Virology principles and applications

1 Viruses are Ubiquitous on Earth 1.2 Reasons for Studying Viruses 1.3 The Nature of V iruses 1.4 The Remainder of the Book Learning Outcomes Sources of Further Information Chapter 2: Me

Principles and Applications

Tai Lieu Chat Luong

Contents

Preface to Second Edition

Preface to First Edition

Abbreviations Used in This Book

Greek Letters Used i n This Book

Color Coding for Molecules

Chapter 1: Viruses and Their Importance

1 1 Viruses are Ubiquitous on Earth

1.2 Reasons for Studying Viruses

1.3 The Nature of V iruses

1.4 The Remainder of the Book

Learning Outcomes

Sources of Further Information

Chapter 2: Methods Used in Virology

2.1 Introduction to Methods Used in Virology

Sources of Further Information

Chapter 3: Virus Structure

3 1 Introduction to Virus Structure

Sources of Further Information

Chapter 4: Virus Transmission

4 1 Introduction to Virus Transmission

4.2 Transmission of Plant Viruses

4.3 Transmission of Vertebrate Viruses

4.4 Transmission of Invertebrate Viruses

4.5 Permissive Cells

Learning Outcomes

Sources of Further Information

Chapter 5 : Attachment and Entry of Viruses into Cells

5.1 Overview of Virus Replication

5.2 Animal Viruses

5.3 Bacteriophages

Learning Outcomes

Sources of Further Information

Chapter 6: Transcription, Translation, and Transport

6 1 Introduction to Transcription, Translation, and Transport 6.2 Transcription of Virus Genomes

6.3 Transcription in Eukaryotes

6.4 Translation in Eukaryotes

6.5 Transport of Molecules in Eukaryotic Cells

6.6 Transcription and Translation in Bacteria

Learning Outcomes

Sources of Further Information

Chapter 7 : Virus Genome Replication

7 1 Overview of Virus Genome Replication

7.2 Locations of Virus Genome Replication in Eu karyotic Cells

7 .3 Initiation of Genome Replication

7 4 Polymerases

7.5 DNA Replication

7 6 Double-Stranded RNA Replication

7.7 Single-Stranded RNA Replication

7.8 Reverse Transcription

Learning Outcomes

Sources of Further Information

Chapter 8 : Assembly and Exit of Virions from Cells

8 1 Introduction to Assembly and Exit of Virions from Cells 8.2 Nucleocapsid Assembly

8.3 Formation of Virion Membranes

8.4 Virion Exit From the Infected Cell

Learning Outcomes

Sources of Further Information

Chapter 9 : Outcomes of Infection for the Host

9 1 Introduction to Outcomes of Infection for the Host

9.2 Factors Affecting Outcomes of Infection

9.3 Non-Productive Infections

9.4 Productive Infections

Learning Outcomes

Sources of Further Information

Chapter 10: Classification andl Nomenclature of Viruses

10 1 Hi story of Virus Classification and Nomenclature

10.2 Modern Virus Classification and Nomenclature

10.3 Baltimore Classification of Viruses

Learning Outcomes

Sources of Further Information

Chapter 11: Herpesviruses (and Other dsDNA Viruses)

1 1 1 Introduction to Herpesviruses

1 1 2 The Human Herpesviruses

11.3 The Herpesvirus Virion

11.4 HSV-1 Genome Organization

1 1 5 HSV-1 Rep I ication

1 1 6 Latent Herpesvirus Infection

11.7 Other dsDNA Viruses

Learning Outcomes

Sources of Further Information

Chapter 12: Parvoviruses (and Other ssDNA Viruses)

Sources of Further Information

Chapter 13: Reoviruses (and Other dsRNA Viruses)

13.l Introduction to Reoviruses

13.2 Rotavirus Virion

13.3 Rotavirus Repl ication

13.4 Other dsRNA Viruses

Learning Outcomes

Sources of Further Information

Chapter 14: Picornaviruses (and Other Plus-Strand RNA Viruses)

14 1 Introduction to Picornavi ruses

14.2 Some Important Picornaviruses

14.3 Picornavirus Virion

14.4 Picornavirus Replication

14.5 Picornavirus Recombination

14.6 Picornavirus Experimental Systems

14.7 Other Plus-Strand RNA Vi ruses

Learning Outcomes

Sources of Further Information

Chapter 15: Rhabdoviruses (and Other Minus-Strand RNA Viruses)

15 1 Introduction to Rhabdoviruses

15.2 Some Important Rhabdoviruses

15.3 The Rhabdovirus Virion and Genome Organization

15.4 Rhabdovirus Replication

15.5 Other Minus-Strand RNA Viruses and Viruses with Ambisense Genomes 15.6 Reverse Genetics

Learning Outcomes

Sources of Further Information

Chapter 16: Influenza Virus

16.1 Introduction to Influenza Viruses

16.2 The Influenza Virion

16.3 Influenza A Virus Replication

16.4 Importance of Influenza Viruses

Sources of Further Information

Chapter 18: Human Immunodeficiency Viruses

18.6 Progression of HIV Infection

18.7 Prevention of HIV Transmission

Learning Outcomes

Sources of Further Information

Chapter 19: Hepadnaviruses (and Other Reverse-Tra nscribing DNA Viruses) 19.1 Introduction to Hepadnaviruses

