19th cen­tury (Fig. 1.7). nized mi­cro­or­gan­ism, but also they could only re­pro­duce in
From these be­gin­nings, in­ves­ti­ga­tion into the causes of in­fec­ their host or­gan­isms. For ex­a m­ple, ex­tracts of an in­fected
tious dis­ease was placed on a se­cure sci­en­tific foun­da­tion, the to­bacco plant di­luted into ster­i le so­lu­tion pro­duced no ad­di­
first step to­ward ra­tio­nal treat­ment and ul­ti­mately con­trol. tional in­ tious agents un­
fec­ til in­tro­ duced into leaves of
Furthermore, dur­ing the last de­cade of the 19th cen­tury, fail­ healthy plants, which sub­se­quently de­vel­oped to­bacco mo­
ures of the par­a­digm that bac­te­rial or fun­gal agents are re­ saic dis­ease. The se­rial trans­mis­sion of in­fec­tion by di­luted
spon­si­ble for all­ dis­eases led to the iden­ti­fi­ca­tion of a new ex­tracts es­tab­lished that these dis­eases were not caused by a

B OX 1.4
D I S C U S S I O N
New meth­ods amend Koch’s prin­ci­ples
While it is clear that a mi­crobe that ful­fi lls The most rev­o­lu­tion­ary ad­vances in our jects and (ii) in­oc­u ­lat­ing a healthy in­di­v id­ual
Koch’s pos­tu­lates is al­most cer­tainly the abil­ity to link par­tic­u ­lar vi­ruses with dis­ease with a sam­ple from a dis­eased sub­ject re­sults
cause of the dis­ ease in ques­ t ion, we now (or ben­e­fit) come from the more re­cent de­vel­ in trans­mis­sion of the dis­ease as well as the
know that mi­crobes that do not ful­fi ll such op­ment of high-throughput nu­cleic acid se­ mo­lec­u ­lar mark­ers.
cri­te­ria may still rep­re­sent the eti­o­log­i­cal quenc­ing meth­ods and bioinformatics tools
Falkow S. 1988. Molecular Koch’s pos­t u­lates ap­plied to
agents of dis­ease. In the lat­ter part of the that al­low de­tec­t ion of vi­ral ge­netic ma­te­rial mi­cro­bial path­o­ge­nic­i­t y. Rev Infect Dis 10(Suppl
20th cen­t ury, new meth­ods were de­vel­oped to di­rectly in en­v i­ron­men­tal or bi­o­log­i­cal sam­ 2):S274–S276.
as­so­ci­ate par­tic­u ­lar vi­ruses with dis­ease based ples, an ap­proach called vi­ral metagenomics. Fredricks DN, Relman DA. 1996. Sequence-based
on im­mu­no­log­i­cal ev­i­dence of in­fec­tion, for Based on these de­vel­op­ments, al­ter­na­t ive iden­t i­fi­ca­t ion of mi­cro­bial path­o­gens: a re­con­sid­er­a­
ex­a m­ple, the pres­ence of an­ti­bod­ies in blood. “metagenomic Koch’s pos­tu­lates” have been tion of Koch’s pos­tu­lates. Clin Microbiol Rev 9:18–33.
Mokili JL, Rohwer F, Dutilh BE. 2012. Metagenomics
The avail­abil­ity of these meth­ods led to the pro­posed in which (i) the de­fin­i­tive traits are and fu­t ure per­spec­t ives in vi­r us dis­cov­ery. Curr
pro­posal of mod­i­fied “mo­lec­u ­lar Koch’s pos­ mo­lec­u ­lar mark­ers such as genes or full ge­ Opin Virol 2:63–77.
tu­lates” based on the ap­pli­ca­tion of mo­lec­u ­lar nomes that can uniquely dis­t in­g uish sam­ples Racaniello V. 22 Jan­u­a ry 2010. Koch’s pos­t u­lates in the
tech­niques to mon­i­tor the role played by vir­u­ ob­tained from dis­eased sub­jects from those 21st cen­t ury. Virology Blog. http://​w ww.​v irology.​ws​
lence genes in bac­te­ria. ob­tained from matched, healthy con­trol sub­ /​2010/​01/​22/​kochs-​postulates-​i n-​t he-​21st-​century/​.​
12 Chapter 1

60

Fungi (17)
50 Bacteria (50)
Protozoa (11)
Cumulative number of discoveries

Filterable viruses (19)

40

30

20 Koch's introduction of Discovery

efficient bacteriological of TMV
methods

10

0
1835 1845 1855 1865 1875 1885 1895 1905 1915 1925 1935
Year

Figure 1.7 The pace of dis­cov­ery of new in­fec­tious agents in the dawn of vi­rol­o­gy. Koch’s in­tro­duc­tion of ef ­fi­cient bac­te­ri­
o­log­i­cal 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 vi­r us. Data from Burdon KL. 1939. Medical Microbiology (Mac­mil­lan Co, New York, NY).

A B bac­te­rial toxin pres­ent in the orig­i­nal prep­a­ra­tions de­rived

from in­fected to­bacco plants or cat­tle. The fail­ure of both
Bacteria path­o­gens to mul­ti­ply in so­lu­tions that read­i ly sup­ported the
+ virus growth of bac­te­ria, as well as their de­pen­dence on host or­
gan­isms for re­pro­duc­tion, fur ­t her dis­tin­g uished these new
agents from path­o­genic bac­te­ria. Beijerinck termed the sub­
mi­cro­scopic agent re­spon­si­ble for to­bacco mo­saic dis­ease
con­ta­gium vi­vum fluidum to em­pha­size its in­fec­tious na­ture
and dis­tinc­tive re­pro­duc­tive and phys­i­cal prop­er ­ties. Agents
pass­ing through fil­ters that re­tain bac­te­ria came to be called
ultrafilterable vi­ruses, ap­pro­pri­at­ing the term vi­rus from the
Latin for “poi­son.” This term was sim­pli­fied even­tu­a lly to
Virus “vi­rus.”
The dis­cov­ery of the first vi­rus, to­bacco mo­saic vi­rus, is
of­ten at­trib­uted to the work of Ivanovsky in 1892. However,
Figure 1.8 Filter sys­tems used to char­ac­ter­ize/purify vi­rus he did not iden­tify the to­bacco mo­saic dis­ease path­o­gen as a
par­ti­cles. (A) The Berkefeld filter, invented in Germany in 1891, was a dis­tinc­tive agent, nor was he con­vinced that its pas­sage through
“candle”-style filter comprising diatomaceous earth (called Kieselguhr), bac­te­rial fil­ters was not the re­sult of some tech­ni­cal fail­ure. It
pressed into a hollow candle shape. The white candle in the upper cham- may be more ap­pro­pri­ate to at­tri­bute the found­ing of the field
ber is open at the top to receive the liquid to be filtered. The smallest pore
size retained bacteria and let virus particles pass through. Such filters were of vi­rol­ogy to the as­tute in­sights of Beijerinck, Loeffler, and
probably used by Ivanovsky, Loeffler, and Frosch to isolate the first viruses. Frosch, who rec­og­nized the dis­tinc­tive na­ture of the plant and
(B) Modern-day filter systems are made of disposable plastic with the an­i­mal path­o­gens they were study­ing more than 120 years ago.
upper and lower chambers separated by a biologically inert membrane,
The pi­o­neer­ing work on to­bacco mo­saic and foot-and-
available in a variety of pore sizes. Such filtration approaches may have
limited our detection of giant viruses. Image courtesy of EMD Millipore mouth dis­ease vi­ruses was fol­lowed by the iden­ti­fi­ca­tion of
Corporation. vi­ruses as­so­ci­ated with spe­cific dis­eases in many other or­gan­
 Foundations 13

