PART V

VIROLOGY

The Short Textbook ofMedical

Microbiology(Including Parasitology)

The Short Textbook ofMedical

Microbiology(Including Parasitology)

Tenth Edition

Satish Gupte MD

Professor and HeadDepartment of Microbiology

Gian Sagar Medical College and HospitalRamnagar (Banur), Distt. Patiala. (Punjab)

Ex-Professor and HeadDepartment of Microbiology

Adesh Institute of Medical Sciences and Research CentreBathinda (Punjab)

andGovt. Medical College and Associated Hospitals

Jammu 180001, J & K, India

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The Short Textbook of Medical Microbiology (Including Parasitology)

© 2010, Mrs Jyotsna Gupte

All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmittedin any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the priorwritten permission of the author and the publisher.

This book has been published in good faith that the material provided by author is original. Every effort ismade to ensure accuracy of material, but the publisher, printer and author will not be held responsible forany inadvertent error(s). In case of any dispute, all legal matters are to be settled under Delhi jurisdictiononly.

First Edition : 1982Second Edition: 1984Third Edition: 1986Fourth Edition: 1989Fifth Edition: 1993Indonesian Edition: 1990Sixth Edition: 1995Seventh Edition: 1999Eighth Edition: 2002Ninth Edition: 2006Tenth Edition : 2010

ISBN : 978-81-8448-840-1

Typeset at : JPBMP typesetting unitPrinted at :

Foreword

Dr Satish Gupte has done wonderful job in updating his book to tenth edition. Thesimplicity of language and scholarly work done in the book shows the effort of authorfor updating the book.

I wish more undergraduate and postgraduate students will read this book forupdating their knowledge and skills.

I wish the book to be a landmark in subject of Microbiology.

Dr AS Sekhon MDPrincipal

Gian Sagar Medical College and HospitalRam Nagar (Banur)

Distt. Patiala, PunjabIndia

GIAN SAGAR MEDICAL COLLEGE & HOSPITALRam Nagar (Banur), Distt. Patiala, Punjab-140601 (India)

Mobile : 9876900006, Telefax : 01762-507759

Preface to the Tenth Edition

Over 27 years back first edition of The Short Textbook of Medical Microbiology was brought out in 1982.Since then the volume is galloping while passing through silver jubilee year landing right into revised,enlarged and updated tenth edition decorated elegantly and colorfully with entirely fresh and new getup. Indonesian edition of this volume was published in 1990 with collaboration of Binarupa Aksaraunder the title MICROBIOLOGI DASAR earning fame and name in that region.

It has been kept in mind to condense and include the latest major meaningful advances in medicalmicrobiology in this volume. Distinct feature of this volume is inclusion of updated Medical Parasitologywith Entomology. All the chapters are thoroughly revised and updated as highlighted by esteemedreaders from time to time. New chapters are included namely; microbiology in the service of humanbeing, molecular techniques in microbiology, emerging and re-emerging infections and laboratory andhospital wastes. Also included in this volume: Biofilm, new methods of disinfection and sterilization,super antigen, Ogawa medium, adenosine deaminase activity, immunological investigations ofMycobacterium tuberculosis, Napah virus, pandemic of swine flu (H1N1) breakthrough 2009, laboratorydiagnosis of septicemia/bacteremia and infective endocarditis and Nobel prize in Medicine etc.

The challenge was to keep the volume comprehensive, profusely colored, beautifully illustrated andstill handy maintaining emphasis on the needs of MBBS and BDS students. Several colored figures andphotographs, tables and colored illustrations (adjusted appropriately between texts) are included toenhance and intensify the utility of this volume. The chapters pertaining to clinical microbiology areespecially addressed to budding doctors (interns and house surgeons/physicians) who usually faceproblems in collection and transportation of clinical samples for laboratory investigations.

It is the privilege to offer my sincere gratitude to my respected teachers: Professor TD Chugh, ProfessorUma Sabharwal, Professor RK Arya and Professor DR Arora for their blessings, encouragement, inspirationand good will. My special thanks to Professor Aruna Aggarwal, Professor BL Sherwal, Professor JagdishChander, Professor Prem Narwan, Professor Raj Kumar, Professor Anjum, Professor Bella Mahajan andmany others for their best wishes and valuable suggestions in upbringing the current edition of thisvolume.

I am thankful to Dr GM Warke of Hi Media Laboratory, Mumbai, for kind permission to use somephotographs.

I acknowledge the help rendered by faculty of Department of Microbiology, Gian Sagar MedicalCollege and Hospital, (Banur), Patiala in the preparation of this edition. Thanks to Mr Gurpreet Singh fortaking responsibility of secretarial work of this volume.

Professor AS Sekhon, Dean and Principal, Gian Sagar Medical College, (Banur), Patiala has encouragedme and enriched me with his good wishes to accomplish the commitment of bringing up this edition tothe contentment and expectations of readers. From the core of my heart, I am grateful to him indeed.

My sincere thanks to Shri Jitendar P Vij (Chairman and Managing Director), Jaypee Brothers MedicalPublishers (P) Ltd., New Delhi and his dedicated and dynamic team for publishing this book elegantlyand speedily.

In true sense I derived much inspiration and blessings from my mother, respected Late Smt. KaushalyaGupte and respected father Late Shri Charanjit Gupte. Also, I take opportunity to express my thanks toall my family members especially to my wife Mrs Jyotsna Gupte and dear son Anubhav Gupte whoactually helped me sincerely while I was simply engrossed in preparing and also during finalizing thismanuscript.

Comments, suggestions and constructive criticisms towards improvements of future editions of thisvolume are very much welcomed. In fact, they underline the shortcomings which are looked into andrectified in the interest of our esteemed readers.

“Gupte House” Satish Gupte60, Lower Gumat,Jammu 180016September, 2009

In India as also in other countries of the Third World, despite advances in the field of microbiology, therehas been an acute dearth of well-written textbooks on the subject for the undergraduate medical students.

The Short Textbook of Medical Microbiology attempts to fill that long-left gap. In spite of great deal ofexpansion in all branches of microbiology, I have tried to make it concise yet comprehensive, handy andup-to-date. The stress has been on contemporary medical microbiology’s relevance for today’s needs inour set-up. A large number of line diagrams and tables are included to simplify the points. Also, thelanguage has been simple, easy and straightforward. In line with our requirements more emphasis hasbeen laid on practical aspects. A strong point of the digest is a special stress on the mechanism of productionof disease by the microbes and laboratory diagnosis.

In writing this volume, I have considerably counted on the superb teaching of my teachers, namelyProf TD Chugh, Prof RK Arya and Prof (Mrs) Uma Sabharwal. The goodwill and wise counsels of Dr DRArora, Dr Ashok Malik, Dr (Mrs.) Santhosh Saini, Dr Satish Walia, Dr RL Kaul, Dr (Mrs) SudershanKumari Gupta and Dr Satish Sharma proved a source of much stimulation.

Lastly, I express my appreciation for the various favors extended by the family folk at various stagesof publication.

With this, let me hope that this little book makes its modest contribution by presenting the subject ofmedical microbiology in an easy, simple and straightforward form helping the undergraduates to imbibebasic and applied things on the subject.

“Gupte House” Satish Gupte60, Lower Gumat,JammuJuly 1982

Preface to the First Edition

Contents

Part IGeneral Bacteriology

1. Glossary of Microbiology .................................................................................................... 32. History and Scope ................................................................................................................ 103. Morphology of Bacteria ...................................................................................................... 164. Nutritional Requirements of Bacteria .............................................................................. 305. Bacterial Metabolism .......................................................................................................... 356. Media for Bacterial Growth ............................................................................................... 377. Classification and Identification of Bacteria .................................................................. 448. Sterilization and Disinfection ........................................................................................... 509. Infection................................................................................................................................. 57

10. Antimicrobial Therapy ....................................................................................................... 60

Part IIBacterial Genetics

11. Bacterial Genetics ................................................................................................................ 67

Part IIIImmunology

12. Immunity ............................................................................................................................... 8313. Antigen .................................................................................................................................. 9214. Antibodies—Immunoglobulins ........................................................................................ 9515. Antigen and Antibody Reaction ..................................................................................... 10116. The Complement System ................................................................................................. 10717. Structure and Functions of Immune System ................................................................ 11118. The Immune Response ..................................................................................................... 11719. Hypersensitivity ................................................................................................................. 12220. Immunohematology .......................................................................................................... 12921. Miscellaneous ..................................................................................................................... 132

Part IVSystematic Bacteriology

22. Staphylococcus ................................................................................................................... 14523. Streptococcus ...................................................................................................................... 15424. Pneumococcus ..................................................................................................................... 16225. Neisseria .............................................................................................................................. 16626. Corynebacterium................................................................................................................. 17127. Mycobacteria....................................................................................................................... 17628. Bacillus ................................................................................................................................ 190

x The Short Textbook of Medical Microbiology (Including Parasitology)

29. Clostridium.......................................................................................................................... 19430. Enterobacteriaceae ............................................................................................................. 20231. Pseudomonas ...................................................................................................................... 21632. Vibrio .................................................................................................................................... 21833. Campylobacter .................................................................................................................... 22234. Helicobacter Pyloridis ....................................................................................................... 22535. Brucella ................................................................................................................................ 22736. Pasteurella, Yersinia and Francisella .............................................................................. 23037. Hemophilus .......................................................................................................................... 23438. Bordetella ............................................................................................................................ 23739. Spirochaetes ........................................................................................................................ 23940. Miscellaneous Bacteria ..................................................................................................... 24641. Rickettsiae ........................................................................................................................... 25342. Chlamydiae ......................................................................................................................... 25843. Newer Bacteria ................................................................................................................... 26344. Microbiology of Oral Cavity ........................................................................................... 267

Part VVirology

45. General Characteristics of Viruses ................................................................................. 27146. Chemotherapy of Viral Diseases .................................................................................... 28047. Classification of Viruses .................................................................................................. 28648. Oncogenic Viruses ............................................................................................................. 28949. DNA Viruses....................................................................................................................... 29550. RNA Viruses ....................................................................................................................... 30251. Severe Acute Respiratory Syndrome (SARS) ............................................................... 31152. Avian Influenza (Bird Flu) ............................................................................................... 31353. Acquired Immune Deficiency Syndrome (AIDS) ....................................................... 31454. Miscellaneous Viruses ...................................................................................................... 320

Part VIMycology

55. Mycology ............................................................................................................................. 331

Part VIIParasitology

56. Introduction ........................................................................................................................ 35357. Protozoa ............................................................................................................................... 35758. Helminths ............................................................................................................................ 38359. Medical Entomology ......................................................................................................... 399

Part VIIIClinical Microbiology

60. The Normal Flora ............................................................................................................... 40961. Collection and Transport of Clinical Specimens ......................................................... 41162. Collection and Preliminary Processing of Specimens ................................................ 413

Contents xi

63. Diagnostic Microbiology—An Approach to Laboratory Diagnosis ........................ 41564. Rapid and Automation Methods in Diagnostic Microbiology ................................. 42765. Molecular Techniques in Microbiology ........................................................................ 42966. Serological and Skin Tests ............................................................................................... 43167. Microbiology in the Service of Human being .............................................................. 43468. Community Microbiology ............................................................................................... 43669. Emerging and Re-emerging Microbial Diseases ......................................................... 43870. Nosocomial Infections ...................................................................................................... 44171. Hospital and Laboratory Waste ....................................................................................... 44472. Diagnostic Virology .......................................................................................................... 44773. Emergency Microbiology ................................................................................................. 44974. Bacteriology of Milk, Air and Water .............................................................................. 451

Part IXAppendices

Appendices 1-15 ................................................................................................................... 459

Index ........................................................................................................................................ 467

45. General Characteristics of Viruses46. Chemotherapy of Viral Diseases47. Classification of Viruses48. Oncogenic Viruses49. DNA Viruses50. RNA Viruses51. Severe Acute Respiratory Syndrome (SARS)52. Avian Influenza (Bird Flu)53. Acquired Immune Deficiency Syndrome (AIDS)54. Miscellaneous Viruses

Virology

45 General Characteristicsof Viruses

Viruses are unicellular, ultramicroscopic par-ticles containing either RNA or DNA, whichreproduce inside living cells, pass throughfilters that retain bacteria and are covered bya protein coat.The general properties of viruses are (Table45.1):

i. Do not possess cellular organization.ii. Contain one type of nucleic acid, either

RNA or DNA but never both.iii. Lack enzymes necessary for protein and

nucleic acid synthesis and so dependupon synthetic machinery of host cells.

iv. They multiply by complex process andnot by binary fission.

v. They are unaffected by antibiotics.vi. They are sensitive to interferon.