19.9 Prevention and Treatment of HBV Infection

19.10 Other Reverse-Tra nscri bing DNA Viruses

Learn ing Outcomes

Sources of Further Information

Chapter 20: Bacterial Viruses

20.1 Introduction to Bacterial Viruses ( Bacteriophages)

20.2 Single-Stranded RNA Phages

20.3 Double-Stranded RNA Phages

20.4 Single-Stranded DNA Phages

20.5 Double-Stranded DNA Phages

Learning Outcomes

Sources of Further Information

Chapter 2 1 : Origins and Evolution of Viruses

21.1 Introduction to Origins and Evolution of Viruses

21.2 Origins of Viruses

21.3 Evolution of Viruses

Learning Outcomes

Sources of Further Information

Chapter 22: Emerging Viruses

22.1 Introduction to Emerging Viruses

22.2 Viruses in New Host Species

22.3 Viruses in New Areas

22.4 Viruses in New Host Species and in New Areas

22.5 New viruses

22.6 Recently Discovered Viruses

22 7 Re-Emerging Viruses

22.8 Virus Surveil lance

22.9 Dealing with Outbreaks

Learning Outcomes

Sources of Further Information

Chapter 23: Viruses and Cancer

23 1 Introduction to Viruses and Cancer

23.2 Papillomavi rus-Linked Cancers

23.8 Virus-Associated Cancers in Animals

23.9 Cell Lines Derived From Virus-Associated Cancers

23 1 0 How Do Viruses Cause Cancer?

23 1 1 Prevention of Virus-Induced Cancers

Learning Outcomes

Sources of Further Information

Chapter 24: Survival of Infectivity

24.1 Preservation of Virus Infectivity

24.2 Destruction of Virus Infectivity

24.3 Inactivation Targets in Virions

24.4 Inactivation Kinetics

24 5 Agents that Inactivate Virus Infectivity

Learning Outcomes

Sources of Further Information

Chapter 25: Virus Vaccines

25 1 Introduction to Virus Vaccines

25.2 Live Attenuated Virus Vaccines

25.3 Inactivated Virus Vaccines

25.4 Virion Subunit Vaccines

25.5 Live Recombinant Virus Vaccines

25.6 Mass-Production of Viruses for Vaccines

Sources of Further Information

Chapter 26: Anti-viral Drugs

26.1 Introduction to Anti-viral Drugs

26.2 Development of Anti-viral Drugs

26.3 Examples of Anti-viral Drugs

26.4 Drug Resistance

26.5 Anti-vi ra l Drug Research

Lea rn i ng Outcomes

Sou rces of Fu rther Information

Chapter 27: Pri ons

27 l Introd uction to Prions

27.2 Tra nsm issi ble Spongiform Encephal opathies

27 3 The Nature of Prions

27.4 Pri on Diseases

27.5 Pri on Stra i ns

27 6 Pri on Tra nsm ission

27 7 The Protei n-Only Hypothesis

Lea rn i ng Outcomes

Sou rces of Fu rther Informati on

Vi rologistss1> T M Vocabulary

[S ormRG]

KATI TPB I ET I h33

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This edition first published 2013 by John Wiley & Sons Ltd

Copyright © 2013, 2007 John Wiley & Sons Ltd

Cover image: Glass Sculpture of HIV virus courtesy of Wellcome Images

All effo rt has been made to trace and acknowledge ownership of copyright The publisher would be glad to hear from any copyright holders whom it has not been possible to contact

The rights of John Carter and Venetia Saunders to be identified as the authors of this work has been asserted in accordance with the U K Copyright, Designs and Patents Act 1988

All rights reserved No part of this publication may be reprod uced, stored in a retrieval syste m , or transm itted, in any form o r by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher

Wiley publishes in a variety of print and electronic formats and by print-on-demand Some material included with standard print versions of this book may not be included i n e-books or in print-on-demand If this book refers to media such as a

CD or DVD that is not included in the version you purchased, you may download this material at http ://books upport.wiley.com For more information about Wiley products, visit www.wiley.com

Designations used by companies to distinguish their products are often claimed as trademarks All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners The publisher is not associated with any product or vendor mentioned in this book This p u blication is designed to provide accurate and authoritative information in regard to the subject matter covered It is sold on the understanding that the publisher is not engaged in rendering professional services If professional advice or other expert assistance is req u i red, the services of a com petent professiional should be sought

Library of Congress Catalog ing-in-Pu blication Data

Carter, John B.,

1944-Virology : principles and applications / John Carter, Venetia Saunders - 2nd ed p.; cm

Includes bibliographical references and index

ISBN 978-1-119-99 143-4 (cloth) - ISBN 978- 1-1 19-99142-7 (pbk.)

I Sau nders, Veneti a A , 1949- II Ti tl e

[DN LM : 1 Viruses 2 Vi rus Diseases QW 160]

Assistant Ed itor : Kathari ne Earwaker

Ma rketi ng Manag ers : Fran Hunt and Jo U nderwood

Project Editor: J u l iet Booker

To Myra, Robert, Jon, and Mark

Preface to Second Ed iti on

Vi rology conti nues to be an exciti ng subject and to develop at a ra pid pace The intro d u ction of new la borato ry tec h n iq u e s a nd th e conti n u ed a p p l i cati on of esta bl ished techni ques are prod ucing a wealth of new information There has been

an explosion in the publication of vi rology papers reporting the di scovery of new viruses and providi ng deeper i nsights i nto many facets of the subject We have tried to reflect these developments i n the second edition, which contai ns m uch new materi a l , i ncl u d i ng a n add itional chapter (on i nfl u enza vi rus) In a n attem pt to

i m prove cl arity, sections of the text have been rewritten

In the diagra ms, many of which have been red rawn for incl usion i n this ed ition, there is a sta ndard color code to d ifferentiate va rious types of n uclei c acid and protei n molecu les Please note that, in the interests of cla rity, there have been some mod ifications to the color code used in the fi rst ed iti on There is a key to the

c o l o r c o d e o n p a g e x x i x I n a d d i t i o n , t h e b o o k h a s a w e b s i t e ( w w w w i l e y co m / co l l eg e/ca rte r) w ith a cco m p a n y i n g tea ch i n g a n d l e a r n i n g resources, includ i ng ani mations of vi rus replication cycles