isms. Important land­marks from this early pe­riod in­clude for in­ves­ti­ga­tors to ob­tain im­ages of vi­ruses, es­pe­cially as they
the iden­ti­fi­ca­tion of vi­ruses that cause leu­ke­mias or solid tu­ ap­pear to be re­mark­ably el­e­gant (Fig. 1.9). Images of many dif­
mors in chick­ens by Vilhelm Ellerman and Olaf Bang in 1908 fer­ent vi­rus par­ti­cles con­firmed that these agents are very
and Peyton Rous in 1911, re­spec­tively. The study of vi­ruses as­ small (Fig. 1.10) and that most are far sim­pler in struc­ture than
so­ci­ated with can­cers in chick­ens, par­tic­u­larly Rous sar­coma any cel­lu­lar or­gan­ism. Many ap­peared as reg­u­lar he­li­cal or
vi­rus, even­tu­a lly led to an un­der­stand­ing of the mo­lec­u­lar ba­ spher­i­cal par­ti­cles. The de­scrip­tion of the mor­phol­ogy of vi­rus
sis of can­cer (Volume II, Chapter 6). par­ti­cles made pos­si­ble by elec­tron mi­cros­copy also opened
The fact that bac­te­ria could also be hosts to vi­ruses was the way for the first ra­tio­nal clas­si­fi­ca­tion of vi­rus­es.
first rec­og­nized by Fred­er­ick Twort in 1915 and Félix d’Hérelle
in 1917. d’Hérelle named such vi­ruses bac­te­rio­phag­es be­ The Intracellular Parasitism of Viruses
cause of their abil­ity to cause their bac­te­rial host cells to Organisms as Hosts
rup­t ure (a phe­nom­e­non called ly­sis; “phage” is de­r ived from A de­fi n­i ng char­ac­ter­is­t ic of vi­r uses is their ab­so­lute de­
the Greek for “eat­i ng”). In an in­ter­est­i ng twist of ser­en­d ip­ pen­dence on a liv­ing host for re­pro­duc­tion: they are ob­li­gate
ity, Twort made his dis­cov­ery of bac­te­rial vi­r uses while test­ par­a­sites. Transmission of plant vi­ruses such as to­bacco
ing the small­pox vac­cine vi­r us to see if it would grow on mo­saic vi­r us can be achieved read­i ly, for ex­a m­ple, by ap­ply­
sim­ple me­d ia. He found bac­te­rial con­tam­i­nants, some of ing ex­tracts of an in­fected plant to a scratch made on the
which proved to be in­fected by a bac­te­rio­phage. As dis­cussed leaf of a healthy plant. Furthermore, as a sin­g le in­fec­tious
be­low, in­ves­t i­ga­t ion of bac­te­rio­phages es­tab­lished not only par­t i­cle of many plant vi­r uses is suf ­fi­cient to in­duce a char­
the foun­da­tions for the field of mo­lec­u ­lar bi­­ol­ogy but also ac­ter­is­t ic le­sion (Fig. 1.11), the con­cen­t ra­t ion of the in­fec­
fun­da­men­tal in­sights into how vi­r uses in­ter­act with their tious agent could be mea­sured. Plant vi­r uses were there­fore
host cells. the first to be stud­ied in de­tail. Some vi­r uses of hu­mans and
other spe­cies could also be prop­a­gated in lab­o­ra­tory an­i­
The Defining Properties of Viruses mals, and meth­ods were de­vel­oped to quan­t ify them by de­
Throughout the early pe­riod of vi­rol­ogy when many vi­ruses of ter­min­i ng the le­t hal dose. The trans­mis­sion of yel­low fe­ver
plants, an­i­mals, and bac­te­ria were cata­loged, ideas about the vi­r us to mice by Max Theiler in 1930 was an achieve­ment
or­i­gin and na­ture of these dis­tinc­tive in­fec­tious agents were that led to the iso­la­t ion of an at­ten­u­ated strain, still con­sid­
quite con­t ro­ver­sial. Arguments cen­tered on whether vi­ruses ered one of the saf­est and most ef­fec­t ive ever pro­duced for
orig­i­nated from parts of a cell or were built from unique com­ the vac­ci­na­t ion of hu­mans.
po­nents. Little prog­ress was made to­ward re­solv­ing these is­sues After spe­cific vi­ruses and ap­pro­pri­ate host or­gan­isms were
and es­tab­lish­ing the de­fin­i­tive prop­er­ties of vi­ruses un­til the iden­ti­fied, it be­came pos­si­ble to pro­duce suf ­fi­cient quan­ti­ties
de­vel­op­ment of new tech­niques that al­lowed their vi­su­al­i­za­ of vi­rus par­ti­cles for study of their phys­i­cal and chem­i­cal
tion or prop­a­ga­t ion in cul­tured cells. prop­er­ties and the con­se­quences of in­fec­tion for the host.
Features such as the in­cu­ba­tion pe­riod, symp­toms of in­fec­
The Structural Simplicity of Virus Particles tion, and ef­fects on spe­cific tis­sues and or­gans were in­ves­ti­
Dramatic con­fir­ma­tion of the struc­tural sim­plic­ity of vi­rus gated. Laboratory an­ mals re­
i­ main an es­ sen­tial tool in
par­ti­cles came in 1935, when Wendell Stan­ley ob­tained crys­ in­ves­ti­ga­tions of the path­o­gen­e­sis of vi­ruses that cause dis­
tals of to­bacco mo­saic vi­rus. At that time, noth­ing was known ease. However, real prog­ ress to­ ward un­ der­ stand­ing the
of the struc­tural or­ga­ni­za­tion of any bi­o­log­i­cally im­por ­tant mech­a­nisms of vi­rus re­pro­duc­tion was made only with the
mac­ro­mol­e­cules, such as pro­teins and DNA. Indeed, the cru­ de­vel­op­ment of cell cul­ture sys­tems. The first and the sim­
cial role of nu­cleic ac­ids as ge­netic ma­te­rial had not even plest, but cru­cial to both vi­rol­ogy and mo­lec­u ­lar bi­­ol­ogy,
been rec­og­nized. The abil­ity to ob­tain an in­fec­tious agent in were cul­tures of bac­te­rial cells.
crys­tal­line form, a state that was more gen­er­a lly as­so­ci­ated
with in­or­ganic ma­te­rial, cre­ated much won­der and spec­u ­la­ Lessons from Bacteriophages
tion about whether a vi­rus is truly a life form. In ret­ro­spect, In the late 1930s and early 1940s, the bac­te­rio­phages, or
it is ob­v i­ous that the rel­a­tive ease with which this par­tic­u ­lar “phag­es,” re­ceived in­creased at­ten­tion as a re­sult of con­tro­
vi­rus could be crys­tal­lized was a di­rect re­sult of its struc­tural versy cen­ ter­
ing on how they might have arisen. John
sim­plic­i­t y. Northrup, a bio­chem­ist at the Rocke­fel­ler Institute in Prince­
The 1930s saw the in­tro­duc­tion of the in­stru­ment that rap­ ton, NJ, cham­pi­oned the the­ory that a phage was a met­a­bolic
idly rev­o­lu­tion­ized vi­rol­o­g y: the elec­tron mi­cro­scope. The prod­uct of a bac­te­rium. On the other hand, Max Delbrück, in
great mag­ni­f y­ing power of this in­stru­ment (even­tu­ally more his work with Emory El­lis and later with Sal­va­dor Luria, re­
than 100,000-fold) al­lowed di­rect vi­su­al­i­za­tion of vi­rus par­ti­ garded phages as au­ton­o­mous, sta­ble, self-rep­li­cat­ing en­ti­ties
cles for the first time. It has al­ways been an ex­cit­ing ex­pe­ri­ence char­ac­ter­ized by her­i­ta­ble traits. According to this par­a­digm,
14 Chapter 1

A B C D

50 nm 50 nm 100 nm 100 nm

Figure 1.9 Electron mi­cro­graphs of vi­rus par­ti­cles fol­low­ing neg­a­tive stain­ing. (A) The com­plex, nonenveloped vi­r us bac­
te­r io­phage T4. Note the in­tri­cate tail and tail fi­bers. Reproduced with per­mis­sion from Dr. Rob­ert L. Duda, University of Pitts­burgh,
Pitts­burgh, PA. (B) The he­li­cal, nonenveloped par­t i­cle of to­bacco mo­saic vi­r us. Courtesy of Plant Resistance Gene Wiki (http://​prgdb.​
crg.​eu/​w iki/​Species:Tobacco_​mosaic_​v irus), li­censed un­der CC BY-SA 3.0. (C) Enveloped par­ti­cles of the rhab­do­v i­rus ve­sic­u ­lar sto­ma­ti­tis
vi­rus. Courtesy of CDC/Dr. Fred. A. Mur­phy (CDC PHIL ID#5611). (D) Nonenveloped, ico­sa­he­dral hu­man ro­ta­v i­rus par­ti­cles. Courtesy
of F. P. Wil­liams, U.S. Environmental Protection Agency, Wash­ing­ton, DC.