Evolutionary Origin of Viruses

The origin of virus is not known. Three hypo-theses have been proposed:1. Viruses became parasites of primitive cells

and the two evolved together. This maybe the reason that many viruses todaycause no host cell damage and remainlatent in the host.

2. Viruses evolved from parasitic bacteria.While this possibility exists for other

obligatory intracellular organisms, e.g.chlamydiae, there is no evidence thatviruses evolved from bacteria.

3. Viruses may be the component of host cellsthat become autonomous. They resemblegenes that escape the regulatory control ofhost cell. There is evidence that some tumorviruses exist in host cells as unexpressedgene. The likelihood of it is great becausesome small viruses are evolved in thisfashion. However, large viruses (poxand herpes group) show very limitedresemblance to host cell DNA.

Rivers’ postulates: At the time Koch’s postu-lates were formulated true viral pathogenswere unknown. In 1937, TM River created asimilar group of rules to establish causativerole of viruses in disease Rivers’ postulates areas under.1. The viral agent must be found in the host’s

body fluid at the time of the disease or inthe cells showing lesions.

2. The viral agent obtained from the infectedhost must produce specific disease in asuitable healthy animals or plant or provideevidence of infection in the form of anti-bodies against the viral agent. It isimportant to note all host material used forinoculation must be free of any bacteria orother microorganisms.

TABLE 45.1: Properties of viruses with comparison to prokaryotes

Cellular Growth in Binary Both Ribo- Sensitivity Sensitivityorgani- inanimate fission RNA somes to tozation media and DNA antibiotics interferon

Bacteria + + + + + + –Mycoplasma + + + + + + –Rickettsiae + + + + + + –Chlamydiae + + + + + + –Virus – – – – – – +

272 Virology

3. Similar material from such newly infectedanimals or plants must in turn be capableof transmitting the disease in question toother hosts.

Morphology

Size: Viruses vary widely in size. The largestamong them is pox virus measuring about 300nm. The smallest viruses is foot and mouthdisease virus measuring 20 nm.

The methods of estimating the size of virusparticles are:

i. Collodion membrane filter of gradedporosity.

ii. Ultracentrifugation.iii. Electron microscope.

Shapes: Some viruses have characteristicshape, e.g. rabies virus has bullet shape, poxviruses are brick-shaped, tobacco mosaic virusis rod-shaped, bacteriophage has head and tail,like sperm, influenza or polio viruses arespheroidal and so on.

Structure and symmetry: Viruses have centralcore of nucleic acid which is either RNA orDNA but never both. This central core ofnucleic acid is covered by protein coat calledcapsid. The capsid itself is composedof number of subunits called capsomere(Fig. 45.1). The capsomere may be arranged asunder:

i. Around coiled nucleic acid which isknown as helical arrangement.

ii. As cubes around spheroidal nucleic acidknown as icosahedral arrangement.

iii. Some viruses do not fit either helical oricosahedral symmetry due to comple-xity of these structure, e.g. pox virus,bacteriophage, etc.

Virion may be enveloped or nonenveloped.The envelope is derived from host cellmembrane when virus is released by budding.Envelope is lipoprotein in nature.

Protein subunits may be seen as projectingspikes on the surface of the envelope. Theseare called peplomers. A virus may have morethan one type of peplomer, e.g. influenza virushas two peplomers:

i. Triangular spike, i.e. hemagglutinin.ii. Mushroom-shaped, i.e. neuraminidase.

Reaction to Physical andChemical Agents

i. Heat and cold: Viruses are mostly des-troyed by heating at 60°C for 30 minutesexcept hepatitis virus, adeno-associatedvirus, scrapie virus. Viruses may bepreserved by storage at –20 to –70°C indeep freezer (except poliovirus).

ii. pH: They are usually stable at pH 5 to 9.iii. Ether susceptibility: Arbo, myxo and

herpes viruses are destroyed by etherwhereas entero, reo and adeno areresistant to the action of ether.

iv. Radiation: UV light, X-rays and heavyparticles inactivate viruses, the UV raysdimerizes the pyrimidine bases ofnucleic acid strand and gamma rayscause lethal break in the genome.

v. Vital dyes: Toluidine blue, neutral redand acridine orange penetrate virusparticles. These dyes unite with nucleicacid making viruses susceptible toinactivation by visible light.

vi. Glycerol: Viruses remain viable in50 percent glycerol whereas bacteria die.

vii. Stabilization by salt: Magnesium chloride,magnesium sulfate stabilize some of theviruses so that they are not inactivatedby heating at 50°C in one hour.

viii. Disinfectants: Lysol, dettol, are ineffec-tive against viruses. Higher concentra-tion of chlorine, iodine may kill viruses.Dilute formaldehydes and beta propio-lactone, hydrochloric acid, KMnO4,H2O2 are most useful disinfectantsagainst virus.

Fig. 45.1: Structure of virus

General Characteristics of Viruses 273

ix. Antiviral agents: I-methylisatin β-thio-semicarbazone are used against smallpox, amantidine against influenza,rubella and respiratory syncytial virus.

Viral Multiplication (Fig. 45.2)

Virus depends on the synthetic machinery ofhost cell for replication because it lacksbiosynthetic enzymes. The sequence of eventsare as under:1. Adsorption: The virus is adsorbed at a

particular site on the host cell which iscalled receptor. In case of poliovirus thereceptor is lipoprotein present on thesurface of primate. The host cell receptorfor influenza virus are glycoproteinspresent on the surface of respiratoryepithelium. Adsorption or attachment isspecific and is mediated by binding ofvirion surface structure known as legands,to receptors on cell surface. In HIV surface,glycoprotein (gp120) acts as a legand andit binds to the CD4 60 kD glycoprotein onthe surface of mature T lymphocyte.

2. Penetration: Virus particles may be engul-fed by animal cell by the mechanism calledviropexia. Viropexia is like phagocytosis.In case of enveloped virus, viral envelopemay fuse with plasma membrane andrelease nucleocapsid into the cytoplasm.

3. Uncoating: This is a process by which thevirus lose its outer layer and capsid. In some

cases, uncoating is effected by lysozomalenzyme of host cell. For example, in poxvirus, uncoating occurs in two steps. Outercoating is removed by lysozyme present inphagocytic vacuole of host cell. This is thefirst step. In second step, internal core ofvirus (nucleic acid and internal protein) isreleased into cytoplasm and is effected byviral uncoating enzyme. Thus DNA isreleased.

4. Biosynthesis: There is synthesis of viralnucleic acid and capsid protein. There issynthesis of regulator protein which shutsdown the normal cellular metabolism anddirect sequential production of viralcomponent. DNA viruses synthesize theircomponents in host cell nucleus except poxvirus which synthesize their componentsin cytoplasm. Likewise RNA virusessynthesize their components in cytoplasmof host cell except orthomyxoviruses, para-myxovirus and leukoviruses. Biosynthesisconsists of following steps:i. Transcription of messenger RNA from

viral nucleic acid.ii. Translation of mRNA into early pro-

teins. They initiate and maintain thesynthesis of virus component and shutdown the host protein and nucleic acidsynthesis.

iii. Replication of viral nucleic acid.iv. Synthesis of late proteins, which are the

components of daughter virion capsids.

Fig. 45.2: Viral multiplication

274 Virology

Transcription: Mechanism of transcriptionand nucleic acid synthesis differs in differenttypes of viruses:a. In single stranded (SS) nucleic acid, comple-

mentary strand is first synthesized pro-ducing double stranded (DS) replicativeforms.

b. Double stranded (DS) viral DNA acts astemplate and for its replication RNA virususes various methods for replication:i. In poliovirus, SS RNA acts directly on

mRNA.ii. SS RNA parenteral (positive strand) acts

as template for production of comple-mentary strands (negative strand) whichact as template for progeny viral RNA.

iii. In SS RNA (e.g. influenza) parenteralRNA produces complementary negativestrands which act both as mRNA and astemplate for synthesis of progeny viralRNA.

iv. Oncogenic RNA viruses (leukovirus)exhibit unique replicative cycle. Thevirus genome is SS RNA which is con-verted into RNA-DNA hybrid by viralenzyme reverse transcriptase (RNAdirected DNA polymerase) from itshybrid DS DNA is synthesized whichis integrated into host cell genome(provirus). Provirus acts as template forthe synthesis of progeny viral RNA.

This integration of provirus with host cellgenome may cause transformation of the celland development of neoplasm.5. Maturation: Assembly of daughter virion

follows synthesis of viral nucleic acid andproteins. It may take place in nucleus(herpes, adeno) or cytoplasm (picorna,pox). Enveloped virus gets envelope fromthe cell membrane of the host duringa process of budding. Nonenvelopedviruses are present intracellularly as fullydeveloped virion.

6. Release: In bacterial viruses release takeplace by lysis of infected bacterium. Inanimal, viruses release occur without lysis(myxo). Some viruses like polio may causecell lysis during their release.

Cycle of replication:a. Fifteen to thirty hours in animal virus.

b. Fifteen to thirty minutes in bacterialphage.

Eclipse phase is the time from stage of pene-tration of virus into host cell till appearance ofmature daughter viruses. In this phase, viruscannot be demonstrated in host cell.

Abnormal Replicative Cycles

Magnus phenomenon: When virus yield willhave high hemagglutinin titer but low infec-tivity. They are also called incomplete viruses.

Abortive infection: Sometimes in host cellsviral component may be synthesized butmaturation or assembly is defective. Either norelease occur or the progeny is noninfectious.

Defective viruses: They are viruses which aregenetically deficient and so incapable ofproducing infectious daughter virion.

Virines: They are made up of small amountof RNA complexed with protein of host cellorigin.

Prions

They are small proteinaceous infectious parti-cles which resist inactivation by proceduresthat modify nucleic acid. In short infectiousagents which lack nucleic acid genome arecalled prions. They are about 5 nm in diameter,resistant to heat, ultraviolet rays and nuclease.However, they are sensitive to proteases. Somefeatures of prions are:1. They cause diseases which are confined to

central nervous system.2. The diseases caused by prions have pro-

longed incubation period.3. The diseases due to prions show slow,

progressive and fatal course.4. Prion diseases may show spongiform

encephalopathy and vacuolation ofneurons.

Immune response: Prions do not cause aninflammatory response. They do not inducethe formation of interferons. There is noantibody response against prions. Obviously,it is not possible to screen people for exposureto prions by demonstrating antibodies.

Prion diseases are often called spongiformencephalopathies because of the postmortem

General Characteristics of Viruses 275

appearance of the brain large vacuoles in thecortex and cerebellum. Specific examples ofprion diseases in mammals include:

Scrapie Sheep

TME (Transmissible mink encephalopathy) MinkCWD (Chronic wasting disease) Muldeer,

ElkBSE (Bovine spongiform encephalopathy) Cows

Humans are susceptible to several priondiseases too some of which are:

CJD Creutzfeld-Jacob diseaseGSS Gerstmann-Straussler-Scheinker syndromeKuru Alper’s syndrome

Humans might be infected by prions bytwo ways as under:1. Acquired infection through diet and fol-

lowing medical procedures like surgery,growth hormones injections, cornealtransplants, etc.

2. Apparent hereditary mendelian transmis-sion where it is autosomal and dominanttrait.Thus prion diseases are distinct in the sense

that they are both infectious as well ashereditary.

Characters of Prions

• Resistant to chemicals, radiation and heat.• Proteinaceous and filterable• Do not produce inflammatory reaction in

host.• Do not produce antibody in host• Abnormal fibers and plaques may be

formed in the brain of host by prions.• Difficult to treat.