We are grateful to the many people who p rovided feed back on the fi rst edition a nd

to those who made suggestions for the second edition We have i ncorporated many

of the suggestions for new material, thoug h space constrai nts prevented us from

i ncorporati ng them a l l Aga i n , we would be g rateful to receive feed back

We thank the team at John Wi ley & Sons Ltd for all thei r help with this new ed ition and to our fa mil ies for their conti nuing support

John B

Carters€-John Ca rter147@ntlworld com

Venetia A Sau

nderss€-s€-Authors are now reti red; both were previously at School of Pharmacy & Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK

Preface to First Edition

Virology is a fascinating and rapidly developing subject, and is worthy of study purely because viruses are interesting! Furthermore, virology is a branch of science that is of immense relevance to mankind for a host of reasons, not least of which are the threats to human health caused by viruses, such as HIV, hepatitis B virus, papillomaviruses, measles, and influenza vi ruses, to mention just a few There is a continuing need for trained virologists and it is hoped that this book will play a small role in helping to fulfill that need To a large extent the material in the book is based on virology taught at Uverpool John Moores University

This is not a textbook of fundamental virology, medical virology, veterinary virology, plant virology or of bacteriophages, but a bit of each of these! The general pattern

of the book is that principles of virology are covered earlier and applications are covered later There is no strict demarcation between the two, however, so the reader may be made aware of important applications while principles are being introduced

The first 10 chapters cover basic aspects of virology A chapter on methods used in virology comes early in the book, but could be skimmed to gain an overview of its contents and thereafter used for reference There is one chapter on each of the seven Baltimore classes, concentrating mainly on animal viruses There is a chapter devoted entirely to HIV and an extended chapter on phages, reflecting the renewed interest i n their biology and applications After a chapter on origins and evolution of viru ses, there follow five chapters covering various aspects of applied virology, including vaccines and antiviral drugs The final chapter is on prions, which are not viruses but are often considered along with the viruses

Each chapter starts with s"Dt-bAt a glance,s"DK a brief summary with the dual aim of giving a flavor of what is coming up and providing a revision aid Each chapter ends with a list of learning outcomes and a guide to further reading in books and journals The references are mainly from the twenty-first century, but there is a selection of important papers from the last century

The book has a web site (www.wiley.com/go/carter) where you can find:

• many references additional to those in the book;

• links to the journal references (to the full text where this is freely available, otherwise to the abstract);

• links to virology web sites;

• self-assessment questions and answers for each chapter, to reinforce and extend concepts developed in the book

A key feature of our book is a standard color code to differentiate various types of nucleic acid and protein molecules in the diagrams The color code is explained in

the following pages It is appreciated that color coding may be of limited value to individuals who have difficulty i n d ifferentiating colors, so we have also labeled many of the molecules

A number of virus replication cycles are described and the reader should be aware that these are models based on evidence to date; the models may have to be modified in the light of future evidence We present the virus replication cycles as fitting within a general framework of seven steps:

1 Attachment of a virion to a cell

2 Entry into the cell

3 Transcription of virus genes into mRNAs

4 Translation of virus mRNAs into virus proteins

5 Genome replication

6 Assembly of the virus proteins and genomes into virions

7 Exit of the virions from the cell

We hope that this helps in appreciating how virus replication fits into a general pattern, and i n comparing the replication cycles of different types of virus For some groups of viruses the framework has to be modified, and we make clear when that is the case

If you come across an unfam iliar term please consult the Virolog istss"f> ™ Vocabulary at the back of the book This glossary i111cludes not only virology-specific terms, but also a selection of terms from cel l biology, m olecular biology, immunology, and medicine

A list of abbreviations that are used throughout this book appears on the following pages

We wish to thank the many people who have made the production of this book possible We thanl< all those who supplied images and those who gave permission for the use of their images; we are especially g rateful to David Bhella, Tom Goddard, Kathryn Newton, and Jean-Yves Sgro Thanks also to Robert Carter for assistance with images We acknowledge the contributions of the many students who have acted as guinea pigs for our teaching materials and who have provided

us with feedback Grateful thanks also to those who reviewed material for the book and provided valuable feedback We are sorry that we were unable to include all the topics suggested, but if we had done so the book would have run to several volumes! Many thanks to Rachael Ballard and all at John Wiley & Sons Ltd who helped the book come to fruition Finally, thanks to our families for their support and for their patience during those many hours we spent ensconced in the study

We hope you find the book useful and we would be interested to hear what you think of it We have tried to ensure that there are no errors, but it is probable that some h.ave slipped through; if you come across any errors please inform us

John B Carter

John.Carter! 47@ntlworld.com

Venetia A Sau nders

School of Pharmacy & Biomolecular Sciences, Liverpool John Moores Un iversity, Byrom Street, Liverpool, L3 3AF, UK

Abbreviations Used in This Book

(+) DNA plus strand (positive strand) DNA

( - ) DNA minus strand (negative strand) DNA

(+) RNA plus strand (positive strand) RNA

( - ) RNA minus strand (negative strand) RNA

AIDS acquired immune deficiency syndrome

AP-1 activator proteii n 1

C term inus carboxy terminus

cccDNA cova lently closed circular DNA

CPE cytopathic effect

DIP defective interfering particle

DNA deoxyribose nucleic acid

DTR direct terminal repeat

E early

E F elongation factor

ERV endogenous retrovirus

E coli Escherichia coli

Ff F-specific filamentous

GFP green fluorescent protein

gp (1) g lycoprotein (as in HIV-1 gp120)

(2) gene product (as in phage T4 gpS)

HAART highly active anti-retroviral therapy

HAV hepatitis A virus

HSV herpes simplex virus

HTLV- 1 human T-lymphotropic virus 1

ICSO 50°/o inhibitory concentration

ICTV International Committee on Taxonomy of Viruses

IRES internal ribosome entry site

ITR inverted terminal repeat

N terminus amino terminus

NF-KB nuclear factor kappa B

NK cell natural killer cell

NSP non-structural protein

ORF open reading frame

ori origin (replication)