A NMR
X ray
Cryo-EM
Electron microscope
Light microscope

(1 cm) (1 mm) 10–4 (1 µm) (1 nm) (1 Å)

10–2 10–3 10–5 10–6 10–7 10–8 10–9 10–10
Meters

Plant Animal Small

cells cells Bacteria Viruses Ribosomes Proteins molecules Atoms

Herpesvirus
Ribosomes (200 nm)
(20 nm)
Poliovirus (30 nm)

Figure 1.10 Size mat­ters. (A) Sizes of an­i­mal and plant cells, bac­te­ria, vi­ruses, pro­teins, mol­e­cules, and at­oms are in­di­cated. The re­
solv­ing pow­ers of var­i­ous tech­niques used in vi­rol­ogy, in­clud­ing light mi­cros­copy, elec­tron mi­cros­copy, cryo-electron mi­cros­copy (Cryo-
EM), 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 above 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]). (B) Illustration of the size dif­fer­ences among two an­i­mal vi­ruses and a
typ­i­cal eu­kary­otic host cell.
 Foundations 15

and suf ­fi­cient 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 Mar­t ha Chase showed that
this hy­poth­e­sis was in­cor­rect; DNA, not pro­tein, car­ries the
in­for­ma­tion for vi­rus re­pro­duc­tion (Box 1.5).
Bacteriophages were orig­i­nally thought to be le­thal agents,
in­vari­ably kill­ing their host cells af­ter in­fec­tion. In the early
1920s, a pre­v i­ously un­k nown in­ter­ac­tion was dis­cov­ered, in
which the host cell not only sur­v ived the in­fec­tion but also
sta­bly in­her­ited the ge­netic in­for­ma­tion of the vi­rus. It was
also ob­served that cer­tain bac­te­rial strains could lyse spon­
ta­ne­ously and pro­duce bac­te­r io­phages af­t er a pe­r iod of
Figure 1.11 Lesions in­duced by to­bacco mo­saic vi­rus on an in­ growth in cul­t ure. Such strains were called ly­so­gen­ic, and
fected to­bacco leaf. In 1886, Adolph Mayer first de­scribed the char­ the phe­nom­e­non, ly­sog­e­ny. Studies of ly­sog­eny re­vealed
ac­ter­is­tic pat­terns of light and dark green ar­eas on the leaves of to­bacco
plants in­fected with to­bacco mo­saic vi­r us. He dem­on­strated that the many pre­v i­ously un­rec­og­nized fea­tures of vi­rus-host cell in­
mo­saic le­sions could be trans­mit­ted from an in­fected plant to a healthy ter­ac­tions (Box 1.6). Recognition of this phe­nom­e­non came
plant by aque­ous ex­tracts de­rived from in­fected plants. Following ap­ from the work of many sci­en­t ists, but it be­gan with the el­e­
pli­ca­tion of the prep­a­ra­tion to healthy plant leaves, the num­ber of char­ gant ex­per­i ­ments of André Lwoff and col­leagues at the In-
ac­ter­is­t ic le­sions con­tain­i ng dead cells is di­rectly pro­por ­t ional to the
num­ber of in­fec­t ious par­t i­cles in the test sam­ple. Courtesy of USDA stitut Pas­teur in Paris. Lwoff showed that a vi­r al ge­nome
Forest Service, under license CC BY 3.0. ex­ists in ly­so­genic cells in the form of a si­lent ge­netic el­e­ment
called the pro­phage. This el­e­ment de­t er­m ined the abil­ity
of ly ­s o­genic bac­te­r ia to pro­duce in­fec­t ious bac­te­r io­phages.
Subsequent stud­ies of the E. coli bac­te­r io­phage lambda es­
phages were seen as ideal tools with which to in­ves­ti­gate the tab­lished a par­a­digm for one mech­a­nism of ly­sog­eny, the in­te­
na­ture of genes and he­red­ity. Probably the most crit­i­cal early gra­tion of a phage ge­nome into a spe­cific site on the bac­te­r ial
con­t ri­bu­t ion of Delbrück and El­lis was the per­fec­t ion of the chro­mo­some.
“one-step growth” method for syn­chro­ni­za­tion of the re­pro­ Bacteriophages be­came in­ex­t ri­ca­bly as­so­ci­ated with the
duc­tion of phages, an achieve­ment that al­lowed anal­y­sis of a new field of mo­lec­u­lar bi­­ol­ogy. Their study es­tab­lished
sin­gle cy­cle of phage re­pro­duc­tion in a pop­u ­la­tion of bac­te­ many fun­da­men­tal prin­ci­ples: for ex­a m­ple, con­t rol of the
ria. This ap­proach in­tro­duced highly quan­ti­ta­tive meth­ods to de­ci­sion to en­ter a ly­so­genic or a lytic path­way is en­coded in
vi­rol­ogy, as well as an un­prec­e­dented rigor of anal­y­sis. The the ge­nome of the vi­r us. The first mech­a ­nisms dis­cov­ered
first ex­per­i­ments showed that phages in­deed mul­ti­plied in the for the con­trol of gene ex­pres­sion, ex­em­pli­fied by the el­e­
bac­te­rial host and were lib­er­ated in a “burst” fol­low­ing dis­ gant op­eron the­ory of No­bel lau­re­ates François Ja­cob and
rup­tion of the cell. Jacques Monod, were de­duced in part from stud­ies of ly­sog­
Delbrück was a zealot for phage re­search and re­cruited tal­ eny by phage lambda. The bi­­ol­ogy of phage lambda pro­
ented sci­en­tists to pur­sue the fun­da­men­tal is­sues of what is vided a fer­tile ground for work on gene reg­u ­la­tion, but
now known as the field of mo­lec­u ­lar bi­­ol­ogy. This cadre of study of vir­u ­lent T phages (T1 to T7, where T stands for
sci­en­tists fo­cused their at­ten­tion on spe­cific phages of the “type”) of E. coli pa­ved the way for many other im­por­tant
bac­te­rium Escherichia coli. Progress was rapid, pri­mar­ily be­ ad­vances. As we shall see, these sys­tems also pro­v ided an
cause of the sim­plic­ity of the phage in­fec­tious cy­cle. By the ex­ten­sive pre­v iew of mech­a­nisms of an­i­mal vi­r us re­pro­duc­
mid-1950s it was ev­i­dent that vi­ruses from bac­te­ria, an­i­mals, tion (Box 1.7).
and plants share many fun­da­men­tal prop­er­ties. However,
the phages pro­v ided a far more trac­ta­ble ex­per­i­men­tal sys­ Animal Cells as Hosts
tem. Consequently, their study had a pro­found im­pact on the The cul­ture of an­i­mal cells in the lab­o­ra­tory was ini­tially
field of vi­rol­o­g y. more of an art than a sci­ence, re­stricted to cells that grew out­
One crit­i­cal les­son came from a de­fin­i­tive ex­per­i­ment that of or­gans or tis­sues main­tained in nu­tri­ent so­lu­tions un­der
es­tab­lished that vi­ral nu­cleic acid car­ries ge­netic in­for­ma­ ster­ile con­di­tions. Cells so ob­tained from liv­ing tis­sues, called
tion. It was known from stud­ies of the “trans­form­ing prin­ci­ pri­mary cells, have a fi­nite life span. Their de­pen­dence for
ple” of pneu­mo­coc­cus by Oswald Avery, Colin MacLeod, and growth on nat­u­ral com­po­nents in their me­dia such as lymph,
Maclyn McCarty (1944) that nu­cleic acid was both nec­es­sary plasma, or chicken em­bryo ex­tracts, and the tech­ni­cal de­mands
16 Chapter 1

B OX 1.5
E X P E R I M E N T S
The Hershey-Chase ex­per­i­ment
By dif­fer­en­t ially la­bel­ing the nu­cleic acid and of the in­fect­ing vi­r us could be re­moved soon con­di­tions. Because such blended cells pro­
pro­tein com­po­nents of vi­r us par ­t i­cles with af­t er in­fec­t ion by ag­i­tat ­i ng the bac­te­r ia for a duced a nor­mal burst of new vi­r us par­t i­cles,
ra­d io­ac­t ive phos­pho­r us (32P) and ra­d io­ac­t ive few min­utes in a blender. In con­t rast, 32P-la- it was clear that the DNA con­tained all­of the
sul­f ur (35S), re­spec­t ively, Al­f red Hershey and beled phage DNA en­tered and re­mained as­ in­for­ma­t ion nec­es­sary to pro­duce prog­eny
Mar­tha Chase showed that the pro­tein coat so­ci­ated with the bac­te­r ial cells un­der these phag­es.