Nobel Prize in Medicine 1997

Stanley Prusiner won this prestigious prizethrough his discovery of prion, a diseasecausing agent like bacteria or virus. The prionprotein can manifest itself as two proteins, i.e.innocent protein and dangerous or diseasecausing proteins. Stanley Prusiner solved theriddle of prion’s properties.

Prions exist normally as innocuous cellularproteins. However, prions possess an innatecapacity to convert their structures. Thatultimately results in the formation of harmful

particles, the causative agents of severaldeadly brain disease like dementia type inhumans and animals.

As a matter of fact this discovery provideskey insight into dementia related diseases likeAlzheimer’s and mad-cow disease. It givesopportunity to understand the biologicalmechanisms underlying these diseases andalso establishes a foundation for drug deve-lopment and new type of medical treatmentstrategies.

INTERACTIONS AMONG VIRUSES

When same host is infected by two or morevirus particles they may interact as under:1. Recombination: Here the nucleic acid strands

break and part of the genome of one parentis joined to part of the genome of the secondparent. The recombinant virus is gene-tically stable and produce progeny likeitself on replication. Double stranded DNAgenomes recombine efficiently whereassingle stranded RNA genomes which areunsegmented do not recombine.

2. Genetic reactivation: When an inactive virusparticle is rendered active by interactionwith other inactive virus particles in thesame cell, it is called multiplicity reacti-vation. Here recombination occursbetween the damaged nucleic acids of theparents producing a viable genome thatcan replicate.Sometimes, a portion of genome of theinactivated virus recombines with that ofactive parent so that certain markers ofinactivated parents are rescued and appearin viable progeny. They are geneticallystable. This is called marker rescue.

3. Complementation: This means the inter-action of viral gene products in cellsinjected with 2 viruses, one or both ofwhich may be defective. The basis forcomplementation is that one virus providesa gene products in which the second isdefective, allowing second virus to grow.However, the genotypes of the two virusesremain unchanged.

4. Phenotype mixing: It usually occurs betweendifferent members of the same virus family.The intermixed capsid proteins must beable to interact correctly to form a struc-

276 Virology

turally intact capsid. In short, phenotypicmixing is the association of a genotype withheterologous phenotype.

5. Interference: It means infection with2 viruses leading to an inhibition ofmultiplication of one of these two viruses.Thus it is called interference. Themechanisms involved in interference are:i. One virus may inhibit the ability of the

second to adsorb to the cell by blockingthe receptors (enteroviruses) or bydestroying the receptors (orthomyxo-viruses).

ii. One virus may compete with the secondvirus for components of the replicationapparatus (polymerase).

iii. The first virus may cause the infected cellto produce an inhibitor (interferon) thatmay prevent replication of the secondvirus.

Interference has been used as a basis forcontrolling outbreaks of infection with viru-lent strains of poliovirus by introducing intothe population an attenuated poliovirusthat interferes with the spread of the virulentvirus.

Cultivation Viruses

Since they are obligate intracellular parasitesand cannot grow on inanimate culturemedium, 3 methods are used for their culti-vation:a. Animals inoculation.b. Chick embryo.c. Tissue culture.a. Animal inoculation: It is one of the oldest

methods for the cultivation of viruses. Thepoliomyelitis virus after intraspinal orintracerebral inoculation in monkeyscauses typical paralytic disease and soisolation of viruses. Suckling mice issusceptible to Cox-sackie viruses withmanifestation of severe myositis and para-lysis. Smallpox virus may be inoculated inthe scarfied skin or cornea of rabbit. Braintissue of rabied dog when inoculated intra-cerebrally in mice or rabbit developencephalitis.Growth of virus in animals may be knownby the disease, visible classical lesions ordeath. Sometimes immunity in experi-mental animal may interfere with thegrowth of viruses in that animal. It is not

out of place to mention the other utility ofanimal inoculation, i.e. to study patho-genesis, immune response, and epide-miology.

b. Chick embryo: They are better than animalinoculation because of following reasons:i. They are clean and bacteriologically

sterile.ii. They do not have immune mechanism

like animals to counteract virus infec-tion.

iii. They do not need feeding and caging.iv. Chick embryo offers several sites for

cultivation of viruses, i.e. chorioallantoicmembrane (CAM) for variola or vacci-nia and herpes viruses, allantoic cavityprovides rich yield of influenza andsome paramyxoviruses, amniotic sacmay be used for the isolation of influenzavirus and yolk sac for the cultivation ofchlamydiae, rickettsiae and some viru-ses (Table 45.2). Allantoic inoculationmay be used for growing influenza virusfor vaccine purposes. Yellow fever (17D strain) and rabies (flury strain) areother vaccines produced from chickembryo (Fig. 45.3).

TABLE 45.2: Growth of viruses in chick embryo

Route of Virus Lesion on Hem-inoculation CAM agglutination

Chorioallantoic Poxviruses + –

Membrane (CAM) Herpes simplex + –Herpes virus B + –

Amniotic cavity Influenza virus – +Mumps virus – +

Allantoic cavity New castle – +diseaseInfluenza – +Mumps – +

Yolk sac JE Virus, + –West Nile virusNile virus + –

Fig. 45.3: Structure of chick embryo

General Characteristics of Viruses 277

Disadvantages of Egg Inoculation

1. Eggs may be contaminated with myco-plasma and latent fowl viruses which mayinterfere with the growth of other viruses.

2. The susceptibility of chick embryo is limi-ted to a few viruses only.

3. Even slight amount of bacterial contami-nation in the inoculum may kill theembryo.

c. Tissue culture: Tissue culture of human oranimal cells are frequently used for thecultivation of viruses. There are mainlythree types of tissue culture:1. Organ culture, e.g. tracheal ring organ

culture is employed for the isolation ofcoronavirus.

2. Explant culture: Minced tissue may begrown as explant embedded in plasmaclots. This is not useful in virology. Inthe past adenoid tissue explant culturewere used for adenovirus.

3. Cell culture: This is very popular anduseful technique routinely used forcultivation of viruses. From tissue,fragments cells are dispersed byproteolytic enzymes like trypsin andmechanical shake. After washing thecells, they are suspended in growthmedium and distributed in petridishes,test tubes or bottles. The cells adhereto glass surface and grow out to forma monolayer sheet and can be seen insitu under low power.

Cell Cultures in Use

Primary cell cultures• Rhesus monkey kidney cell culture• Human amnion cell culture• Chick embryo fibroblast cell culture

Diploid cell strains• WI-38 (Human embryonic lung cell strain)• HL-8 (Rhesus embryo cell strain)

Continuous cell lines• HeLa (Human carcinoma of cervix cell line)• HEP–2 (Human epitheloma of larynx cell line)• Vero (Vervet monkey kidney cell line)• McCoy (Human synovial carcinoma cell line)• KB (Human carcinoma of nasopharynx cell line)

There are three types of cell cultures (Table45.3):a. Primary cell cultures: When normal cells

freshly taken from body grown for the first

time, they are called primary cell culture.They can be maintained in serial culture.They are useful for isolation and cultivationof viruses for vaccine production, e.g.rhesus monkey kidney cell culture, humanamnion cell culture, chick embryo fibro-blast culture, etc.

b. Diploid cell strains: They are capable of100 divisions in culture. They are useful forthe isolation of fastidious pathogens andalso for the production of viral vaccines.Examples are human embryonic lung cellstrain (WI-38) and rhesus embryo cellstrain (HL-8).

c. Continuous cell lines: They are single typeof cells mainly derived from cancer cells.These also can be grown in successivegeneration by transferring them from onetest tube to another without change incharacter of cells. These are used only forthe isolation of virus. Vaccine preparationon these cells is not safe for human use,e.g. HeLa (human carcinoma of cervix cellline).

TABLE 45.3: Isolation of viruses from cell lines

Cell line Virus isolation

Primary• African green monkey • HSV, RSV, mumps,

rubella• Chick embryo fibroblast • Rabies, pox-viruses

Diploid CellHuman fetal lung Rabies, adeno, CMV(WI-38, MRC-5)

ContinuousHe La Polio, pox, reo RSVHEp-2 Adeno, RSVMDCK InfluenzaRD Polio, enterovirusesVero Polio, rabies, measles

Other examples of continuous cell lines are:KB (human carcinoma of nasopharynx cell

line), HEP 2 (human epitheloma of larynx cellline), McCoy (human synovial carcinoma cellline), BAK 21 (baby hamster kidney cell line)and Detroit-6 (sternal marrow cell line).

Detection of virus growth on cell cultures:Viruses multiplying in tissue culture manifesttheir presence by producing:1. Changes in the cells called cytopathogenic

effects (CPE), e.g. measle virus producessyncytium formation and SV40 producesprominent cytoplasmic vacuolation.

278 Virology

2. When viruses grow in cell culture, cellmetabolism is inhibited and there is no acidproduction. In normal cell culture becauseof active metabolism there is active acidproduction. Phenol red (indicator) candetect the presence of acid formation bychanging its color into yellow.

3. Hemadsorption: When influenza and para-influenza viruses grow in cell culture theirpresence may be detected by addition ofguinea pig erythrocytes to the culture. Ifthe viruses are multiplying in culture,erythrocytes will adsorb on the surface ofcell.

4. Interference: Growth of first virus willalways check infection by the second virusby interference.

5. Transformation: Oncogenic or tumor pro-ducing viruses cause cell transformationand loss of contact inhibition.

6. Fluorescent antibody straining is also amethod of detecting viral multiplication.

7. Hemagglutination test may be performedby using tissue culture fluid, e.g. ortho-myxoviruses and paramyxoviruses.

INCLUSION BODIES

During multiplication of virus in host cells,virus specific structures are produced and theyare called inclusion bodies. Sometimes, theymay become larger than the individual virusparticles. They have distinct size, shape,location and staining properties. The size ofinclusion bodies may be from 1 to 30 µ. Theyare rounded, oval, pyriform or irregular inshape. They can be demonstrated under lightmicroscope. Acidophilic inclusion bodies canbe seen as pink structure when stained withGiemsa, or eosin methylene blue stain. Someviruses produce basophilic inclusion bodies.Inclusion bodies are believed to be the site ofdevelopment of viruses.

Vaccinia infected cells show small multipleintracytoplasmic inclusion (Guarnieri’sbodies) (Table 45.4). Large intracytoplasmicinclusions (Bollinger’s bodies) are seen infowlpox. Again Molluscum bodies areintracytoplasmic, quite large about 30 µ andseen in Molluscum contagiosum. Negri bodiesare intracytoplasmic seen in rabies virusinfection.

Intranuclear bodies are Cowdry type A(seen in herpes, yellow fever virus) in granularform and variable in size. On the other hand,Cowdry type B are circumscribed and multi-ple. They are found in adeno- and poliovirus.

Inclusion bodies which are both intra-nuclear and intracytoplasmic are encounteredin measles virus.

LABORATORY DIAGNOSIS

The appropriate specimen is collected,preserved and transported using propertechniques along with clinical information. Thefollowing approach is used for diagnosis ofviral disease:

Microscopic Examination

Viruses can be demonstrated and identified bydirect microscopic examination of clinicalspecimens. The various procedures involvedinclude:

Light microscopy: It can reveal characteristicssuch as inclusion bodies (e.g. Negri bodies) ormultinucleated giant cells. Tzanck smearshowing herpes virus induced multinucleatedgiant cells in vesicular skin lesions can be easilyobserved under light microscopy.

Electron microscopy: It is clinically being usedfor viruses that are difficult to culture.Rotavirus and hepatitis A virus in feces areincreasingly being detected by electronmicroscopy.

Immunoelectron microscopy: Addition of specificantibody to the specimen enhances thesensitivity of electron microscopy. The added

TABLE 45.4: Cell membrane receptors for viruses

Viruses Receptors membrane

Influenza Sialic acid on glycoproteinsincluding glycoprotein A molecule

Rabies Acetylcholine receptorsHIV CD 4 molecule on T cellsEpstein Barr C3d receptor on B cellsVaccinia Epidermal growth factor receptorReovirus Beta-adrenergic hormone receptortype 3Rhinovirus Intercellular adhesion molecule

General Characteristics of Viruses 279

antibody aggregates with the virus particlethereby making its demonstration easier.