PBS primer binding site

PCR polymerase chain reaction

pfu plaq ue-form ing unit

RF rep l i cative form

RI rep l icati ve i ntermed iate

RNA ri bose nucleic acid

RN Ai RNA interference

RNase H ri bonuclease H

rRNA ri bosom al RNA

RT-PCR reverse tra nscri ptase-polymerase cha i n reaction

s Sved berg unit

SARS severe acute respi ratory synd rome

SIV si m ia n i m m unodeficiency vi rus

S p l sti mul atory protein 1

SS si ng le-stra nded

T thymi ne

T antigen tu mor antigen

TCI DSO vi rus dose that i nfects 50°/o of tissue cultures

TK thymidine ki nase

tRNA transfer RNA

TSE transmissi ble spongiform encephal itis

vCJ D vari ant Creutzfeldt-Ja kob d isease

VP vi rus protei n

VPg vi rus protein, genome l i nked

Greek letters used i n th is book

Color Coding fo r Molecules

With the ai m of maxi m i zi ng cla rity of the d iagrams the following standard color code is used to depict mol ecu les

o ia

CHAPTER 1

Vi ruses a nd Thei r Im porta nce

CHAPTER 1 AT A GLANCE

Photogra phs reproduced with perm ission of

1 World Health Organ ization

2 From U mesha ppa et al (201 1 ) Veteri nary I m m u nol ogy and I m m unopathol ogy,

14 1 , 230 Reproduced by permission of Elsevier and the authors

3 Ma cfarlane a nd Rob i nson ( 2004) Cha pter 1 1 , M i crobe-Vector Interactions i n Vector-Borne Diseases, 63rd Sym posi u m of the Society for General M i crobiology,

Ca mbridge U niversity Press Reprinted with perm ission

1 1 VI RU SES ARE U BIQUITOUS ON EARTH

Vi ruses i nfect al l cel l ular l ife forms : eu karyotes (vertebrate ani ma ls, i nvertebrate

an i mals, plants, fun g i ) and prokaryotes ( bacteria and archaea) The vi ruses that

i nfect bacteri1a are often referred to as bacteriophages, or phages for short

The presence of viruses is obvious in host organ isms show i n g sig n s of d isease Many healthy organisms, however, are hosts of non-pathogenic vi rus i nfections, some of which are active, while some are q uiescent Furtherm ore, the geno mes of many organ isms contain remnants of ancient virus genomes that integ rated i nto thei r host genomes long ago As well bei ng present with i n the i r hosts, vi ruses are

al so fou nd i n soi l, ai r and water Many aq ueous envi ronments contai n very high concentrations of viruses that i nfect the organ isms that l ive i n those environments There is a strong correlation between how i ntensively a species is stud ied and the

n u m ber of vi ruses found in that species Our own species is the subject of most attention as we have a vested i nte rest i n learn i n g about agents that affect ou r health It is not surprisi ng that there are more vi ruses known that i nfect man ki nd than any oth e r speci es, and n ew h u m an vi ruses co nti n u e to be fo u n d Th e

i ntestinal bacteri u m Escherichia coli has also been the su bject of m uch study and many viruses have been found in th is species If other species received the same

a mou nt of attention it is l i kely that many would be found to be hosts to si mi lar

n u m bers of viruses

It is undou bted ly the case that the vi ruses that have been di scovered represent only a ti ny fracti on of the vi ruses on the Ea rth Most of the know n pla nts, ani malls, fungi, bacteria and a rchaea have yet to be i nvestigated for the presence of vi ruses, and new potential hosts for vi ruses conti nue to be discovered Furthermore, the

a na lysis of D N A fro m n a tu ra l e nviron m e n ts poi nts to the existe n ce of m a ny bacterial species that have not yet been isolated i n the laboratory; it is l i kely that these Bbf-bnon-cultiva ble bacteria s"bK: are also hosts to vi ruses

1 2 IREASONIS FOR STU DYIN G VI RUSES

1.2 1 Some viru ses ca use d isease Viru ses a re i m porta nt agents of many human d iseases, ra n g i ng from the trivia l (e.g com mon colds) to the letha l (e g ra bies), and vi ruses also play roles i n the develop ment of several types of cancer As wel l as ca usi ng i nd ividuals to suffer,

vi rus di seases ca n also affect the wel l-bei ng of societi es Smallpox had a g reat

i m pact in the past a nd AI DS is having a g reat i m pact today

There is therefore a req u i rement to und ersta nd the nature of vi ruses, how they replicate and how they ca use disease Th is knowledge perm its the devel opment of effective means for prevention, d iag nosis, and treatment of virus diseases th rough the production of vaccines, diag nostic reagents and a nti-vi ral d rugs Vacci nes, such

as rotavirus a nd measl es vacci nes, have saved mill ions of l ives and i m proved the

q u a lity of l ife fo r m i l lions m o re S m a l lpox has bee n eradicated as a res u lt of vacci nation Anti-vi ral d rugs, such as those used agai nst H IV a nd herpes si m plex

vi rus, play m ajor roles i n the treatment of i nfectious disease Med i cal appl ications therefore constitute major aspects of the science of vi rology

Ma ny vi ruses cause disease i n do mestic a n i m a l s (such as cattle, sheep, dogs,

po ultry, fish, a nd bees ) a nd in wild a n i m a l s (such as red sq u i rrels a n d seals) Vacci nes are used to control some of these diseases, for exa m ple foot and mouth disease and bl uetongue Crop pla nts are also hosts to l a rge n u m bers of vi ruses, such as ri ce yel low mottle vi rus and cucu m ber mosaic vi rus, a vi rus with a very wide host ra nge These, and other plant viruses, can cause devastati ng outbreaks

of d i sease i n crop plants, with signifi cant i m pact on the q uantity and q ual ity of food produced