Infection Blending/separation
Blen n Centrifugation/detection
Centri

Viral protein
labeled with
radioactive sulfur

Radioactivity predominantly No radioactivity detected

in the supernatant fraction in next generation of phage

Viral DNA labeled with

radioactive phosphorus

Radioactivity predominantly Radioactive DNA is

in the cell pellet detected in progeny phage

of ster­ile cul­ture prior to the dis­cov­ery of an­ti­bi­ot­ics, made to in­ves­t i­gate the re­pro­duc­t ion of vi­r uses. Viral in­fec­t ious
re­pro­duc­ible ex­per­i­men­ta­tion very dif ­fi­cult. However, by cy­cles could be stud­ied un­der pre­cisely con­trolled con­di­tions
1955, the work of many in­ves­ti­ga­tors had led to a se­ries of im­ by em­ploy­ing the an­a ­log of the one-step growth cy­cle of bac­
por­tant meth­od­o­log­i­cal ad­vances. These in­cluded the de­vel­ te­rio­phages and sim­ple meth­ods for quan­ti­fi­ca­tion of in­fec­
op­ment of de­fined me­dia op­ti­mal for growth of mam­ma­lian tious par­ti­cles de­scribed in Chapter 2.
cells, in­cor­po­ra­tion of an­ti­bi­ot­ics into cell cul­ture me­dia, and Our cur­rent un­der­stand­i ng of the mo­lec­u­lar ba­sis of vi­
de­vel­op­ment of im­mor­tal cell lines such as the mouse L and ral par­a­sit­ism, the fo­cus of this vol­u me, is based al­most en­
hu­man HeLa cells that are still in wide­spread use. These ad­ tirely on an­a ­ly­ses of one-step growth cy­cles in cul­tured
vances al­lowed growth of an­i­mal cells in cul­ture to be­come a cells. Such stud­ies es­tab­lished that vi­ruses de­pend ab­so­
rou­tine, re­pro­duc­ible ex­er­cise. lutely on the bio­syn­t hetic ma­chin­ery of their host cells for
The avail­abil­ity of a va­ri­ety of well-characterized an­i­mal syn­t he­sis of the com­po­nents from which prog­eny vi­ral par­
cell cul­tures had sev­eral im­por­tant con­se­quences for vi­rol­ogy. ti­cles are built. In con­trast to cells, vi­r uses are not re­pro­
It al­lowed the dis­cov­ery and prop­a­ga­tion of new hu­man vi­ duced by growth and di­v i­sion. Rather, the in­fect­i ng ge­nome
ruses, such as ad­e­no­v i­rus, mea­sles vi­rus, and ru­bella vi­rus, con­tains the in­for­ma­t ion nec­es­sary to re­d i­rect cel­lu­lar sys­
for which an­i­mal hosts were not avail­­able. In 1949, John tems to the pro­duc­tion of many cop­ies of all­the com­po­
Enders and col­leagues used cell cul­tures to prop­a­gate po­lio­ nents needed for the de no­vo as­sem­bly of new vi­r us par­t i­cles.
vi­rus, a feat that led to the de­vel­op­ment of po­lio vac­cines a few It is re­mark­able, how­ever, that while vi­r uses lack the com­
years later. Cell cul­ture tech­nol­ogy rev­o­lu­tion­ized the abil­ity plex en­er­g y-generating and bio­syn­t hetic sys­tems nec­es­sary
 Foundations 17

B OX 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 (ly­so­
genic) re­la­tion­ships with their host cells. Such
bac­te­r io­phages were called tem­per­ate. In a
ly­so­genic bac­te­r ial cell, vi­ral ge­netic in­for­
ma­tion per­sists but vi­ral gene ex­pres­sion is
re­pressed. Such cells are called ly­so­gens, and
the qui­es­cent vi­ral ge­nome, a pro­phage. By
anal­ ogy with the pro­ phage, an in­ te­
grated
DNA copy of a ret­ro­v i­ral ge­nome in an an­i­
mal ge­nome is termed a pro­v i­rus.

Propagation as a Prophage
For some bac­te­rio­phages like lambda and
Pioneers in the study of ly­sog­e­ny: No­b el lau­re­ates François
Mu (Mu stands for “mutator”), pro­ phage
Ja­cob, Jacques Monod, and Andr é Lwoff, 1965. Courtesy of the
DNA is in­te­g rated into the host ge­nome of U.S. National Library of ­Medicine.
ly­so­gens and pas­sively rep­l i­c ated by the host.
Virally en­coded en­zymes, known as inte-
grase (lambda) and transposase (Mu), me­d i­ rupt­ing host DNA se­quences. This pro­cess is Transduction of Host Genes
ate the co­va­lent in­ser­t ion of vi­ral DNA into called in­ser­tional mu­ta­gen­e­sis and is a phe­ Bacteriophage ge­nomes can pick up cel­lu­lar
the chro­mo­some of the host bac­te­r ium, es­ nom­e­non ob­served with ret­ro­v i­rus­es. genes and de­liver them to new cells (a pro­cess
tab­lish­ing it as a pro­phage. The pro­phage known as trans­duc­tion). For ex­a m­ple, oc­ca­
DNA of other bac­te­r io­phages, such as P1, ex­ Gene Repression and Induction sional mis­takes in ex­ci­sion of the lambda pro­
ists as a plas­m id, a self-rep­l i­c at­i ng, au­ton­o­ Prophage gene ex­ pres­sion in ly­ so­
gens is phage from its host chro­mo­some af­t er in­duc­tion
mous chro­mo­some in a ly­so­gen. Both forms turned off by the ac­tion of vi­ral pro­teins called re­sult in pro­duc­tion of un­usual prog­eny phages
of prop­a­ga­tion have been iden­ti­fied in cer­ re­pres­sors. Expression can be turned on when that have lost some of their own DNA but have
tain an­i­mal vi­r uses, for ex­a m­ple, ret­ro­v i­ re­pres­sors are in­ac­ti­vated (a pro­cess called in­ ac­quired the bac­te­rial DNA ad­ja­cent to the pro­
ruses and a le­t hal her ­pes­v i­r us. duc­tion). The dis­cov­ery that genes can be reg­ phage. The acute trans­form­i ng ret­ro­v i­r uses
u­lated by such trans-act­ing pro­teins, and also arise via cap­ture of genes in the vi­cin­ity of
Insertional Mutagenesis elu­ci­da­tion of their mech­a­nism, set the stage their in­te­gra­tion as pro­v i­ruses (Volume II,
Bacteriophage Mu in­ serts its ge­
nome into for later in­ves­ti­ga­tion of the con­trol of gene Chapter 6). These can­cer-inducing cel­lu­lar
many ran­dom lo­ca­t ions on the host chro­mo­ ex­pres­sion with other vi­ruses and their host genes are then trans­duced along with vi­ral
some, caus­ing nu­mer­ous mu­ta­tions by dis­ cells. genes dur­ing sub­se­quent in­fec­tion.