Fluorescent microscopy: Direct or indirectfluorescent antibody technique is useful fordetection of viruses or viral antigens in clinicalspecimens. Some of the common virusesdetected by fluorescent microscopy includerabies virus, paramyxovirus, orthomyxovirus,adenovirus and herpes virus.

Identification in Cell Culture

The viruses can be grown by inoculation intoanimals, eggs or cell cultures. The presence ofa virus in the clinical specimen can be detectedby observing a “cytopathic effect” in cellculture, hemadsorption etc. A definitivediagnosis of the virus in cell culture is madeusing known antibody by tests like comple-ment fixation, hemagglutination inhibitionand neutralization of the cytopathic effects.Other procedures that can be used are ELISA,fluorescent antibody, radioimmunoassay andimmunoelectron microscopy.

Detection of Viral Antigens

ELISA, radioimmunoassay and latex aggluti-nation may be useful for detecting viralantigens.

Detection of Viral Nucleic Acids

The detection of viral DNA or RNA isincreasingly becoming the “gold standard” inviral diagnosis. Labeled nucleic acid probes arehighly specific with rapid results. Polymerasechain reaction (PCR) technique allows rapidamplification of target DNA sequence so thatit can be readily identified using labeled probesin a hybridization assay. The detection of HIV-1, HIV-2, human papillomavirus, hepatitis Bvirus, hepatitis C virus, enterovirus andEpstein-Barr virus has been simplified usingthe above technique.

Serology

A rise in antibody titer against the virus duringthe course of viral infection is a definitiveevidence of its etiology. However, an antibodytiter in a single specimen does not distinguishbetween a previous infection and a currentone. Paired serum samples, collected duringthe acute and convalescent phases arerequired. Presence of IgM specific antibodiesis meaningful in certain viral infections. Theantibody titer can be determined by theimmunological tests mentioned above. Othernonspecific serologic tests include heterophilantibody test (Monospot) used for diagnosisof infectious mononucleosis.

280 Virology

46 Chemotherapy ofViral Diseases

Sometime back close relationship betweencellular metabolic process required for viralmultiplication and those needed for survivalof vertebral cells, was thought to be the genuinereason not to search for antiviral agents.

Now a number of chemicals are availablewhich inhibit viral replication to greater extentthan cellular metabolism. Reason may be theiraction on synthesis or functions of viralenzymes or nucleic acids. Very few of themare found useful in practice, e.g.1. Pyrimidine nucleoside.2. Methisazone.3. Amantadine.

All these three have negligible effect onhuman morbidity or mortality from viraldiseases.

Problems of Viral Chemotherapy

Three broad problems to effective antiviralchemotherapy are:

i. Obligatory dependence of viruses uponmetabolism of the host cell.

ii. The fact that viral production is welladvanced by the time symptomsappear.

iii. Drug-resistant mutant.All substances which inhibit viral repli-

cation also interfere with cellular processes,e.g.a. Puromycin or cycloheximide inhibits both

viral and cellular protein synthesis.b. Fluorodeoxyuridine or cytosine arabinoside

inhibits the DNA synthesis.c. Actinomycin D inhibits DNA dependent

RNA synthesis.At virostatic concentration, these are toxic

for normal cells. These are lethal drugs andhence not useful for human consumption

except iododeoxyuridine (toxic drug) which isapplied topically to treat localized infection,e.g. herpes simplex of the cornea.

Ideal antiviral agent must block viralproduction without causing lethal damage touninfected cells. However, there are four keypoints that should theoretically be susceptibleto antiviral attack:1. Attachment of virus may be dependent on

the interaction of specific chemicalgrouping on the surface of each.

2. Transcription of mRNA from DNA ofdeoxyribose viruses require virus specifiedDNA dependent RNA polymerase andsome riboviruses utilize a virus specificpolymerase.

3. Viral mRNA molecules are apparentlydifferent in some fundamental way fromRNA of mammalian cells.The mechanism of action of antiviral

chemotherapeutic agent is given in Table 46.1.

TABLE 46.1: Mechanism of action of antiviralchemotherapeutic agent

Drug Probable point Principalof action viruses inhibit

Amantadine Penetration or Influenza Auncoating

Iododeoxyuridine DNA replication HerpetovirusAra—A 1, Arac

Rifamycins Reverse transc- Retrovirusesriptase assembly Poxviruses

Thiosemicarbazones Translation of late Poxvirusviral mRNA

Interferon Transcription or All virusestranslation

Poly I, poly C Induction ofStatolon interferon andPyran copolymer stimulation of All virusesCOAM reticuloendothelialTilorone systemPropanediamine

Chemotherapy of Viral Diseases 281

4. Virus coded enzymes essential to thereplication of viral nucleic acid butirrelevant to the cell are synthesized in theinfected cell.

Rational Approach to the Search forAntiviral Agents

The approach which led us to discovery ofantibacterial antibiotics has produced verypoor results for antiviral drugs. Approachshould be based upon the search for agentscapable of inhibiting biochemical reactionsknown to be unique to the multiplication ofviruses.

Attachment and Penetration

It requires apposition to specific complemen-tary receptor on the surface of virus and cellrespectively. Effective antiviral agents shouldbe able to interfere with any one, i.e. receptoror cell and can block infection, e.g. neuramini-dase destroys glycoprotein receptors in lungsof mice and render them refractory to influ-enza infection till new receptors appear aftersome hours. Amantadine and derivatives donot inhibit attachment but appear to havesome effect on the process of penetration.

Transcription

RNA transcriptase Transcriptase essential forviruses transcription of early mRNAPoxviruses from uncoated viral coat.

All trans- are viral All except May be somecription specific Pox are specific chemical

RNA inhibits thedependent enzymes without

effecting DNAdependent cellularRNA polymerases.

Imidazole and interferon block transcription.

Retro- Reverse Rifamycinviruses transcriptase (experimentally)

may render them may be usefulto attack at this point

Post-transcriptional cleavage (large molecules) ofpolycistronic mRNA.

Transcribed from DNA and some-RNAvirus and subsequent addition of poly(A) may be susceptible to selectivechemotherapy.

Translation

Translation of Affected by:

Protein from (1) Methisazone block the trans-viral RNA (2) Thiosemi- lation of late

carbazone vaccinia mRNAInterferon Blocks translation of all viral mRNA.

Replication of Viral Nucleic Acid

It occurs rapidly and often in resting cell.

5 iodo, 2 deoxy- Inhibit Systematically toxicuridine, adenine DNA but locally effectivearabinoside synthesis against DNA viruses,and cytosine e.g. multiplication inarabinoside cornea where only

few cells divide.

Benzimidazole and Inhibits RNA replication byderivatives, e.g. 2 α picorna viruses in culturedhydroxymethyl and cells.benzimidazole (HBB)

Post-transcriptional Cleavage of Proteins

Only in PICORNA viruses and ProteinToga (probably) arisen

Cleavage is important step Inhibition of cleavagein assembly of other viruses by Rifamycin.

Assembly

Guanidine Disrupt hydrogen bond andmature virion are not assembled.

Regulation of Gene Expression

Above mentioned stages may be under controlof viral protein.

Guanidine may act to throw out the wholeof the cycle out of gear.

INTERFERON

Interferons are host-coded glycoproteinswith molecular weight 20,000 to 40,000 thatinhibit virus replication and are produced byintact animals or cultured cells in response tovirus infection or other inducers. They are non-toxic, nonantigenic, inactivated by proteolyticenzymes. They can withstand heating at 37°Cfor 30 minutes. They are believed to be thebody’s first line of defence against viralinfection. It is ideal antiviral chemotherapeuticagent for the use in man. In man, it is non-toxic, non-allergic and active against broadspectrum of viruses. Interferon differs fromantibodies (Table 46.2).

282 Virology

There are multiple species of interferonsthat fall into 3 general groups named IFNα,IFNβ and IFNδ. Out of these IFNα family islarge being coded by at least 14 genes whereasIFNβ and IFNδ are coded by one or few geneseach. These interferons are similar in size butthe 3 groups are antigenically distinct. IFNαand IFNβ are resistant to low pH. IFNβ andIFNδ are glycosylated.

Synthesis of interferons: Normal cells do notgenerally synthesize interferon until they areinduced to do so. Interferons are produced byall vertebrate species. RNA viruses arestronger inducers of interferon than DNAviruses. Interferon also can be induced by DSRNA, bacterial endotoxin and small moleculeslike tilorone. However, IFNδ is not producedin response to most viruses but is induced bymitogen stimulation.

IFNα class of interferons is synthesized byleukocytes, IFNβ by fibroblasts and IFNδ onlyby lymphocytes.

Diploid strain of human embryonic fibro-blast, e.g. WI-38 could provide adequatesupply of cells. Precaution for inactivation ofviruses used as interferon inducer must beundertaken for purification of interferon.Human skin fibroblast stimulated with poly I,Poly C to produce 5 hours’ burst of interferonabout once in 24 hours may be used to obtainhuman interferon.

Synthetic Polynucleotides and OtherInferferon Inducers

Interferon levels in human being followingnatural infection or artificial stimulation arelow as compared to those found in mice butthese low levels are protective.

Synthetic chemicals are preferred tolive attenuated viruses, e.g. polynucleotide(Poly I and Poly C).

Poly I and Poly C

In human beings they protect against widevariety of viral infection. When administeredintramuscularly, one day before infection until4 days after challenge, with rhino type-13,inoculated by same route, produce lessnumber of virion and less severity of commoncold. Topical applications in respiratory tracthave some value in human medication withouttoxic effects.

Drawbacks: Systematic administration is con-traindicated except in Grave illness, becauseof side effect on hemopoiesis and liver function.Following single dose, animals becometemporarily refractory to interferon inductionby 2nd dose. This drawback is seen with allvarieties of interferon inducers.

Mechanism of action: It is poorly understood.It is clear that interferon induces an antiviralstate by prompting the synthesis of otherproteins that actually inhibit virus replication.

Interferon molecules bind to cell surfacereceptors, with IFNα and IFNβ sharing acommon receptor and IFNδ choosing a distinctreceptor. This binding triggers the synthesisof several enzymes believed to be responsibleof antiviral state. These cellular enzymes sub-sequently block viral replication by inhibitingthe translation of viral mRNA into viralprotein. Atleast three enzymatic pathwaysseems to be involved.1. Protein kinase phosphorylates inactivates

a cellular initiation factor and thus prevents

TABLE 46.2: Differences between interferons and antibodies

Interferons Antibodies

1. Are produced by any microbe infected cells 1. Are produced by B lymphocytes2. Leave the infected cell and enter a nearby 2. Circulate in blood and lymph to dispose of

healthy cell to dispose of microbes antigens3. Induce the healthy cell to synthetize antimicrobial 3. Selectively bind to antigens that are immobilized

proteins to check microbial multiplication for easy attack by phagocytes4. Are quick in action but offer a temporary immunity 4. Are slow in action but offer long-lasting immunity

against microbes against antigens5. Act inside the cells 5. Act outside cells

Chemotherapy of Viral Diseases 283

formation of initiation complex requiredfor viral protein synthesis.

2. An oligonucleotide synthetase, 2,5-oligo A,that is required for oligoadenylic acidformation activates a cellular endonucleaseRNase, which degrades viral mRNA.

3. Phosphodiesterase that leads to inhibitionof peptide elongation. Besides this, inter-ferons may affect viral assembly perhapsas a result of changes at plasma membrane.

Clinical use: Interferon treatment may behelpful in certain severe viral infections likerabies, hemorrhagic fever, herpes encephalitis,persistent virus infections like Hepatitis B,Herpes zoster, etc. Topical interferon in the eyemay suppress herpetic keratitis and enhancehealing.

All interferons are being tested but inter-feron IFNδ would be expected to be quiteeffective due to its higher anticellular activityas anticancer agent.

In short, human interferon can preventdissemination of Herpes zoster and Herpessimplex in immunosuppressed patients, sup-press viremia of chronic active Hepatitis B. Ifgiven before trigeminal ganglion surgery forneuralgia, it can temporarily suppress theappearance of lesions of herpes simplex.