Another a rea where vi ruses can ca use econo m i c da mage is i n those i n dustries where the prod ucts resu lt from bacterial fermentatio n In the dairy i ndustry phages can d estroy the lactic acid bacteria used to prod uce cheese, yog urt, and other m i l k prod ucts, while other phages can destroy Corynebacteri u m species used i n the

i nd ustri al prod ucti on of am i no acids

1 2 2 Some vi ruses are usefu ll

S o m e v i ru se s a re st u d i e d b e cau se th ey have u sefu l c u rre n t o r pote n tial applications

• P h a g e ty p i n g of bacteria So m e g ro u ps of bacte ria, such as so m e Sal monella speci es, are classified into strai ns on the basi s of the spectru m of phag es to wh ich they are suscepti ble Identificati on of the phage types of bacte rial isolates can p rovi de usefu l e p id e m i olog ical i nfo rmation d u ri n g outb reaks of disease caused by these bacteria

• Sources of enzymes A number of enzymes used in mol ecular biology are

vi rus enzymes Exam ples i nclude reverse transcriptases from retrovi ruses and RNA polymerases from phages

• Pesticid es So m e i n sect pests are control led w ith bacu l ovi ruses, and myxoma virus has been used to control rabbits

• Anti-bacterial agents In the mid-twentieth century phages were used to treat some bacterial infections i n humans Interest waned with the di scovery

o f a n t i b i o t i c s , b u t h a s b e e n re n e w e d w i t h t h e e m e r g e n c e o f anti biotic-resistant strai ns of bacteria

• Anti -cancer ag ents Genetical ly modified strai ns of vi ruses, such as herpes

si m p lex vi rus and vacci n ia vi rus, are being i nvestigated for treatment of cancers These strai ns have been modified so that they are abl e to infect and destroy specific tumor cel ls, but are unable to i nfect normal cells

• Gene vectors for protein prod uction Vi ruses, such as certai n baculovi ruses and adenovi ruses, are used as vectors to take genes i nto an i mal cells

g rowi ng i n cu ltu re Thi s tech nology is used to make cel l s prod uce usefu l protei ns, such as vacci ne com ponents Some genetical ly modified cel ls are used for mass prod uction of proteins

• Gene vectors for treatment of genetic diseases Ch i l d ren with the genetic

d isease Severe Combi ned I m m unodeficiency ( baby in the bubble synd rome) have been s uccessfu l l y treated using retrovi ruses as g e ne vecto rs The

vi ruses i ntroduced i nto the ch ildrensT.>™s stem cells a non-mutated copy of the m utated gene (Section 17 5)

1 2 3 Virus studies have contri buted to knowledge

M uch basic knowledge o f molec u lar biology, cel l biology, a n d cancer has been derived from stud ies with vi ruses Here are a few examples

publ i shed in 1952, used phage T2 and E coli to provi1de strong1 evi dence that genetic material is com posed of DNA

• The fi rst enhancers to be characteri zed were in genes of si mian vi rus 40 ( SV40)

• The fi rst transcri pti on factor to be characterized was the transplantation (T) antigen of SV40

• The fi rst nuclear local izati on si g nal of a protein was identifi ed in the T antigen of SV40

• Introns were discovered d u ring stud ies of adenovirus transcri ption

• The role of the cap structu re at the SsnI end of eu karyotic messenger RNA ( m RNA) was discovered d uri ng stud ies with vacci nia vi rus and a reovi rus

• The fi rst i nternal ri bosome entry site to be d iscovered was fou nd in the RNA of pol iovi rus

• The fi rst RNA pseudoknot to be discovered was in the genome of tu rn ip yel low mosaic virus

l 3 TH E NATU RE OF VI RUSES

1 3 1 Vi ruses a re sma l l pa rticles

Evidence for the existence of very smal l i nfecti ous agents was fi rst provided i n the late n i n ete enth ce ntury by two scie n tists w o rki n g i n d e p e n d e n tly : M arti n us Beijeri nck in Hol land and D i m itri Ivanovski i n Russi a They made extracts fro m diseased plants, wh ich we now know were i nfected with tobacco mosaic vi rus, and passed the extracts throug h fi ne filters The filtrates contai ned an agent that was able to i nfect new plants, but no bacteria could be cu ltu red from the fi ltrates The agent remai ned i nfective through several transfers to new plants, el i m i nati ng the possibility of a toxi n Beijeri nck cal led the agent a snl-bvi russni< and the term has been i n use ever si nee

At around the same ti me, Friedrich Lr�effler and Pau l Frosch transmitted foot and mouth disease fro m an i mal to an i mal i n i noculum that had been hig hly d i l uted A few years later Walter Reed and James Carroll demonstrated that the causative agent of yel low fever is a fi lterable agent

Fig ure 1 1 g ives some i ndication of the size of these agents, which are known as

vi rus particl es or vi rions The vi rion of a herpesvi rus, which is a fai rly large virus, is about ten m i l l ion ti mes smaller than a large bal loon, while the bal loon is smal ler than the Earth by the same factor The vi rions of most vi ruses are too small to be seen with a l i g ht microscope and can be seen only with an electron microscope ( Fi g u re 1 2 )

Fig ure 1 1 Com parative sizes o f a herpesvi rus particle, a bal loon, and the Earth A larg e bal loon is about ten m i llion ti mes larger than a herpesvi rus particle, wh i l e the Earth is larger than the balloon by the same factor

XI

0

Fig ure 1 2 Transm ission el ectron microsco pe Th is is a microscope i n wh ich the

i mage is formed by electrons transmitted through the speci men

Sou rce : Photograph courtesy of J EOL

The un its i n which vi rions are normally measu red are nanometers ( 1 n m = 10s€'9

m ) Although vi rions are very small, their d i mensions cover a large range Amongst the smal lest are parvoviruses ( Figure 1 3), with diameters about 20 n m , while the megav i rus and m i m ivirus ( m icrobe-m i m icki n g virus), isolated from ame bae, are amongst the largest