B OX 1.7
T E R M I N O L O G Y
The epi­some
In 1958, François Ja­cob and Elie Wollman re­ duce in two al­ter­na­tive states: while in­te­g rated
al­ized that lambda pro­phage and the E. coli F in the host chro­mo­some or au­ton­o­mously.
sex fac­tor had many com­mon prop­er­ties. This However, this term is now most com­monly F
re­mark­able in­sight led to the def­i­ni­tion of the ap­plied to ge­nomes that can be main­tained in Integrated
F
epi­some. cells by au­ton­o­mous rep­li­ca­tion and never in­
An epi­some is an ex­og­e­nous ge­netic el­e­ te­grate, for ex­am­ple, the DNA ge­nomes of
ment that is not nec­es­sary for cell sur­v ival. Its cer­tain an­i­mal vi­rus­es.
de­fi n­i ng char­ac­ter­is­t ic is the abil­ity to re­pro­
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peacock; he contended with the lark, the crane and the eagle in
flight.
This was a day on which horses were overcome by asses, and
lions by oxen, a day in which the dog was stronger than the bear and
the cat than the leopard, a day in which the weak confounded the
strong, a day in which slaves were raised on high and nobles
brought to the ground, a day in which the terror of God’s wrath came
upon all, such a day as no chronicle records in time past. May such
a day never come again in our age!
Cap. IX. When all this multitude was gathered together like the
sand of the sea, one, a Jay skilled in speech, took the first place
among them and addressed them thus: ‘O wretched slaves, now
comes the day in which the peasant shall drive out the lord; let
honour, law and virtue perish, and let our court rule.’ They listen and
approve, and though they know not what ‘our court’ means, what he
says has for them the force of law: if he says ‘strike,’ they strike, if he
says ‘kill,’ they kill. Their sound was as the sound of the sea, and
from terror I could scarcely move my feet. They strike a mutual
compact and declare that all those of gentle blood who remain in the
world shall be overthrown.
Then they advance all together; a dark cloud mingled with the
furies of hell rains down evil into their hearts; the earth is wetted with
the dew of the pit, so that no virtue can grow, but every vice
increases. Satan is loose and among them, the princes of Erebus
draw the world after them, and the more I gaze, the more I am
terrified, not knowing what the end will be.
Cap. X. Furious rage there was, they were greedy for slaughter
like hungry wolves. The seven races derived from Cain were added
to them. The prophets spoke of them, Gog and Magog is their name,
they neither fear man nor worship God. Moreover those companions
of Ulysses, whom Circe transformed, are associated with them:
some have the heads of men and others of brute beasts.
Cap. XI. There is Wat, Tom and Sim, Bet and Gib followed by
Hick; Coll, Geff and Will, Grigge, Dawe, Hobbe and Lorkin, Hudd,
Judd, Tebb and Jack, such are their names;68 and Ball teaches them
as a prophet, himself having been taught by the devil.
Some bray like asses, others bellow like bulls, they grunt, they
bark, they howl, the geese cackle, the wasps buzz; the earth is
terrified with their sound and trembles at the name of the Jay.
Cap. XII. They appoint heralds and leaders, and they order that
all who do not favour them shall suffer death. They are armed with
stakes and poles, old bows and arrows, rusty sickles, mattocks and
forks; some have only clods and stones and branches of trees. They
wet the earth with the blood of their betters.
Cap. XIII. These come in their fury to the city of new Troy, which
opens its gates to them, and they surge in and invade the streets
and houses. It was Thursday, the festival of Corpus Christi, when
this fury attacked the city on all sides; they burnt the houses and
slew the citizens. The Savoy burns, and the house of the Baptist falls
to ruin in the flames. They rob and carry away the spoil, and that day
is closed with drunkenness everywhere.
The next day, Friday, is yet worse; no wisdom or courage avails
against them, they rage like a lioness robbed of her young. O, how
degenerate is the city which allows this, how disgraceful that armed
knights should give place to an unarmed mob! There is no Capaneus
or Tydeus, no Ajax or Agamemnon, no Hector or Achilles, to make
defence or attack. Ilion with its towers cannot keep men safe from
the furies.
Cap. XIV. Helenus the chief priest, who kept the palladium of
Troy, was slain in spite of his exhortations. These were deeds worthy
rather of demons than of men. Piety and virtue perished and vice ran
riot. They said ‘Let his blood be upon our heads,’ and slew him
without pity: the curse of Christ shall fall upon them for this deed.
Simon had the same death as Thomas, but at the hands of
greater numbers and for a different cause. Vengeance came for the
death of Thomas; for Simon it daily threatens. It was midday when
this blood was shed, the shepherd was slain by his flock, the father
by his children. He died untimely; but though taken away from us, he
lives in heaven. This is the foulest of all the deeds done: these men
are worse than Cain, who only slew his brother. O cursed hand that
struck the severed head! Wail for this, all ye old and young, the evils
prophesied by Cassandra come down on this city. The king could not
rescue Helenus, but he mourned for him in his heart.
Cap. XV. The chief citizens also perished, there was death and
sorrow everywhere. If a son pleaded for his father, both were slain.
No place of safety can be found by those of gentle condition; they
flee to the forests in vain, and move vaguely hither and thither,
neither city nor field affords them protection. Death is everywhere,
and spares not even the women and the children. There is no
remedy, and neither lamentation nor prayers are of any avail.
Cap. XVI. When I saw all this, horror seized me and I fled. I left
my own house and wandered over the fields, I went from place to
place in search of safety; the enemy pressed after me; I hid in caves
of the woods, and was without hope at evening of what the morrow
might bring. My dreams terrified me and my heart melted like wax in
the fire. I lay hid during the day and trembled at every sound, the
tears that I shed were my sole subsistence. I was alone and in terror
of the wrath of God, my mind was sick and my body was wasted.
Hardly ever did I meet a companion, and those friends whom I had
trusted in prosperity failed me now. I dared scarcely speak a word,
lest I should betray myself to an enemy.
Then, when I saw nothing but death about me, I desired to die,
and yet I was unwilling to perish in so desolate a state. While I wept,
lo, Wisdom came to me and bade me stop my tears, for grief would
at some time cease. I stood amazed and in doubt; death was life to
me and life was death, and wondrous visions passed before me.
Cap. XVII. I saw not far off a Ship, and I ran towards it and
climbed up its side. In it were almost all those of gentle birth,
crowded together and terrified, seeking refuge from the furies. I
prayed that we might have a favourable voyage. The ship left the
shore, but my hopes were vain: the sky grew dark and the winds
lashed the waves into storm, the ship was driven before them amid
thunder and rain. There was confusion among the sailors, and the
captain in vain endeavoured to direct the ship’s course.
Cap. XVIII. At length the storm so increased that all were in
despair of safety. A huge monster of the sea, Scylla and Charybdis
both in one, appeared as if to destroy the ship and all who were in it.
We prayed to heaven for help.
(The Tower of London was like this ship, shaken by the storm, its
walls giving way to the fury of the mob. In vain it offered hopes of
safety; it was stained with foul parricide, and the den of the leopard
was captured by assault.)
When I saw these things I was terrified in my sleep, and I prayed
to God for help. ‘Thou Creator and Redeemer of the human race,
thou who didst save Paul from the sea, Peter from prison and Jonah
from the whale’s belly, hear my prayer, I entreat thee. Help me and
grant that I may be cast up on a favourable shore!’
As I prayed, the monster struck the ship, and it was almost
swallowed up by the fury of Scylla.
Cap. XIX. Yet our cries and tears were not unheard. When the
storm raged most furiously, there was one William, a Mayor, who
was moved to high deeds: he struck down that proud Jay, and with
his death the storm abated, Scylla restored its prey, and the ship
once more rode upright upon the water. The sailors regained their
courage and hoisted a little sail, peace returned and the sky became
clear. I then with all the rest gave thanks to Christ.
Cap. XX. Still my dream went on, and still I seemed to see that
ship, which now with broken oars was drifting in search of a landing-
place. It was driven to that port where all this evil raged; it had
escaped Scylla, but it came to an Island more dangerous than
Scylla. I landed, and asked one of those whom I met, ‘What island is
this, and why is there so great a concourse of people here?’ He
replied: ‘This is called the Island of Brute, and the men who dwell
here are of fair form but of savage condition. This people lays law
and justice low by violence; strife and bloodshed reign here ever. Yet
if they could love one another, no better people would there be from
the rising to the setting of the sun.’
I was saddened and terrified by his answer, I knew not whether
sea or land were more to be feared. The heavenly voice which I had
heard before said to me, ‘Lament not, but take heed to thyself. Thou
hast come to a place where wars abound, but do thou seek peace
within by God’s assistance. Be cautious and silent; but when thou
hast leisure, record these dreams of thine, for dreams often give a
presage of the future.’ The voice was heard no more, and at that
moment the cock crew and I awoke from my sleep, scarce knowing
whether what I had seen was within me or without.
Cap. XXI. Then I returned thanks to God for having preserved me
upon the sea and from the jaws of Scylla. The rustic goes back to his
labours, but in his heart there remains hatred of his lords; therefore
let us be forewarned and provide against future evils. As for me, God
has set me free from the danger, and for this I thank him; and I would
that my country, preserved from destruction, might render due
thanks to God. While the memory of these things is fresh in me, I will
write that which I experienced in my sleep, that waking slumber
which brought to me no mere vision but a dream of reality.

Prologus Libri Secundi.

Many things did I see and note, which my pen shall write, but first
I invoke, not the Muses, but the true Spirit of God, and I will let down
my nets in the name of Christ and for his glory. The style and the
verses are poor, but the meaning is good. I will give that which my
poor faculties can attain to; and may he be my helper who produced
speech from the mouth of an ass. I prefer to do a little good than
none.
The words which follow are not spoken from myself; they are
gathered from various sources, as honey from various flowers or
bright shells from various shores. The name of the book is Vox
Clamantis, because it is the utterance of a fresh sorrow.