Human interferon can:1. Prevent dissemination of Herpes zoster and

Herpes simplex in immunosuppressedpatients.

2. Suppress viremia of chronic activeHepatitis B.

3. If given before trigeminal ganglion surgeryfor neuralgia, it can temporarily suppressthe appearance of recurrent lesions ofHerpes simplex.

Current Research

Attempts to synthesize new polynucleotideswith higher antiviral potency and lowertoxicity in man, e.g. N.N. dioctadecyle N, Nbis (2 hydroxyethyl) propanediamine adminis-tered as oil in water emulsion intranasallyinduce titer higher of interferon.

Interferon may be used in the treatment ofacute life-threatening viral illness. Dosagesrecommended are 5 million units intramus-cularly daily for 14 days (Herpes simplex

type I), 5 million units intramuscularly dailyfor one week (Hepatitis B), 5 million units3 times a week for 10 months (papillomatosis)and one million units intramuscularly for3 days in varicella encephalitis. Now recombi-nant DNA technology has been applied togreatly increase the yields of severalinterferons for clinical trials. Cloned interferongenes are being expressed in large amount inbacteria and yeasts and availability ofgenetically engineered interferon is made.

ANTIVIRAL AGENTS

Mechanism Anti viruses Viruses killed

A. Inhibition of I. Acylovir • Herpes simplexviral DNA II. Ganciclovir Varicella zosterpolymerase III. Ribavirin • Cytomegalovirus

IV. Trisodium • RSVphosphono- • Lassa virusformate • Herpes virus

• Hepatitis B virus

B. Inhibition of • ZidovudinTranscriptase • Dideoxycytidine

• Dideoxyinosine HIV• Trisolium

phosphono-formate

C. Inhibition of I. Indinavirprotease II. Nelfinavir HIV

III. RitonavirIV. Saqnivir

D. Blockage in Amantadine Influenza viruspenetration ofvirus into cells

E. Protein synthesis Interferon All virusesinhibition

Immunomodulators: They are the agents whichreverse immunologic effects of the virus andso can be used as adjuvants to antiviral drugs,e.g. interferon, interleukin II, isopronosinethymic humoral factors, etc.1. Nucleosides derivatives:

a. Halogenated nucleoside:i. Halogenated pyrimidine is inhibitor

of cellular DNA synthesis.ii. 5-iodo deoxyuridine is topically

used in man in keratoconjunctivitisor dendritis ulcer due to Herpes sim-plex virus. Five percent iododeoxy-uridine in dimethyl sulfoxide solu-tion is used in Herpes labialisincreases penetration. Systemati-cally used in Herpes simplex encep-halitis cases but value is not confir-

284 Virology

med. Otherwise it is mutagenic andshould not be given parenterally.

b. Thymidine analogue:Trifluorethymidine is superior to iodo-deoxyuridine for human cellularinfection with Herpes simplex virus.

Acyclovir (9-[2-hydroxyethoxymethyl]guanine acyloguanosine): It stronglyinhibits Herpes simplex virus but haslittle or no effect on other DNA virusesor on host cells. It has been effective intopical application in the control ofherpetic eye lesions in humans and inthe healing of only primary lesions.Latent infections in the ganglia are notcured. Parental administration mayprevent the reactivation of latentherpes virus infections. However,mutants of herpes virus that lackthymidine kinase fail to phosphorylatethe drug and are resistant to it.

Ganciclovir: It is structurally relatedto acyclovir and is specially usefulbecause of its anticytomegalovirusproperty in transplant, AIDS patientsand immunocompromised patients.

2. Arabinofuranosyl nucleoside:It is more useful than halogenated nucleo-side in human medicine. It interferes withboth cellular and viral DNA synthesis buttheir chemotherapeutic index is quitehigher.

Cytosine arabinoside (Ara C) has simi-lar antiviral spectrum as iododeoxyuridine,equally effector in herpetic and vaccinalinfection especially keratitis in man andanimal.

Recently synthesized, adenine arabino-side and 9B-D rabinofuran osyladenine(ara A) have high chemotherapeutic indexthan Ara C or iododeoxyuridine againstherpetic and vaccinal viruses.

Ribavirin: This synthetic nucleoside ana-logue inhibits both RNA and DNA viruseslike influenza, parainfluenza, respiratorysyncytial, adeno, arena and lassa viruses.

Vidarabine (Ara-A): It is purine nucleo-side analogue with activity against Herpessimplex, varicella, Epstein-Barr viruses, etc.

It inhibits viral DNA synthesis. In combi-nation with interferon. It is useful inchronic Hepatitis B infection.

Vidarabine monophosphate (Ara-Amp): It may be used in Herpes simplexencephalitis, genital herpes and varicellazoster infections.

Rimantidine: It is structurally similar toamantidine and is better tolerated. It ismore effective and less toxic.

3. Thiosemicarbazones:I-methylisatin β-thiosemicarbazone(methisazone) inhibits multiplication ofpox virus. It interacts with perhaps viralor cellular proteins to prevent translationof late viral mRNA. It may have role intreatment of complication of smallpox vac-cination such as eczema vaccinatum orvaccina gangrenosa. Drug is not a substi-tute of smallpox vaccination.

4. Amantadine:I-amantanamine hydrochloride blocksmultiplication of influenza A viruses at astage of penetration or uncoating. Effi-ciency and safety in man is controversial.Otherwise also it has prophylactic impor-tance only. It is not effective after infection.1-methyl spiromaleate has been said toshow promise in early clinical trial.

5. Some other compounds of potential value:Rifamycin derivative:Rifamycin inhibits bacterial RNA synthesisby binding to DNA dependent RNA poly-merase. No such effect on mammalianpolymerases is known.

Studies show that rifamycin inhibitsreverse transcriptase of retroviruses at theconcentration slightly lower than thoseblocking cellular DNA dependent DNApolymerase.Benzimidine:Enviroxime (2, amino-1-[isopropylsulfonyl] benzimidazole phenyl-ketoneoxime) inhibits rhinovirus.

Derivatives 2 (α-hydroxybenzyl)benzimidazole also called HBB inhibits themultiplication of picorna viruses notableCoxsackie B and ECHO viruses at non-cytocidal concentration in cultured cell butof no value at all in vivo.

Chemotherapy of Viral Diseases 285

Guanidine hydrochloride behaves like-wise. Drawbacks are:1. Drug dependence2. Drug resistance.

AGENTS INHIBITING REVERSETRANSCRIPTASE

Zydovudine (AZT): Structurally, it resemblesthymidine. It is a nucleoside analogue ofthymidine having more affinity for reversetranscriptase than natural substrate. Hencebinding of natural nucleoside by reversetranscriptase is blocked.

Zydovudine is phosphorylated by cellularkinase and so preferentially bound to thereverse transcriptase during transcription ofviral DNA. When zymidine is added insteadof thymidine to nascent DNA chain elongationis terminated. Thus, the synthesis of DNA isinterrupted immediately after incorporation ofzydovudine.

In HIV infection, zydovudine is quiteeffective therapy. It is more useful in reducingtransplacental and perinatal transmission ofHIV to neonates. Side effects of this drug areanemia, neutropenia, etc.

Dideoxylnosine (DDC): They act as chainterminators. They are useful in HIV infectionthat are resistant to zydovudine. Side effectsare painful sensorimotor peripheral axonalneuropathy (20 to 30%) and pancreatitis (15%).

Protease inhibitors: HIV protease is essentialto the assembly of virion and productionof infectious virus particles. The inhibitorsHIV protease include saquinavir, ritonavir,indinavir. They inhibit the processpolyproteins. They are competitive inhibitorsof the HIV. However, use of single drugtherapy by protease inhibitors appears todevelop drug resistance.

BROAD SPECTRUM NUCLEOSIDEANALOGUES

1. Phosphonoformic acid (Foscarnet): Theyare classical inhibitors of herpes viruspolymerase. It inhibits DNA chain elonga-tion. It is effective in Herpes simplex andcytomegalovirus infections. It also inhibitsHIV reverse transcriptase.

2. Ribavirin: It has action on cellular enzymeswhich are important in viral replication byblocking protein synthesis. It is useful inrespiratory syncytial virus infection inneonates and infants. It has been usedsuccessfully in early stages of Hantavirusinfection.

Suramine: This naphthalene derivative isfound useful in the treatment of AIDS. Itinhibits reverse transcription.

Other upcoming antiviral drugs are AL-721, HPA-23, ddA, ddL, ddC and D4T.

NOBEL PRIZE IN MEDICINE-1988

Professor Discoveries ofGertrude Elion important principlesDr George USA that have resultedH Hitchings in the developmentSir James Black UK of a series of new drugs.

Professor Gertrude Elion and Dr GeorgeH Hitchings, both from USA won for theirdiscoveries including trimethoprim, acyclovir(the first successful drug against the Herpesvirus), 6 mercaptopurine and thioguanine, twoanticancer drugs. Aridothymidine or AZT, adrug used for treating AIDS was developedfrom their ideas. Thus, their research led to thedevelopment of drugs for the treatment ofleukemia, malaria, to fight rejection of trans-planted organs and AIDS. However, Sir JamesBlack won his share of this Nobel prize fordesigning the world’s first beta blockerpropranolol and the first H2 antagonist.

286 Virology

Rapid progress in the field of virology withnew information enforces the review andrevision of virus nomenclature. Seven or eightschemes of classification for viruses have beenproduced in the past.

Till 1950 little was known about the viru-ses. Viruses may affect animals, insects, plantsand bacteria. Attempt was made to group orclassify the viruses on the basis of their affinityto different systems or organs of the body, e.g.1 Those producing skin lesion (smallpox,

chickenpox, measles).2. Those affecting nervous system (polio,

rabies).3. Respiratory tract-involving viruses (inf-

luenza, common cold).4. Viruses causing visceral lesions (yellow

fever, hepatitis).It was also suggested that viruses should

be classified based on epidemiological criteria.Some of the examples are as under:1. Enteric virus:

a. Picornavirus.b. Adenovirus.c. Reovirus.d. Hepatitis virus.

2. Respiratory:a. Orthomyxovirus.b. Paramyxovirus.c. Coronavirus.d. Rhinovirus.e. Adenovirus.f. Reovirus.

3. Arbo (arthropod borne):a. Togavirus.b. Bunyavirus.c. Rhabdovirus.d. Orbovirus.

47 Classification ofViruses

It is not out of place to enumerate thecriteria that have been used in forming groupsof animal viruses:1. Type of nucleic acid.2. Chemical composition (Table 47.1).3. Susceptibility to physical and chemical

changes.4. Size measurement.5. Design and construction.6. Antigenic characters.

Nowadays viruses are classified into twogroups depending on the type of nucleic acidthey possess; those containing RNA are calledribovirus and those containing DNA aredeoxyriboviruses (Fig. 47.1). They may befurther classified on the basis of followingcharacters:

i. Strands of nucleic acid.ii. Symmetry of nucleocapsid.

iii. Presence of envelope.iv. Number of capsomers.

Further discussion is based on above men-tioned characters. Deoxyribose (DNA) virusesare at present placed in five groups andribovirus (RNA) into nine groups.

Major Groups of DNA Viruses (Fig. 47.1)

1. Poxvirus: They are large brick-shapedparticle 230 to 300 nm × 200 to 250 nm,visible by light microscope. They maycause smallpox, vaccinia, molluscumcontagiosum, cowpox and milker nodes.Examples of pox virus are variola, vaccinia,molluscum contagiosum, avian pox, etc.

2. Herpes virus: They are enveloped andicosahedral. They multiply within nucleus.They are covered by ether sensitiveenvelope. They may cause vesicular skin

Classification of Viruses 287

Fig. 47.1: Different types of viruses

TABLE 47.1: Chemical composition of viruses

Family Configuration Molecular weight Protein Transcription(Dalton) (Polypeptide)

DNA:Parvo SS 2 3 –Papova DS 3-5 6 –Adeno DS 20-25 9 –Herpetic DS 100 12-27 –Pox DS 160 730 +

RNA:Picorna SS 2-3 4 –Toga SS 4 3 –Bunya SS 6 3 +Arena SS 6 ? ?Corona SS 9 16 +Retro SS 10-12 7-8 +Ortho SS 5 7 +Paramyxo SS 7 6 +Rhabdo SS 4 7 +Reo DS 15 7 +

lesions, encephalitis, chickenpox, etc.Examples are Herpes simplex virus,varicella zoster virus, cytomegalovirus, etc.