Fig ure 1 3 Vi rions of m i m ivi rus, one of the largest vi ruses, and a parvovi rus, one of the smal lest vi ruses

Sou rce : El ectron m icrograph of m i m ivi rus from Cl averie et al ( 2009) J ou rnal of Invertebrate Pathology, 101, 172 Reproduced by perm ission of Elsevi er and the authors Electron microg raph of parvov1i ru s by permissi on of Professor M Stewart McN ulty and The Ag ri- Food and Biosci ences Institute

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P M'O\ ' ir

Vi rology is therefore concerned with very smal l particles, though often with very large n u m bers of those particles ! A concentrated suspension of vi rio ns m i g ht contai n 1 0 1 2 vi rions/ml A sing le vi rus-infected cell might produce 1 05 vi ri ons A person infected with HIV might produce 101 1 virions i n a day

Vi rions are not cel l s They d o not contai n organelles, except for the vi rions of arenaviruses, which contain cel l ri bosomes that were packaged when the vi rions were assem bled

1 3.2 Vi ruses have genes The vi rion contains the genome of the vi rus Whereas the geno mes of cel ls are com posed of doubl e-stranded DNA, there are four possi bil ities for a vi rus genome:

• double-stranded DNA ( dsDNA);

• single-stranded DNA ( ssDNA);

• double-stranded RNA ( dsRNA);

• single-stranded RNA (ssRNA)

The genome is encl osed i n a protein coat known as a capsi d The genome plus the caps id , p l u s other co m ponents i n many cases, constitute the v i rion A m ajor function of the capsid is to protect the genome unti l it can be del ivered into a cel l in which it can replicate

General ly, v i rus genomes are m uch smal ler than cel l genomes and the q uestion arises as to how vi ruses encode al l their req u i rements in a smal l genome Vi ruses achi eve th is in a number of ways

• Vi ruses use host cel l p roteins The g enomes of large viruses d u pl i cate some fu ncti ons of the host cel l , but the smal l vi ruses rely very heavi ly on host cel l fu ncti ons There is, however, one functi on that an RNA vi rus must

e n co d e , no m atte r h o w s m a l l its g e n o m e T h at fu n ction i s an R N A polymerase, because cel ls d o not encode enzymes that can replicate vi rus RNA A significant proportion of an RNA vi rus genome is taken u p with the gene for an RNA polym erase

• Vi ruses cod e effi ciently There m ay be overla p p i n g genes a n d g e nes encoded with i n genes The s m a l l genome of hepatiti s B vi rus is a g ood exa mple (see Secti on 19.6) In vi ruses most, or all, of the genome encodes

p rotei ns, i n contrast to ma m mals where only a bout 1 5°/o of the genome encodes protei ns

• Many vi rus proteins are multifunctiona l Some vi rus proteins have m u ltiple enzyme activities, for example the rhabdovirus L protei n repl icates RNA, ca ps

a nd polyadenylates m RNA, and phosphoryl ates another virus protei n Some

vi rus proteins can bind to cel l proteins with roles in i m mune responses, a nd hence i nterfere with i m munity of the host

1 3 3 Viruses are pa rasites

Vi ruses differ from cells in the way in which they m u lti ply A new cell is always formed di rectly from a pre-existi ng cel l, but a new virion is never formed d i rectly fro m a pre-existi ng virion New virions are formed by a process of replication, which takes place i nside a host cel l and i nvolves the synthesis of components fol lowed by thei r assem bly i nto vi rions

Vi ruses a re therefore parasites of cel ls, and are dependent on their hosts for most

of their req uirements, i ncl uding :

• buildi ng-blocks such as amino acids and nucleosides;

• protei n-synthesizing machi nery ( ri bosomes);

• energy, in the form of adenosine triphosphate

A vi rus mod ifies the intracellu lar envi ron ment of its host in order to enhance the effi ciency of the repl icati on process It does this by prod ucing protei ns, a nd in some cases smal l RNAs, that i nteract with cel l co mponents It has been demonstrated that the p roteins of a vi rus ca n i nte ract with h u n d red s of host p roteins i n a n

i nfected cel l The expression of thousands of host genes ca n be affected, with some genes up-regulated and some genes down-reg ul ated

Viru s- i n d uced m odifications to a h ost cel l m ig h t i n c l ude the form ation of n ew membranous structu res or a red uced i m m une response So me larg e phages of photosynthetic bacterial h osts encode p rotei ns that e n h a nce photosynthesis, thereby proba bly boosti ng the yields of vi rus fro m the cells

A poi nt has now been reached where the nature of vi ruses can be su m marized in a concise defi nition ( see the box)

VIRUS DEFI N ITIO N

A vi rus is a very smal l, non-cel lular parasite of cells Its geno me, wh ich is com posed

of either DNA or RNA, is enclosed i n a protein coat

1 3 4 Some vi ruses a re dependent on other viruses

Some viruses, known as satel lite vi ruses, are u nable to replicate unless the host cel l

is i nfected with a second vi ru s, referred to as a hel per vi rus The hel per virus

provides one or more functions missing from the satellite virus, thereby enabling the latter to complete its replication cycle Examples are given in Table 1 1

Table 1 1 Examples of satellite/helper viruses

Bnt-bViruses belong to biology because they possess genes, replicate, evolve, and are adapted to particular hosts, biotic habitats, and ecological niches However, they are nonliving i nfectious entities that can be said, at best, to lead a kind of borrowed life s'Di<

Marc van Regenmortel and Brian Mahy (2004)

s'Dt-bltB'D™s life, Jim, but not as we know it!s'DK

Dr McCoy speaking to Captain Kirk of the Starship E nterprise, Star Trek

1 3 5 Are viruses living or nonliving?