Liber Secundus.
 Cap. I. Tears shall be the ink with which I write. All is vanity
except the love of God, and man has cause for lamentation from his
birth.
Yet if any people in the world could be happy, God granted this
boon to us; we were blessed above all other nations. Now our former
glory is extinguished and our prosperity is destroyed.
Why is our condition thus changed? Nothing on earth happens
without a cause, yet all deny that they are the cause of this and find
fault with Fortune, who turns all things upside down.
Cap. II. O thou who art called Fortune, why dost thou thus
depress those whom thou didst once exalt? Once our country was
everywhere honoured, all desired to be at peace with it: now our
glory has departed and enemies attack us from all quarters. Reply,
Fortune, and say if thou art the cause of this change. I think not, for I
believe in God and not in Fortune; yet I will describe thee, as men
think that thou art.
Cap. III. Fortune, hear what men say of thee, that thou hast a
double face, and goest by double paths, that nothing in thee is stable
or secure. No gifts may keep thee faithful, thou art lighter than the
dead leaves which fly before the wind: now thou art bright and fair,
now dark and lowering; thy love is more treacherous than that of a
harlot, the prosperity which thou givest is very near to disaster.
Cap. IV. Fortune gives no honey without gall, she changes like
the sphere of the moon. Her wheel is ever turning, and no tears or
prayers will move her. Citizen and husbandman, king and rustic, rich
and poor, all are alike to her. Ah! why was so much power given to
such a one as she is?
Thus men say, believing that Fortune can overthrow the decrees
of God, but in fact she is nothing, fate is nothing, chance has nothing
to do with the affairs of men. Each one makes for himself his own lot:
if the will is good, good fortune follows, if evil, it makes the fortune
bad. Virtue will lead you to the summit of the wheel, and vice will
bring you and your fortune down to the bottom.
 Cap. V. God has said that the man who obeys his commands
shall prosper in wealth and peace: the very elements are subject to
the righteous man. Joshua caused the sun to stand still, Gregory
stayed the plague, Moses divided the sea, Elisha caused iron to
swim, the three children were unhurt by the fire, the earth rose to
give a seat to Hilarius. Wild animals, too, serve the just man, witness
Daniel, Silvester, Moses and Jonah.
Cap. VI. Again, the elements war against sinners: so it was in the
case of the plague caused by David’s sin, in the case of the
Sodomites, Korah, Dathan and Abiram, Lysias and others. The
wicked man cannot enjoy good fortune, nor can the good man be
deprived of it. It was guilt that caused the fall of Pharaoh and of Saul,
the death of Ahab and of Eli with his sons. The Jews always
conquered while they were obedient to God’s law, and were
overcome when they transgressed it.
Cap. VII. It is God Omnipotent, the Three in One, who governs all
things here. As fire, heat and motion are three things combined in
one, so the Father, Son and Holy Spirit are three persons but one
Godhead.
Cap. VIII. Christ, the Son of the Father, became incarnate in man,
and yet remained what he was before, being less than the Father
and yet equal to him, perfect Man and perfect God. As the frailty of
the first Adam brought evil upon us all, so the strength of the second
Adam healed our wound and restored our fallen state.
Cap. IX. We must submit our mind to the faith, for man cannot
understand the things of God, and we must not examine too closely
the mystery which we cannot penetrate. This we know, that life is
given to all through the name of Jesus Christ.
Cap. X. The heathen bows down to figures of wood and stone,
asking help from that which his hands have made. Was not the world
made for man and all things placed in subjection to him? How then
can these idols be of any avail?
As for us, we use images differently, not giving to them the
worship that belongs to God, but by them assisting devotion;
especially the sign of the Cross is to be adored, by means of which
we conquer the powers of evil. Great is the virtue of the Cross, by
which Christ despoiled hell of its prey and ascended into heaven.
Cap. XI. God created the heaven and the earth, and all created
things ought to serve him. As he creates all things, so also he rules
them continually, and he gives his gifts according to men’s merit.
Whatever comes to pass in the world, whether it be good or evil, we
are the cause of it.

Prologus Libri Tercii.

Since good and bad fortune are due to the merits and demerits of
men, I shall examine the various conditions of men and find out
where the fault lies. I shall utter not so much my own words as the
common report of others, and it must be remembered that he who
finds fault with the bad is in effect praising the good. May God assist
me to carry out my task! My abilities are small, and I do not affect
high themes, but I speak of the evils which the common voice of
humanity bewails. Let no envy or calumny attack my work; and do
thou, O Christ, grant that I may avoid falsehood and flattery. With this
prayer I enter on my voyage.