3. Adenovirus: They multiply in nucleus. Theyare ether resistant. They may cause latentinfection of lymphoid tissue, mild

respiratory diseases, conjunctivitis,keratitis, etc. Example is adenovirus, etc.

4. Papova virus: They are icosahedral, multiplyin nucleus and are ether stable. They maycause human warts, papillomata (PA) ofrabbits, dogs, etc. polyoma (PO) in mice.

288 Virology

Some viruses act as vacuolating agents(VA), e.g. SV40. All are potentiallyoncogenic. Examples are papilloma virus,polyoma virus, SV40, etc.

5. Parvovirus: They are very small 18 to 22 nmin diameter and are ether resistant, e.g.minute virus of mice, Kilhamirat virus(RVM) and adeno satellite virus.

Major Groups of RNA Viruses (Table 47.2)

1. Orthomyxoviruses: They are spherical orfilamentous, enveloped with lipoprotein,studded with neuraminidase andhemagglutinin subunits. They may causeepidemics and endemics of influenza, etc.Examples are influenza viruses type A, Band C, etc.

2. Paramyxovirus: They are similar to myxo-virus but are larger and more pleomorphic.They may cause respiratory infections, badcold, measles, mumps, etc. Examples areparainfluenza virus 1 to 4, measles virus,distemper virus, rinderpest virus, mumpsvirus, Newcastle virus, etc.

3. Rhabdovirus: They are large enveloped,bullet-shaped and ether sensitive. Theymay cause rabies in mammals andvesicular stomatitis in cattle, etc. Examplesare rabies virus, vesicular stomatitis virus,etc.

4. Togavirus: They are icosahedral and enve-loped by lipid. They require arthropod vec-tors and may cause meningoencephalitis,lymphadenopathy, bleeding and purpuricrashes, yellow fever, etc. Examples areyellow fever, sindbis and dengue viruses.

5. Arenavirus: They are enveloped and ethersensitive causing benign meningitis andencephalitis, e.g. lymphocytic chorio-meningitis virus, etc.

6. Reovirus: They are ether resistant, nakedicosahedral with double stranded RNAcausing mild respiratory and entericdiseases.

7. Picornavirus: They are small icosahedral,ether and acid resistant causing neuronaldamage with paralysis (polio 1 and 3),aseptic meningitis, etc., e.g. polio virus,echo virus, coxsackie virus, rhinovirus, etc.

8. Leukovirus: They induce malignanttransformation of cells with formation ofnew antigens and enzymes with loss ofcontact inhibition, e.g. leukemia, sarcomain fowls and mice, e.g. Rous sarcoma,murine leukemia, murine mammary tumorvirus, etc.

9. Coronavirus: They are elliptical or spheri-cal and ether sensitive causing cold andacute respiratory infection, mousehepatitis, etc. Examples are human, murineand avian virus.

TABLE 47.2: Morphology of virus

Family Shape Diameter Environment Symmetry No. of(nm) Capsomers

DNAParvovidae Spherical 20 – Icosa 32

PapillomaPapova Spherical 45-55 – Icosa 72

PolyomaAdeno Spherical 70-80 – Icosa 252Herpetic Spherical 150 + Icosa 162Pox Brick 100 × 240 × 300 – Icosa –

RNAPicornavidae Spherical 20-30 – Icosa ? 60Toga Spherical 40-60 + Icosa ?Bunya Spherical 90-100 + Helical –Arena Spherical 85-120 + Helical –Corona Spherical 80-120 + Helical –Retro Spherical 100-120 + Helical –Orth Spherical of filamentous 80-120 + Helical –Paramyxo Spherical of filamentous 100-200 + Helical –Rhabdo Bullet 70-180 + Helical –Reo Spherical 50-80 – Icosa ?

Oncogenic Viruses 289

Viruses that produce tumors in their naturalhosts or in experimental animals or inducemalignant transformation of cells on cultureare known as oncogenic viruses. They may beDNA viruses or RNA viruses (Table 48.1).

DNA Viruses

Papova viridae: They are small double strandedDNA, non-enveloped, 40 to 57 nm in diameterand showing icosahedral symmetry. Allmembers of this family can cause tumor.

Most naturally occurring tumors arebenign, i.e. warts in man and animals. Rabbitpapilloma are initially benign but may becomemalignant.

Polyoma and Simian Virus40 (SV40), in naturecause malignant tumors very rarely but wheninoculated into newborn rodent they maycause malignant tumor.

Polyoma Virus

Discovery of this virus was made in 1956. Itcauses a variety of histologically diversetumors in various parts of the body. Virusescapable of inducing tumors in experimentalanimals can transform cell in vitro. Transfor-mation by polyoma virus is rare event as eveninput with 1000 pfu per cell, only a minorityof cells become transformed.

Though primary virus induced change canproduce cancerous cell, but more commonlyit produces a premalignant cell. Subsequentmutations in the proliferating clone, becomingmore malignant or causing abortive transfor-mations in which, transiently transformed cellsresume their normal growth after few generations.

48 Oncogenic Viruses

Neither tumor cells induced by virusesin vivo, nor cells transformed in cells cultureproduced infectious virus, hence infectiousDNA cannot be extracted from them.Malignant cell contained 2 types of viralantigen quite distinct from structural proteinsof virion (1) T antigen (2) TSTA (tumor specifictransformation antigen):1. T antigen identical with proteins produced

in early stages of cytocidal infections.2. TSTA antigen occurs in cell membrane by

transformed cell.The continued synthesis of these proteins

through an indefinite number of cell genera-tion was suggestive of permanent retention ofviral genetic material within cell. This wasdemonstrated by molecular hybridization test.Malignant cell carries several molecules ofviral DNA integrated cells chromosomes. Theviral DNA is transferred into mRNA speci-fying at least the viral T and transplantationantigen.

Simian Virus 40 (SV40)

Discovered in normal cultures of monkeykidneys cells during production of poliovaccine. Test on baby hamsters showed that itwas oncogenic. Complete viral genome is notrequired for carcinogenesis as happens inpolyoma virus. Transformation is increased bythe use of UV irradiated SV40, or defectivehybrid of SV40 and adenovirus. Completegenome of SV40 is often present in virus trans-formed cells and in virus induced tumors asdemonstrated by induction of infectious virusproduction or, as a result of cell diffusionexperiments.

290 Virology

T and transplantation Ag coded SV40 arevirus specific. They show no cross reactivitywith corresponding antigen of polyoma virusor adenovirus. Temperature sensitive and hostcell dependent mutant as well as compre-hensive range of adenovirus hybrid have beenemployed to pin-point the genes responsiblefor cancer. There is complete map of SV40

genome with function of certain genome aswell as sites of initiation and termination ofDNA replication and transcriptions. Nature ofgenes that determine cell transformation is notknown yet.

Adenoviridae: Adenovirus type 12 producedsarcomas when inoculated in newbornhamster (Trentin 1962). Different adenovirusesof human and animal nature are also onco-

genic. Human type, 12, 18, 31 have high onco-genic potential. No evidence of oncogenicityfor man has emerged. Cell always contain Tand transplantations Ag.

Only 5 percent mRNA recovered frompolyribosomes of transformed cell is virusspecific. Adenoviral DNA molecules integra-ted with cell chromosomes are incomplete.

Herpetoviridae: In lower animal evidence ofherpetovirus oncogenesis is strong. Onco-genesis seems to be associated with incomp-lete expression of viral genome.

Herpes virus samiri is closest analogue tohuman oncogenic herpetovirus isolated frommalignant lymphoma in monkey. In Marekdisease of fowl etiological agent of malignantlymphoma is herpetovirus so much so that

TABLE 48.1: Some characteristics of malignant diseases induced by viruses

Natural Malignant diseases Artificial Malignant cell Transformation of cultured cellVirus host tumor host tumor Virus Species Virus

production product

Adenovirus — — Baby rodent Sarcoma + Rodent *PapovaPolyoma — — Baby rodent Sarcoma + Rodent *SV40 — — Baby rodent Sarcoma + Rodent *

ManRabbit Rabbit Papilloma and Rabbit Papilloma + * *Papilloma carcinoma CarcinomaHerpetovirus(i) Marek’s virus Chicken Lymphomatosis Chicken Chicken * * *(ii) Lucke’s virus Frog Adenocarcinoma Tadpole Adenoma * * *(iii) Herpes virus Monkey Lymphoma Primates Lymphoma * * * samiri Rabbits(iv) EB virus Man Lymphoma — — Man *

Nasopharyngealcarcinoma

Herpes simplex 2 Man Cervical carcinoma — * Man *OncovirusAvian Chicken Leukosis Chicken Leukosis + Chicken +

sarcoma Chicken sarcoma + Chicken +RodentMonkey

Murine Mouse Leukemia Rodent Leukemia Mousesarcoma Sarcoma + Rodent +

ManFeline Cat Leukemia Kitten Leukemia + Cat +

sarcoma DogRabbit Sarcoma + Man +

Mammary Mouse Mammary Mouse Carcinoma + *tumor virus carcinoma

(a) Occasional cells spontaneously yield virus. (b) Virus production can be induced by co-cultivation. (c) Some sarcoma virus strains are defective and cells of foreign species are non-permissive. (d) Transformed cells appear natural. (e) + means viruses are produced.

Oncogenic Viruses 291

immunization in flocks of chicken withattenuated live virus vaccine is successful.

Epstein Barr Virus

Half of all malignancies in children (Burkitt’slymphoma) are a result of oncogenic virusestransmitted by insect. In 1964, Epstein recove-red EB virus from cultured tumor cells. Thereare multiple copies of EB virus DNA integra-ted into the chromosomes of all lines of EBtumor cells. Henle and Henle found EB virusas causative agents of infectious mono-nucleosis. EB virus causes Burkitt lymphoma issupported by the following facts:1. That EB virus causes infectious mononuc-

leosis resembling early stages oflymphomas.

2. That EB virus causes transformation oflymphocytes in vitro.

3. Cases of Burkitt lymphoma show sero-logical evidence of EB virus infection.

4. EB viruses are detectable in Burkittlymphoma and virion can be recoveredafter cultivation of cells in vitro.Association of EB virus and nasopharyngeal

carcinoma is known. EB virus and Hodgkindisease association is also there.

Herpes Simplex Type 2

Association of Cancer cervix and Herpes simplextype 2 is there. In malignant cell demonstra-tion of Herpes simplex antigen may be made.Herpes simplex DNA is detected in the chro-mosomes of malignant cells. UV rays activateHerpes simplex virus type 2 with transfor-mation of cultured cells.

VIRUSES IN HUMAN CANCER

Virus Tumor

DNA virusesEpstein Barr virus • Burkett lymphomas

• Nasopharyngeal carcinomasHerpes simplex May cause carcinoma cervixPapilloma virus • Skin carcinoma

• May cause carcinoma cervixHepatitis B virus Carcinoma liverHepatitis C virus Carcinoma liver

Oncogenic RNA Viruses

Only small number of RNA viruses are onco-genic. They are oncogenic under natural

conditions and are associated with leukemiain several animals. Oncovirus is the largestsubfamily of retroviridae with following 3groups:a. Avian leukosis viruses.b. Murine leukemia and sarcoma viruses.c. Mammary tumor viruses of mice.

Avian Oncoviruses

Naturally occurring avian leukosis are alwaystransmitted genetically as an integrated DNAprovirus transmitted congenitally to producelatent infection. These viruses are related to:1. Hemopoietic systems (lymphomatosis, mye-

loblastosis, erythroblastosis and osteo-petrosis).