There is an ongoing debate as to whether viruses are living or nonliving; the view taken depends on how life is defined Viruses have genes and when they infect cells these genes are replicated, so i n this sense viruses are living They are, however, very different to cellular life forms, so Dr McCoys'D™s stock phrase (see the box) on finding new life forms i n the galaxy could be applied to viruses When viruses are outside their host cells they exist as virions, which are i nert, and could

be described as nonliving, b u t viable bacterial spores are inert and are not considered to be nonliving You might form your own view as to whether viruses are living or nonliving as you progress through this book

When Beijerinck selected the word Bot-bviruss'DK he chose the Latin word for poison This term has now been in use for over a century and virology has developed into a huge subject More recently, the term virus has acquired further meanings Computers are threatened by infection with viruses that can be found in the wild once they have been released by their authors These viruses are specific for certai n file types Infected files may be put on several web sites and a virus epidemic may ensue Another use of the term virus is exem p lified in John Humphryss'D T M book Lost For Words, in which he talks about the deadly virus of management-speak i nfecting language All the italicized terms in this paragraph are also used in the context of the viruses that are the subject of this book

1 4 THE REMAINDER OF THE BOOK

Having outlined the nature of viruses and why they are important, the remainder of the book will examine many aspects of fundamental and applied virology The early

cha pters cover pri nci ples, such as the structu re of virions, virus repl ication, and the classifi cati on of vi ru ses Th e re a re then te n cha pters d evoted to reviews of

pa rticu lar g roups of vi ruses, where both pri nci ples a nd applications of vi rology are covered Towards the end of the book we consider specific applications of virol ogy,

i ncl uding viral vacci nes and anti -vi ral drugs The fi nal chapter is devoted to prions, which are not vi ruses !

It is i m porta nt to poi nt out that m uch of vi rology is concerned with characteristics

of the proteins a nd nucleic acids of vi ruses, and with i nteracti ons between these molecules and the protei ns and n ucleic acids of cel l s ( Figure 1.4 ) Most of these interactions re ly on specific bind i n g between th e m o lecules We s ha l l also be

d iscussi ng cellu lar structures, a nd processes such as transcription, tra nslati on, a nd

D N A re pl icatio n A good backg ro u n d i n m o l ecular biology a nd cel l b i o logy is therefore essential; some useful sources of i nformation for pl uggi ng a ny gaps can

be found under Sources of further information

Fig ure 1 4 Interacti ons between vi rus molecu les and cel l molecu les

Vim pr t in

JI p tei

LEARN I N G OUTCOM ES

After review of th is cha pter you should be able to :

• d iscuss reasons for studyi ng viruses;

• explain how vi ruses differ from cell ular organisms;

• defi ne the term sibt-bvi rus sibi<

SOURCES OF FU RTH E R I N FORMATION

Cell biology and molecular biology books

Alberts, B et a l ( 2008) Molecular Biology of the Cel l , 5th ed ition, Garland

Bo l s o v e r, S R et a l ( 2 0 1 1 ) C e l l B i o l o g y: A S h o rt C o u rs e , 3 rd e d i t i o n ,

Wi ley-Blackwel l

Ka rp, G ( 20 10) Cel l Biol ogy, 6th ed ition, Wiley

Lod ish, H F et a l ( 2008 ) Molecular Cel l Biology, 6th ed ition, Freeman

Pol lard, T D a nd Earnshaw, W C ( 2008 ) Cell Biol ogy, 2nd ed ition, Sa unders

Watson, J D et a l ( 2007) Molecular Biology of the Gene, 6th edition, Pea rson Weaver, R F (20 1 1 ) Molecular Biology, 5th edition, McGraw-H i l l

H i storical paper

Hershey, A D a nd Chase, M ( 1952) Independent functi ons of viral protein a nd nucleic acid in g rowth of bacteri ophage Journal of General Physiology, 36, 39st,," 56 Recent papers

Breitba rt, M and Rohwer, F (2005 ) Here a vi rus, there a vi rus, everywhere the

sa me vi rus? Trends In M icrobiology, 13, 278st,,"284

E n q u i st, L W ( 2009 ) V i rology in the 2 1 st centu ry J o u rn a l of V i ro l ogy, 8 3 , 5296Bn" 5308

Hu nter, W et a l ( 20 1 0) Large-scale field application of RNAi technology red uci ng Israel i acute paralysis virus disease i n honey bees (Apis mel lifera, Hymenoptera :

Ap idae) PLoS Pathogens, 6 ( 1 2) : e 1001 1 60

Ku ta te l a d ze, M a n d A d a m i a , R (2 0 1 0 ) B a cte ri o p h a g e s a s pote n ti a l n e w therapeuti cs to replace or supp lement a nti b ioti cs Trends i n Biotechnol ogy, 28,

Young, L S et al ( 2006) Vi ral gene thera py strateg ies : fro m basi c science to

cl in ical appl ication Journal of Pathol ogy, 208, 299s1J" 3 1 8

CHAPTER 2 Methods Used i n Vi rology CHAPTER 2 AT A GLANCE

1 Plaques formed by i nfl uenza vi rus (large plaq ues) a nd can ine adenovirus- 1 (smal l plaq ues) i n M DCK cell s From M urata et a l ( 20 1 1 ) Vacci ne, 29, 3 1 55 Reprod uced

by perm ission of Elsevier and the a uthors

2 Plaques formed by phage MS2 in Escherichia col i cel ls

3 Separation of reovi rus fu l l vi rions a nd em pty vi rions in a cesi u m chloride g rad ient From Mendez et al ( 2000) Jou rnal of Virologi ca l Methods, 90, 59 Reproduced by permissi on of Elsevier and the a uthors