Liber Tercius.
Cap. I. The order of the world is in three degrees,—Clergy,
Knighthood and Peasantry. I shall deal first with the prelates of the
Church, whose practice is very far removed from the example of
Christ. Riches alone are valued by them, and the poor man is
despised, whatever may be his merits.
Cap. II. Prelates of the Church are now hirelings, whose desire is
to live in luxury and to indulge their appetites. Gluttony and lust
everywhere prevail.
Cap. III. The prelates of the Church aim at earthly honours
instead of heavenly: they desire rather to have the pre-eminence
than to do good. Powerful men escape without rebuke for their sins,
and penance is avoided by payment.
 Cap. IV. As regards the ‘positive law,’ for breach of which
dispensations are granted, I ask first whether Christ gives indulgence
beforehand for sin, or prohibits that which is not sin. If these things
are sins, how can I be free to commit them on consideration of a
money payment; if not, why does the Church forbid them? This is
merely a device for bringing in money to the clergy.
Cap. V. The poison of temporal possessions is still working in the
Church. They no longer war on the pagan, but turn their swords
against their own brother Christians.
Cap. VI. Christ left peace with his disciples, but in our time
avarice and ambition cause prelates to take part in intestine strife,
with swords in their hands and the cross as their ensign. It is not the
part of a soldier to offer incense at the altar or of a priest to bear
arms in war.
Cap. VII. The priest should fight with other than material arms.
David was not permitted to build a house for the Lord, because he
had been a shedder of blood; and those who are stained with the
slaughter of their brethren cannot be the true servants of the altar.
Brotherly love should prevail, and this is opposed to strife and self-
seeking ambition.
Cap. VIII. Worldly men may make wars, but the clergy should not
take part in them; their strength is in their words and prayers, and
they have no need of material arms. Too great prosperity and wealth
is the cause of these evils: they do not see what the end will be.
Cap. IX. The ring and the pastoral staff belong to the Pope, the
sceptre to the Emperor; the one must not usurp the rights of the
other. The Emperor should not claim spiritual power, nor the Pope
temporal. Christ is a lover of peace and his ministers must not
appeal to the sword, but must keep the command, ‘Thou shalt not
kill.’ Let Christ himself lay claim to what is his. Pride is the root of all
evil.
The apostles conquered by prayers and by patience; Peter had
neither silver nor gold, but he healed the lame man; our clergy
abound in wealth, but do no works of healing, either spiritual or
bodily. O thou who art head of the Church, remember that
forgiveness should be until seventy times seven, and that Peter was
commanded by Christ to put up his sword.
Cap. X. The teaching and the writings of the clergy are in favour
of peace and love, and when I wondered why they waged wars, one
answered me in the person of the supreme pontiff and said: ‘Rule on
earth is given to us by divine decree and it pleases us to enjoy all the
good things of this world. Our way is different from that of Christ and
his apostles; we set up the cross as a sign of hatred and vengeance,
we put to death those who will not acknowledge our rule; the
pastoral staff is turned into a spear and the mitre into a helmet, we
can slay with sword as well as with word, and whereas Peter cut off
the ears, we cut off the head.’
Cap. XI. These claim the worship and honour which belong to
God alone, and the goods which they unjustly seize are never
restored. The shepherd preys like a wolf upon his own sheep.
Cap. XII. He who is promoted to dignity in the Church by simony
is like the thief who enters not by the door into the sheepfold. The
Church is a congregation of faithful men, and the clergy are no better
than the laity, except so far as they lead better lives. Yet they lay
burdens upon us which they will not bear themselves, and do not
follow their own precepts. They bear the keys of heaven, but they
neither enter themselves nor allow us to enter: they set no good
example to their flocks.
Cap. XIII. A prelate should be a light to guide his people by
example, and he should encourage them by his voice, and also
reprove and restrain. The oil with which he is anointed is a type of
the qualities that he ought to display.
Cap. XIV. At the Court of Rome nothing can be done without gifts:
the poor man is everywhere rejected. The spirit of Antichrist is
opposite to that of Christ, and there are many signs that he has
already come.
Cap. XV. Our prelates aim at the mere outward show of sanctity
and refuse to bear the burden of Christ. O God, in thy mercy restore
them to the state which they have lost!
Cap. XVI. Rectors of parishes, too, err after the example of the
prelates. They are luxurious in their lives, and many desert their
spiritual cures, in order to frequent courts and great households, with
a view to promotion.
Cap. XVII. Another gets leave from the bishop to leave his parish
on the plea of study at the universities; but there he learns and
teaches only lessons of unchastity. The Church, which is his true
bride, is neglected, and harlots receive the tithe which belongs to
God.
Cap. XVIII. A third rector resides in his parish, but spends his time
in sports, keeps well-fed horses and dogs, while the poor are not
relieved or the sick visited, makes his voice heard more in the fields
and woods than in the church. He lays snares too for the women of
his parish, and if their bodies be fair, he cares not how their souls are
defiled.
Cap. XIX. Another neglects his cure of souls and makes money
by buying and selling. He is liberal of his wealth to none but women;
and if benefices were inherited by the children of those who hold
them, the succession would seldom fail.
Cap. XX. The priests without benefices, who get their living by
‘annuals,’ are equally bad: the harlot and the tavern consume their
gains. Let none admit these to his house, who desires to keep his
wife chaste, anymore than he would admit pigeons to his bed-
chamber, if he wished to keep it clean.
Cap. XXI. These infect the laity by their bad example. The bishop
ought not to ordain such men; and he who might prevent an evil and
does not, is equally guilty with him who causes it.
Cap. XXII. The clergy deny the right of laymen to judge and
punish them; yet the sins of the clergy deeply affect the laity. We are
all brethren in Christ and we are bidden to rebuke our brethren, if
they do wrong, and to cast them out of the Church, if they will not
amend.
 Cap. XXIII. Priests say that in committing fornication they do not
sin more than other men who are guilty of this vice. But their sacred
condition and their vow of chastity makes the evil worse in them than
in a cobbler or a shepherd.
Cap. XXIV. If we consider the office of the priesthood, we shall
find that the vestments and ornaments of priests are all symbolical of
the virtues which they ought to possess.
Cap. XXV. The ceremonies of sacrifices under the old law were
symbols of the virtues required in priests under the new, and as
under the old dispensation the ministers of the altar ought to be
without defect and deformity of body, so the priests of the new law
should be spiritually free from blemish. Uzzah touched the ark with
unclean hands and was punished with death: so he who comes
polluted to the service of the altar is worthy of punishment.
Cap. XXVI. A man must be of mature age before he assumes the
priesthood; for youth is apt to yield to the temptations of the flesh.
The evil impulses cannot be wholly expelled, but they may be kept in
check, as is symbolized by the tonsure of the priest. Let the priest
avoid idleness, whence so many vices spring.
Cap. XXVII. The honour of priests is great, if they live worthily.
They administer to us the sacraments during our lives, they give us
burial when we are dead, they are the salt of the earth and the light
of the world. So much the worse is it when they are ignorant and
bad; the distinction between the good and the bad priest is like that
between the dove and the raven sent out of the ark.
Cap. XXVIII. The young scholars who are being trained for the
priesthood are in these days too often indolent and vicious. If they
are so in youth, they will hardly be good in their later age.
Cap. XXIX. They are induced to undertake the priesthood by
desire to escape from the control of the ordinary law, by dislike of
labour, and by love of good living, seldom by the higher motive,
which once prevailed, of contempt for worldly things and longing
after the highest good. Thus, since the clergy is without the light of
virtue, we laymen wander in the dark.
 Liber Quartus.
Cap. I. Men of Religious Orders are also of various conditions,
some good and others bad. Let each bear his own burden of blame:
I write only what common report tells me.
There are first those who hold temporal possessions, and some
of these live in gluttony and luxury.
Cap. II. Those who leave the world should give up worldly things;
but in these days the monk is known only by his garb. He indulges
himself with the richest food and the choicest drink, he makes haste
when the bell rings for a meal, but he rises very slowly and
reluctantly for midnight prayer. The monks of old were different; they
dwelt in caves and had no luxurious halls or kitchens, they were
clothed in skins, fed on herbs and drank water, and abstained from
fleshly lusts. These men truly renounced the world, but that blessed
state has now perished.
Cap. III. The old monastic rule has given place to gluttony and
drunkenness, and those who live so can hardly be chaste. Pride,
anger and envy prevail among these men, in spite of the restrictions
of their rule.
Cap. IV. There is no brotherly love among them, and the vow of
individual poverty is also broken. They make money in various ways
and spend it on their pleasures and in enriching their children, whom
they call their nephews.
Cap. V. A monk wandering abroad from his cloister is like a fish
out of water; nor are those much better who stay within the walls and
allow their minds to dwell on worldly things.
Cap. VI. Some seek honour and dignity under the cover of the
monastic profession, even though they be of poor and low birth.
Cap. VII. Patience, Chastity and the rest who were once brothers
of religious orders, are now dead or departed, and their contrary
vices have taken their places.
Cap. VIII. So also the regular Canons for the most part neglect
their monastic rule and have only a show of sanctity.
 Cap. IX. Monks who are untrue to their profession are of all men
the most unhappy. They have no real enjoyment of this world and
they lose also the joys of heaven.
Cap. X. Let all members of religious orders perform their vows
and repent of their past sins, of their pride, luxury, avarice, ambition,
gluttony, wrath, envy and strife.
Cap. XI. Above all let them avoid intercourse with women, who
bring death to their souls. Let them labour and study; for idleness is
the great incentive to evil.
Cap. XII. The monk who sets himself to observe his rule will live
hardly and fast often, praying continually and doing penance for sin.
He will submit himself humbly to his prior, and he will not grudge to
perform duties that are irksome. The prior should be gentle with his
younger brethren and not make the yoke too heavy for them.
Cap. XIII. As regards nuns, they too are under the rule of chastity;
but as women are more frail by nature than men, they must not be
so severely punished if they break it. They require meat often on
Fridays for their stomachs’ sake, and this is prepared for them by
Genius the priest of Venus.
Cap. XIV. Where Genius is the confessor of a convent, the laws
of the flesh prevail. The priest who visits nuns too often corrupts
them, and the woman very easily yields to temptation. A wife may
deceive her husband, but the bride of Christ cannot conceal her
unfaithfulness from him: therefore she above all others should be
chaste.
Cap. XV. True virginity is above all praise, and this surpasses
every other condition, as a rose surpasses the thorns from which it
springs. The best kind of virginity is that which is vowed to God.
Cap. XVI. Not all whom Christ chose were faithful, and
everywhere bad and good are mingled together; but the fault of the
bad is not a reason for condemning the good. So when I speak of
the evil deeds of Friars, I condemn the bad only and absolve the
good.
 The number of mendicant friars is too great and their primitive
rule has been forgotten. They pretend to be poor, but in fact they
possess all things, and have power over the pope himself. Both life
and death bring in gains to them.
Cap. XVII. They preach hypocritically against sin in public, but in
private they encourage it by flattery and indulgence. They know that
their gains depend upon the sins which their penitents commit. Friars
do not often visit places where gain is not to be got. They have an
outward appearance of poverty and sanctity, without the reality. I do
not desire that they should be altogether suppressed, but that they
should be kept under due discipline.
Cap. XVIII. Some friars aim at dignity as masters in the schools,
and then they are exempted from their rule and obtain entry into
great houses. The influence of the friar is everywhere felt, and often
he supplies the place of the absent husband and is the father of his
children. Bees, when they wound, lose their stings and are
afterwards helpless: would it were so with the adulterous friar!
Cap. XIX. The order of friars is not necessary to the Church.
Friars appropriate spiritual rights which belong to others; and though
this may be by dispensation of the pope, yet we know that the pope
does not grant such dispensations of his own motion, and he may be
deceived. They ask for the cure of souls, but in fact they are
demanding worldly wealth: not so did Francis make petition, but he
left all and endured poverty.
Cap. XX. This multitude of friars is not necessary for the good of
society. David says of them that they neither take part in the labours
of men nor endure the rule of the law: they toil not, neither do they
spin, and yet the world feeds them. It is vain for them to plead the
merits of Francis, when they do not follow his example. All honour to
those who do as he did.
Cap. XXI. They draw into their order not grown men but mere
boys. Francis was not a boy when he assumed his work; but in these
days mere children are enrolled, caught like birds in a snare: and as
they are deceived themselves, so afterwards they deceive others.
 Cap. XXII. The friar who transgresses the rule of his order is an
apostate and a follower of the apostate fiend. He finds entrance
everywhere, and everywhere he lays snares, encourages hatred,
and fosters impurity. Under a veil of virtuous simplicity he conceals a
treacherous heart. These are ministers of the Synagogue rather than
of the Church, children of Hagar, not of Sara.
Cap. XXIII. They are dispersed over the world like the Jews, and
everywhere they find ease and abundance. Their churches and their
houses are built in the most costly style and adorned with the richest
ornaments. No king has chambers more magnificent than theirs, and
their buildings are a mark of their worldly pride. Unless their souls
are fair within, this outward pomp of religion is of no avail.
Cap. XXIV. Friars differ from one another in the garb of their
order, but all equally neglect their rule. Only the order founded by
brother Burnel still maintains its former state. Two rules of this order I
will set forth, which are almost everywhere received. The first is that
what the flesh desires, that you may have; and the second that
whatever the flesh shrinks from, that you should avoid. So the new
order of Burnel is thought better than those of Benedict or Bernard.
Thus, if bad times come, I shall hold that the error of the Clergy is
the cause. The body is nothing without the spirit: we have darkness
instead of light, death instead of life, and the flock is scattered
abroad without a shepherd.