2. Solid tumor. Best studied variant of avianleukosis virus is Rous sarcoma virus (RSV).RSV produces proliferative foci in the chickfibroblast. All RSV transformed cell aredemonstrable malignant cells. Some strainsof RSV can transform cultured mammaliancells and produce tumors in newborn.

In Rous sarcoma virus (RSV) in vitrosynthesis of DNA provirus occurs.

Murine Oncoviruses

Causative virus of murine leukemia is similarto avian oncovirus in physical, chemical andbiological properties. They produce steady statenoncytocidal infection in mouse fibroblast andhence are difficult to detect. Murine sarcomavirus is defective oncovirus capable of multi-plication only in presence of murine leukemiavirus.

In cultured cell, infection of murine sar-coma virus causes transformation. Culturedcells taken from virus free embryo of highleukemia strain of mice can be induced tosynthesize murine oncovirus by treatment withcertain carcinogens or mutagens.

Mammary Tumor Virus

The mammary tumor virus of mice resemblesleukemia viruses in several properties but mor-phology and morphogenesis of virus infectedcell is different. Mammary tumor virus can bemilk transmitted (1936). Genetic transmissionsof these viruses in normal mouse can takeplace.

292 Virology

Feline Oncoviruses

First isolated in 1964, from cat with lympho-sarcoma and leukemia. Feline leukemiavirus induces leukemia after inoculation intokitten and can be grown in cell culture whereit is produced continuously without anycytopathic effect like murine and avianoncovirus.

Oncovirus particle (Sarcoma virus or Fe LVwith feline sarcoma virus) agents are causativeof solid tumor of cat (fibrosarcoma and lipo-sarcoma). Some preparations of virus inducesarcoma in dogs, rabbits, marmosets, monkeysand also can be transformed to human embryocells. Fe LV may be more relevant to humancancer virus problem. Cats like humans whensubjected to some environmental stress andcircumstances develop leukemia at same rate.

Evidence points to horizontal transfer innature of particulate virus that causes leu-kemia in cats. It suggests endogenous, onco-virus genome copy plays a trivial rolesupported by demonstration of excretion of virusinto environment by infected cat and transmissionby contacts of the infection and disease in naturaland artificial closed environments.

Primate Oncovirus

Oncoviruses

There are two distinct classes in non-humanprimates:1. Baboons carry endogeneous oncoviruses

which are vertically transmitted and areotherwise non-oncogenic.

2. Horizontally transmissible tumorgeniconcovirus shown to induce malignantneoplasm when artificially inoculated intoprimates. These are oncovirus recoveredfrom sarcoma, lymphomas, leukemias ofgibbon, apes or woolly monkeys.

MECHANISM OF VIRAL ONCOGENESIS

The exact mechanism of viral oncogenesis isobscure. However, conclusive evidence existswhich suggests particles identified in humanneoplasm are causative or contributory agentsin these malignancies. Questions to theirorigin, mode of transmission and their relation

to carcinogens, have central issue. However,following hypothesis are suggested:1. Provirus hypothesis: After infection genome

of RNA tumor virus is converted intoRNA-DNA hybrid by the enzyme reversetranscriptase present in the virus. ThisRNA-DNA hybrid produces provirus(DNA form of viral RNA) by DNA directedDNA polymerase. This provirus isintegrated into host cell. Now this provirusacts as template for viral RNA synthesisand also brings about cell transformation.

2. Protovirus hypothesis: Suggests that theregions of DNA in vertebrate cells aretranscribed to RNA and then back againto DNA. This process continues andprovides mechanism for gene amplificationand cellular differentiation. Rarelyabnormal events in this mechanism mayresult in the formation of oncogenic RNAvirus genome.

The hypothesis is supported by the factthat new information (viral sequences) maybe detected only in infected and trans-formed cell which may not be present inuneffected normal cell. The points againstthe hypothesis are: (i) presence of RNAtumor viral sequences in normal uneffected“C” type particles, (ii) the verticaltransmission.

3. DNA oncogenic virus hypothesis suggestsintegration of viral DNA with hostgenome. Under its influence the host cellundergoes malignant changes as a resultof synthesis of viral coded antigens, i.e.tumor antigen and tumor specifictransplant antigen. Virus genome mayrelease the cell from normal regulatorymechanism of morphogenesis such ascontact inhibition.

ONCOGENE VIROGENE HYPOTHESIS

Huebner and Todaro (1969) subsequently revi-sed in 1972 and proposed followinghypothesis:

Every cell of one or all vertebrates containsDNA copy of genome of oncovirus. Virusspecific information is vertically transmitteddepending on complex interplay between hostgenotype and environmental conditions. Viral

Oncogenic Viruses 293

production could be elicited at some stage oflife of individuals.

Recent data indicate that most of oncoviru-ses arising from normal cell are not tumor-genic. Indeed many are xenotropic (incapableof multiplying in the cells of mammalianspecies from which they were originallyinduced).

Implication of oncogenic hypothesis, i.e.spontaneous neoplasm is consequence ofevents within cell shows that naturallyoccurring tumor of vertebrate does not behaveas infectious disease.

This hypothesis is supported by the fol-lowing facts: (i) presence of viral gene productand genetic information in normal uninfectedcell, (ii) by inducibility of C type viruses frommany vertebrate cell clones, and (iii) wellknown vertical transmission of some RNAtumor viruses. This hypothesis does not bearon fact that: (i) some tumors are transmissibleby virus, (ii) artificial production of cancer bytumor viruses in laboratory, (iii) oncogenesisby DNA viruses.

Possible Viral Causation ofHuman Cancer

Human cancer is due to viruses inconsistentwith oncogene hypothesis to suggest that allother carcinogens particularly chemical onlyserve to trigger an endogenesis viral oncogene.Another view is that cancer arises by somaticmutations the incidence of which is known tobe increased by hydrocarbons and radiations.

EB is invariably present in Burkitt’s lymp-homa. Is it an etiological agent or irrelevantpassenger? Likewise association of Herpessimplex and cancer cervix is known.

Koch postulates human tumor cells are beingsearched for evidence, viruses coded non-structural antigen (T Ag), mRNA and inte-grated DNA. All RNA tumor viruses contain aviral RNA-dependent DNA polymerase(reverse transcription). It has stimulated thesearch for enzymes as well as oncovirusparticles in human tumor cells. Oncovirusesrecovery from human leukemia is best studiedfrom myeloid cell of patient of acute myelo-genous leukemia. Virus grows well in several

normal lines of human cells in vivo. Resembledoncovirus associated with woolly monkey sar-coma is indicated by nucleic acid hybridi-zation, serological resemblance of reversetranscriptase and other antigen to virion.

Virus is widespread in normal human beingsas all have antibodies against it and virion can beinduced by chemical treatment of variousmalignant human cell lines. Virus has not beenshown to transform human cell to malignantstate in vitro or in vivo.

Prevention of disease by specific viralvaccine may offer definitive means wherebyviral etiology cancer will be established.

NOBEL PRIZE 1989 MEDICINE

Michael Bishop and Discovery ofHarold Varmus the cellular originUniversity of California of viral oncogenesSan Francisco, USA

In their normal form, cellular oncogenesplay a role in growth and development bytriggering gene expression. Their discoveriessuggest that cancer arises from the malfunc-tioning of normal genes and that damage togenes is likely to be responsible for this.

Present working models in cancer considertumors as generally due to genetic damage.There are several lines of evidence which sup-port this notion, e.g. hereditary predisposition,occurrence of abnormal chromosomes, defi-cient DNA repair, the mutagenicity potentialof a number of carcinogens, etc. (all showingassociation with cancers and these all point todefects in the genetic apparatus).

This case is strengthened by Bishop andVarmus’ discoveries of cellular genes (proto-oncogenes) that are involved in normal growthand development which in altered form(oncogenes) in retroviruses can cause tumorgrowth. The transduction (pick up) ofprotooncogenes RNA by retroviruses and itsincorporation in the latter in an altered formhas oncogenic potential. Infection with suchvirus can set in motion tumorigenic events.

The detection of cellular genes involved innormal growth and development throughviral oncogenes is a big step forward in theunderstanding of cancer. These oncogenes can

294 Virology

now be used as tools and introduced intoexperimental animals to see their sufficiencyindividually or in combination in producingcancer. It seems also a good hypothesis thathormones growth factors act as signals forgene expression. Discovery of proto-onco-genes “C”-Jun and “V”-Jun belonging to amulti-gene family which in cooperation withFOS gene and its product through the forma-

tion of so called leucine zippers apparentlystart gene expression at specific sites on DNA.

The work of Bishop and Varmus has pro-vided not only new insights in ourunderstanding of cancer but also opened theway for the study of normal growth anddevelopment as it appears that several of thesecellular protooncogenes are involved in thesenormal processes.

DNA Viruses 295

POXVIRUS

They are the largest and most complex virusesof vertebrates. They are DNA viruses. Poxviridae family is divided into following groupson the bases of antigenic reactions and mor-phological differences:

Group I (Viruses of Mammals)

1. Variola2. Vaccinia3. Cowpox4. Ectromelia5. Rabbitpox6. Monkeypox

Group II (Viruses of Birds)

1. Fowlpox2. Turkeypox

Group III (Tumor Producing)

1. Myxoma2. Fibroma

Group IV (Miscellaneous)

1. Contagious pustular dermatitis2. Milker nodule3. Bovine pustular stomatitis.

Morphology

It is brick-shaped measuring 300 × 200 × 100nm. It consists of central biconcave DNA core.It is covered by: (a) inner coat adhered tonucleoprotein, and (b) outer irregular layer.On either side of nucleoid is oval structurecalled lateral body (Fig. 49.1).

49 DNA Viruses

In dry state virus may remain infective atroom temperature for one year. In moist statevirus can be destroyed at 60°C in 10 minutes.Acid may destroy the virus in hour time (pH 3to 5). They are susceptible to ultraviolet light,formalin and oxidizing agents.

Antigenic Properties

There are about 8 antigens demonstrated byprecipitations in gel. Some of them are:1. LS antigen: It has 2 components: heat labile

(L) and heat stable (S). Antibodies to L-Santigen is not protective in any way. Theyare responsible for flocculation,precipitation and complement fixationreaction.

2. Agglutinogen: This is responsible foragglutination with specific antiserum.

3. Nucleoprotein antigen (NP): It is responsiblefor neutralization of infectivity andacquired immunity. However, NP antigenis common to poxviruses.

4. Hemagglutinin: It is lipoprotein complex witha molecular weight of 100,000 to 200,000. It

Fig. 49.1: Structure of pox virus

296 Virology

is heat stable and does not exhibit receptordestroying activity. Antibodies to this anti-gen are not protective.

5. Protective antigen has been isolated duringearly stage of virus replication. Their rolein immunity has not been proved so far.Cultivation: They can be cultivated on chick

embryo (11 to 13 days old), tissue culture(monkey kidney, He La and chick embryo cell),animals (monkey, calves, sheep and rabbit).

SMALLPOX (VARIOLA MAJOR)

It is infectious disease manifested as skin lesion(single crop) which are macular to start withand subsequently may pass through papular,vesicular and pustular stages in 10 to 12 days.There may be systemic involvement.

Pathogenesis

Variola viruses may enter through mucosa ofupper respiratory tract. Virus may propagatein regional lymph nodes. They are transportedthrough bloodstream to reticuloendothelialcells. There virus multiplication occurs. Fromhere viruses are again thrown intobloodstream and then they settle at skin andthe mucosa with the start of clinical disease.There is formation of macule, papule, vesicleand pustule. Fever at pustular stage may bebecause of absorption of necrotic cell debrisfrom skin. Since smallpox gives increaseddegree of protection hence there is norecurrence of disease.

Clinical Features

The incubation period is about 12 days. Thepre-eruptive phase is characterized by malaise,fever, vomiting, and headache. After 2 to 3days skin rashes start appearing. Single cropof eruptions appears on the 3rd to 5th daysof onset of illness. The lesion first appearson the buccal mucosa (exanthem) whichmay develop into macule, papule andvesicle. Vesicle is associated with virusshedding through oropharyngeal secretions(infective stage). There may be conjunctivitis.Corneal exanthems may become the cause ofblindness.