4 Fluorescence microscopy of cells expressing a cytomega lovirus protein From Li u

et a l (2012) Vi rus Research, 166, 3 1 Reproduced by perm ission of El sevier

5 An endosom e (labeled with a red fl uorophore) contai ning ra bies vi rus protei n (labeled with 9reen fl uorescent protei n ) i n the cytoplasm of a n i nfected cel l From Finke a nd Conzel mann ( 2005) Virus Research, 1 1 1, 1 20 Reprod uced by permission

of Elsevi er Li m ited and the authors

6 Neg atively stai ned particles of an orbivirus The bar represents SO n m From Atto u i et a l ( 20 0 5 ) J o u rn a l of G e n e ra l Vi ro logiy, 8 6 , 3409 Re p rod u ced by permissi on of the Society for Genera l M i crobiology and the a uthors

7 Reconstructed i magies from cryo-electron m icroscopy : measles vi rus nucleocapsid ( l eft), echovi rus type 12 bound to a frag ment of its cell receptor ( right) Cou rtesy of

Dr David Shella ( M RC Vi rologiy Un it, Glasg ow} Rei nterpretations of data i n Bhel la et

al ( 2004) Journal of Molecular Biology, 340, 3 19 ( by perm ission of El sevi er Li m ited ) and Shella et al ( 2004) Journa l of Bi ological Chemistry, 279, 8325 (by permission

of The Ameri can Soci ety for Biochemistry and Molecu lar Biology)

8 Bl uetongue virus proteins From Bel houchet et al ( 20 1 1 ) PLoS ON E, 6 ( 1 0) : e25697

9 Orbivi rus RNA seg ments separated by electrophoresis through an agarose gel From Atto ui et al ( 2005 ) Journa l of General Vi rology, 86, 3409 Rep rod uced by permissi on of the Society for Genera l M i crobiology and the a uthors

1 0 Analysis o f va ricel la-zoster virus transcri ption using m icroa rrays From Ken nedy

et al ( 2005) Journal of General Vi rology, 86, 267 3 Reproduced by perm ission of the Soci ety for General M icrobiol ogy and the authors

VP1 VP2 VP3 VP4 VPS VP6 VP7

of the m M u ch of the focus of th i s cha pter is on m eth ods that a re u n i q ue to

virology Many of these methods are used not only in virus research but also i n the diagnosis of virus diseases of humans, animals, and plants

Initially this chapter could be skimmed to gain a n overview of its contents and thereafter used for reference Details of the methods outlined here, and of other methods i mportant in virology, fill many volumes, some of which are listed at the end of the chapter

2 2 CU LTIVATION OF VIRUSES

Virologists need to be able to produce the objects of their study, so a wide range of procedures has been developed for cultivating viruses Virus cu ltivation is also referred to as propagation or growth, all terms borrowed from horticulture! A few tech niq ues have been developed for the cultivation of viruses in cel l-free systems, but in the vast majority of cases it is necessary to supply the virus with appropriate cells in which it can replicate

Phages are supplied with bacterial cultures and plant viruses may be propagated using specially cultivated pla nts, tissue cultures or protoplasts (plant cells from which the cell wall has been removed) Animal viruses may be propagated in whole organisms, such as mice, which may be genetically modified Transgenic mice are used in studies of hepatitis B and hepatitis C viruses Some vertebrate viruses are propagated in eggs contain i ng chick embryos ( Figure 2 1 ) , while some insect viruses are propagated in insect larvae For the most part, however, animal viruses are grown in cultured animal cells

Figure 2.1 Cultivation of viruses in eggs containing chick embryos

Source: Photograph courtesy of Taronna Maines and Greg Knobloch, Centers for Disease Control and Prevention

allantoic cavity

chorioal lantoic membrane

2.2 1 Animal cel l culture

Animal cell culture techniques are well developed and most of the cells used are from continuous cell lines derived from huma ns and other animal species Continuous cell lines consist of cells that have been im mortalized, either in the laboratory or in the body (Figure 2.2); they can be subcultured indefinitely The

Hela cel l line is a wi dely used conti nuous cel l l ine that was i niti ated i n the m i ddle of the twentieth centu ry from cel ls ta ken from a cervical ca rci nom a

Fi gure 2 2 Derivation of continuous cell l i nes of human a n d a n i mal cel ls Most types

of cel l taken from the body do not g row wel l in cu lture If cells fro m a pri mary culture can be su bcultured they are g rowing as a cell l ine They ca n be subcultu red only a fi nite n u m ber of ti m es un less they are i m morta l ized, i n which case they can

be su bcu ltu red i n defi n ite ly as a conti n u ous ce l l l i n e Ca ncer cel ls a re a l ready

i m morta l i zed, and conti n u ous cell l i nes m ay be esta bl i shed fro m these without further treatment

Figure 2.3 Cel l cu lture flasks, dishes, and pl ates

Sou rce : Photogra phs of TPP cell culture prod ucts courtesy of MIDSCI

Contami nation with bacteri a and fu ngi can ca use major prob lems i n cel l cu lture work; i n order to m i ni mize these problems work is normally done in a steri le cabi net ( Figure 2 4) a nd most med ia contai n antibiotics Many cel l types req u i re a rel atively high concentrati on of carbon dioxi de, which ca n be su ppl ied in a speci al incu bator Figure 2.4 Cel l cultu re work Preca utions to avoid conta m i nation i nclude working in

a steri le cabi net and weari ng gl oves and mask

Sou rce : Cou rtesy of Sanofi Pasteursn"Copyrig ht

2 3 ISOLATION OF VI RU SES

Many vi ruses can be isol ated as a result of the i r abil ity to form d iscrete visi ble zones ( plaques) in layers of host cel ls If a confl uent layer of cells is inoculated with an amount of vi rus to infect a small proporti on of the cells, then plaq ues may form