Liber Quintus.
Cap. I. I will speak in the second place of the order of Knighthood.
This was established first to defend the Church, then for the good of
the community, and thirdly to support the cause of the widow and
orphan. If a knight performs these duties, he should have praise, but
not if he makes war merely for the sake of glory.
If a knight overcomes his enemies, but is overcome by the love of
a woman, he has no true glory, for he makes himself a slave instead
of free.
Cap. II. If the knight would reflect on the variety and uncertainty of
love, he would not allow himself so easily to be made captive.
 Cap. III. But when he sees beauty in woman decked out with all
its charms, he thinks it divine and marvellous, and he can offer no
effectual resistance. Lovers are blind and are driven by every kind of
unreasonable impulse. Women deceive men, and men also deceive
and betray women.
Cap. IV. The knight has little need to fear bodily wounds, which
may easily be healed; but love is not to be cured by physicians, and
this deprives him both of reason and of honour.
Cap. V. Those who seek fame and worldly honours only, are
hardly better than those who are conquered by women.
Cap. VI. The good woman is one whose praise is above all
things. The bad is a subtle snare for the destruction of men. She
paints her face and uses every art to deceive. The world is
treacherous, but woman is more treacherous still.
Cap. VII. The good knight, who labours neither for gain nor for
glory, and is not conquered by love, obtains the victory over the
enemies of the Church and of his country, and gives us the blessing
of peace.
Cap. VIII. The bad knight is the causer of many evils in the other
orders of society. He deserves to have Leah, not Rachel, as his
bride. Those who follow wars for the sake of the spoils are like
vultures that prey upon the corpses of the dead. Alas, in these days
gold is preferred to honour and the world to God.
Cap. IX. Another estate remains, that of the cultivators of the soil,
who provide sustenance for the human race in accordance with the
divine ordinance laid down for Adam. These at the present time are
lazy and grasping, as well as few in number; one peasant now asks
more wages than two did in past time, and one formerly did as much
work as three do now. We know from recent experience what evil the
peasant is capable of doing. God has ordained, however, that
nothing is to be had without toil; therefore the peasant must labour,
and if he will not, he must be compelled.
Cap. X. There are also the casual labourers, who go from one
employment to another and always find fault with the food that they
get from their masters. These are irrational like beasts, and they
should be disciplined by fear of punishment.
Cap XI. In cities there are chiefly two classes, the merchants and
the craftsmen. The former sin by not regarding festivals and holy
days.
Cap. XII. Usury and Fraud are two sisters, daughters of Avarice,
to whom the dwellers in cities pay honour. Usury is forbidden of old,
but by a gloss on the text it is now approved.
Cap. XIII. Fraud is worse, because it is common to all places.
From the young apprentice to the master all practise it in selling.
Cap. XIV. Craftsmen, who make things, follow the laws of Fraud,
and so do those who sell articles of food, as meat, fish, bread, beer
and so on.
Cap. XV. It is an ill bird that fouls its own nest, and it is shameful
for a citizen to benefit strangers at the expense of his fellow-citizens.
It is an evil thing when one of low condition is exalted to the highest
place in the city. The evil man is a common scourge; but though he
be mounted on high, he shall fall and perish.
Cap. XVI. The man whose tongue is unrestrained is as a
pestilence among the people. The tongue causes strife and many
evils; it breaks through every guard and devours like a flame. None
can say how many evils the tongue of the talkative man brings about
in the city: it causes discord and hatred instead of peace and love;
and where peace and love are not, there God is not. The citizen who
thus plagues his fellows should be put to death or banished: it is
expedient that one should die, lest the whole people should perish.
Thou ruler of the city, labour to bring about harmony and peace,
and above all deal prudently. Great consequences often follow from
small things, and the fire which seems to be extinguished may blaze
up again. Justice and peace, which formerly reigned, must be
restored, so that the ruin which overtook Rome and Athens may be
averted from our city.

Liber Sextus.
 Cap. I. Besides the three degrees of society above described,
there are those who are called ministers of the Law. Of these some
labour for true law and justice, and these I praise; but most practise
an art under the name of law which perverts justice. The advocate
will plead the cause of any man who pays him, and compels his rich
neighbours to give him gifts, for fear that evil should befall them. He
has a thousand ways of making his gains; the great and powerful
break through his snares, but the weak and defenceless are caught
in them. Like the bat or the owl he loves darkness rather than light:
yet sometimes the biter is bitten.
Cap. II. The advocate oppresses and plunders the poor, and
rejoices in discord as a physician in disease. He contrives every
device to enrich himself and his offspring; he joins house to house
and field to field. But his heir dissipates that which he has gathered
together, and a curse comes upon him at the last.
Cap. III. The land is ruined by the excessive number of lawyers.
As a straight stick appears crooked when plunged in water, so does
straightforward and simple law become distorted in the mind of the
lawyer. As clouds conceal the sun, so do advocates obscure the
clear light of the law. Conspiracy, they say, is unlawful, but they
themselves conspire to protect one another, and the law has no
power over these.
Cap. IV. They ascend by degrees from the rank of apprentice to
that of serjeant and so to the office of judge. The administration of
justice is disturbed chiefly by three things, gifts, favour, and fear.
Those who make friends with the judge will hardly lose their case.
Cap. V. O ye who sell justice for gain, learn what end awaits you.
The higher you rise, the greater will be your fall: the more wealth you
gather, the greater will be your misery. O thou judge who seekest
after wealth, why dost thou attend to all things else and neglect
thyself? Thou wilt gain the world, but lose heaven. All worldly power
comes to an end, and so, be sure, will thine.
Cap. VI. As regards the sheriffs, the bailiffs, and the jurymen at
assizes, they are ready to accept bribes and pervert justice. As the
toad cursed the harrow, so I curse these many masters, who are all
unjust.
Cap. VII. Laws, nevertheless, there must be, to punish the
transgressor; and if there are laws there must also be judges. The
worst of evils is when justice is not to be had, and this causes a land
to be divided against itself. Much depends upon the ruler: for the sins
of a bad king the people are punished as well as the king himself.
The higher a man’s place is, the worse is the effect of his evil-doing.
A law is nothing without people, or people without a king, or a king
without good counsel. 69 Complaints are everywhere heard now of
the injustice of the high court, and the limbs suffer because the head
is diseased. The king is an undisciplined youth, who neglects all
good habits, and chooses unworthy companions, by whose influence
he is made worse. At the same time older men give way to him for
gain and pervert the justice of the king’s court. None can tell what
the end will be: I can only mourn over these evils and offer my
counsel to the youthful king.
Cap. VIII. Every subject is bound to serve his king, and the king to
govern his people justly. Hence I shall endeavour to set forth a rule
of conduct for the honour of my king.
First then, I say, govern thyself according to the law, and enforce
on thyself the precepts that are fitting for others. A king is above all
others; he should endeavour to overcome and rise above himself. If
thou art above the laws, live the more justly. Be gentle in thy acts, for
thy wrath is death. Endeavour to practise virtue in thy youth and to
avoid evil communications.
Cap. IX. Avoid false friends and those who stir up war for the
sake of their own profit. Resist those who will tempt thee to evil, O
king. Take vengeance on wrong, and let justice be done without fear
or favour.
Cap. X. Show mercy also, where mercy is fitting, and listen to the
prayer of the poor and helpless. Let fit men of proper age and
sufficient wisdom be appointed to administer justice.