Scab starts forming after 12 days and crustsstart separating after 3 to 6 week of the onsetof disease leaving behind scars. In pre-eruptionphase the disease is non-infective but virus canbe isolated from blood up to 2nd day of fever.From 6 to 9 days saliva becomes infective, maybe because of ulceration of lesions in mouth.In vesicular stage first broken skin lesion andlater dried scabs appear.

Laboratory Diagnosis

It is significant especially in non-endemic areasor in areas from where disease has beeneliminated. A case identified as smallpox mustbe informed to health authorities so thatproper measures could be undertakenpromptly. It becomes still more importantwhen smallpox is declared eradicated from allover the world.

Collection of Specimen

A special kit has been designed by WHO forcollection of specimen containing:1. Hagedorn needle.2. Clean and sterile slides.3. Clean Pasteur pipettes.4. A labeled screw capped container.5. A double container of metal or wood for

dispatching samples to the concernedlaboratory.We can collect specimen like maculopapu-

lar, pustular, crusting stage lesions. In specialcases blood may be collected in pre-eruptivestage.

Demonstration of Virus

1. By light microscope by demonstratingGuarnieri bodies (Fig. 49.2).

2. By electron microscope.3. Viral antigen may be demonstrated by

serological techniques like complementfixation, hemagglutination, immunofluo-rescent and convenient and routinely usedprecipitation in gel (PIG).

4. Isolation of viruses from throat, washing,skin lesions and blood. Specimen may becultured on chorioallantoic membrane ofchick embryo.

DNA Viruses 297

Demonstration of Antibody

Retrospective diagnosis may be made bydemonstration of antibodies rise by testingpaired sera. In smallpox convalescent’s PIGtest is useful for the retrospective diagnosis ofsmallpox.

Prophylaxis

Some of the preventive measures includeimmunization and chemoprophylaxis.

Vaccination

There are three strains available for vaccineproduction:a. Elstree (Lister Institute)b. EM63 (Moscow)c. New York Board of Health strain.

Commercial vaccine may be prepared byinoculating vaccinia virus on scarified skin ofcalves, sheep and buffalo. Alternatively virusmay be cultured in bovine cells or CAM ofchicken embryo. Vaccine thus prepared isstored and dispensed in liquid form or infreeze-dried form.

Method of Vaccination

It consists of introducing intradermallysufficient live vaccinia viruses. Recommendedsites are outer aspect of upper arm at theinsertions of deltoid muscles. The methodsincludes multiple puncture with bifurcatedneedle and multiple pressure with a sharpneedle.

Response of Vaccination

There are 3 types of response:1. Primary reaction: This is manifested 3 to 4

days after inoculation as papule whichrapidly becomes vesicle and it enlargeswith secondary erythema. On 8th to 9thday center is depressed with turbidcontents. It is also associated with axillarylymphadenopathy and fever.

On 10th day pustule dries up and scabis formed which separates in a week’s time.Immunity appears after 10th day andpersists for years.

2. Accelerated reaction (vaccinole): It occurs incase of limited residual immunity from pre-vious vaccination. It is more rapid thanprimary reactions. Vesiculation appearswith maximum intensity between 3 to 7days after vaccination. It enhances waningimmunity.

3. Immediate reaction: It occurs in immunecases. It is most marked on 2nd to 3rd dayas papule. It is hypersensitivity response.Immediate response neither indicates levelof immunity nor it leads to immunity.

Complication of Primary Vaccination

1. Post-vaccinal encephalitis may occur whichmay be due to activation of latent infection,neuro-allergy, and interaction of vacciniavirus. Encephalitis may occur 10 to 12 daysafter vaccination. The mortality is about 50percent.

2. Vaccinia gangrenosa in which primarylesion fails to heal and extend slowly withloss of tissue. Fresh vesicles may appearwith ulceration of nasopharynx. This isassociated with abnormality of immuneresponse and is usually fatal.

3. Abortion may occur due to intrauterineinfection of fetus. Hence, it iscontraindicated in pregnant women.Since 1977 when last case was reported in

Somalia, smallpox has been eradicated world-wide, hence vaccination is unjustified in viewof its several complications.

MOLLUSCUM CONTAGIOSUM

It is human disease with multiple discretenodule 2 mm size, limited to epidermis and

Fig. 49.2: Guarnieri bodies

298 Virology

occurs anywhere in the body except palm andsole. Each lesion at its top carries small open-ing having white core. The incubation periodis 19 to 50 days. Transmission is through abra-sion and swimming pool. It is very uncommonand involves children and adults. Its trans-mission in animals is not successful.

Cowpox: It occurs in cattle as ulcer of teats andcontiguous part of udder. Lesions appear alsoin the hands of man. It produces hemorrhagein chorioallantoic membrane of chick embryoand rabbit skin. Vaccinations with vacciniavirus protects human beings.

Milker nodules: It occurs in hands of manfrom lesions of teats and udder. Warty, non-ulcerating nodules on hand and arm are cau-sed by poxvirus of ORF subgroup. Regionallymph nodes are enlarged. Immunity in mandoes not last long and second attack may occurafter few years.

ORF: Infection of man occurs with virus ofcontagious pustular dermatitis of sheep. Thereis single lesion of hand, forearm or face, aslowly developing papule which become flatand vesicular and ultimately heals withoutscarring. The disease occurs by handling ofsheep. There is no infection to man.

Yaba and tanapox: Yaba is benign tumorunder natural condition in monkeys of Africancountries. Laboratory worker handling theseanimals may develop similar lesions.

Tanapox is isolated from solitary skinlesion in Kenyans. Patient looks quite ill.Perhaps, it is derived from monkey by insecttransmission.

ADENOVIRUSES

It is non-enveloped DNA virus with diameter70 to 90 nm and are spherical. It has icosa-hedral symmetry. It is relatively stable bet-ween 4° and 36°C and can be stored in frozenstate. It is heat labile destroyed at 56°C withinminutes. It resists ether and bile salts.

It is host specific. Human adenovirusesgrow only in tissue cultures of human origin,e.g. human amnion, HeLa or HEp. Cytopatho-genic changes include rounding of cell andaggregations into grape-like clusters. Intranuc-lear inclusion may be demonstrated by

staining (Fig. 49.3). Human adenovirus mayproduce undifferentiated sarcoma in 30 to 90days when inoculated in newborn hamster.

Adenovirus can be classified into two sub-groups based on their ability to agglutinate ratand monkey erythrocytes. Around 33 sero-types are identified of adenovirus infectingman. They cause self-limited infection ofrespiratory tract, eye and may be intestine. Itmay cause pharyngitis and tonsillitis (types 1to 5), pneumonia (types 4 and 7), acute respira-tory disease (types 4, 7 and 21), pharyngocon-junctival fever (types 3 and 7), epidemickeratoconjunctivitis (type 8), acute follicularconjunctivitis (types 3 and 7a), intestinallesions causing gastroenteritis and obesity(type 36). They may cause oncogenesis inhamster (types 12, 18 and 31).

Laboratory diagnosis may be establishedby isolating virus from throat, eye or feces. Thespecimen may be inoculated on A 549 cell line(American type culture collection USA) tissuecultures like HeLa, HEP-2, and then noting thecytopathic effects. Serological techniques likecomplement fixation, hemagglutination,hemagglutination inhibition and neutraliza-tion are useful.a. Adenovirus SV40: Monkey kidney tissue

culture, used in identification ofadenovirus, may be contaminated withSV40 virus. They do not produce cytopathiceffect on rhesus monkey kidney. Hence,both the viruses replicate and producemature viral particles. In the process we

Fig. 49.3: Cowdry bodies (intranuclear)

DNA Viruses 299

may get viral particle containing genomeof SV40 and capsid of adenovirus, whichmay be called hybrid virus.

b. Adeno associated virus: It is defective virusbecause it does not replicate in the absenceof adenovirus. It is non-pathogenic andantigenically different from adenovirus. Itis DNA virus about 20 nm. It is also knownas adeno-satellite virus.

Herpes Virus (Fig. 49.4)

It is double stranded DNA viruses about 100to 150 nm in size, lipid enveloped and sensitiveto ether and chloroform. It replicates in thenucleus of host cell. It produces intranucleareosinophilic inclusion bodies. It does notpossess common antigen. Examples are:1. Herpes simplex types 1 and 2.2. Varicella.3. Herpes zoster.4. Cytomegalovirus.5. EB virus.1. Herpes simplex: It may produce mild vesi-

cular eruption in skin or mucousmembrane. It has two types; type 1 straincauses infection of mouth, eyes, centralnervous system, etc. and type 2 straincauses infection of genitals. Type 1 andtype 2 may be differentiated antigenicallyalso.

It may be cultured on chorioallantoicmembrane producing typical white,shining, non-necrotic pocks. Pocks are lessthan 0.75 mm in type 1 and more than 1mm in type 2 strain. They can also be

grown on rabbit kidney, HeLa, HEp-2 orhuman amnion tissue culture. They mayproduce experimental infection in animalslike rabbit, mice, etc.

It is one of the most common infectionof man. Only man is the natural host andmay produce illness like herpes labialis,eczema herpeticum, keratoconjunctivitis,and meningoencephalitis. The herpestype 2 is venereal infection and producegenital herpes and neonate herpes. Thevirus may remain latent for years perhapsin sensory nerve ganglia and getreactivated.

Laboratory diagnosis is established byisolation of virus from infected materialwhich may be inoculated on chorioallan-toic membrane of chick embryo where weget typical pocks. They may also be isolatedin rabbit kidney and human amniontissue culture where typical intranuclearinclusion bodies appear. The isolate maybe confirmed by neutralization tests.Serological tests like neutralization andcomplement fixation tests are also usefulfor its diagnosis.

For herpetic keratoconjunctivitis, 5-iodo-2 deoxyuridine is beneficial.

2. Varicella: Morphology of this virus isidentical to that of herpes simplex. It causeschickenpox which chiefly affects children.It is characterized by successive crops oferuptions on skin and mucous membrane.High fever is also there. The source ofinfection is chickenpox patient or herpeszoster patient. There are no animalreservoirs. Portal of entry is respiratorytract. Incubation period is 7 to 23 days.Chickenpox is usually uneventful diseasewith complete recovery. Sometimescomplications may occur like secondarybacterial infection, e.g. rash, encephalitisand pneumonia. One attack confers life-long immunity.

The virus does not grow on animal andchick embryo, but may multiply in humanembryonic tissue culture producing intra-nuclear inclusion bodies (Cowdry type A).Laboratory diagnosis may be establishedby examining smear from vesicle. Giemsastain shows multinucleated giant cells andFig. 49.4: Morphology of herpes virus

300 Virology

intranuclear eosinophilic inclusion bodies.Virus may be isolated on human tissueculture. If vesicle fluid is examined underelectron microscope we may see typicalparticles of herpes virus. Serologicaltechniques like agar gel precipitation test,complement fixation and neutralizationtests may be useful. A fluorescent antibodytechnique is also helpful for detection ofvaricella antigen.

Following varicella vaccines (given at15 months of age) are available:i. Takahashi live attenuated vaccine (not

used because of oncogenicity andpossibility of herpes zoster in later life).

ii. Formalin killed vaccine.iii. Live attenuated OKA (varicella strain)

vaccine after prolonged field trial isfound quite effective. It is indicated forprevention of chickenpox in immuno-compromised children including thosewith hematological cancers or solidtumor. This vaccine is being evaluatedfor routine immunization of healthychildren.

iv. Development of subunit or recombi-nant vaccine may eliminate the risk ofherpes zoster.

Convalescent sera from herpes zosterpatients contain much more levels ofantibody than serum from varicellaconvalescents. Hence, the administrationof this sera may confer protection tocontacts of chickenpox patients.

3. Herpes zoster: It is the disease of old age stillit may occur at any age even newborn arenot spared. The disease is characterized byappearance of skin eruptions over thedistribution of sensory nerves.

The portal of entry is unknown but insome cases varicella virus becomes neuro-tropic and is localized in nerve cells. Fromhere infection spreads along posteriornerve root fibers with formation of vesiclesin the segment of skin supplied by that par-ticular nerve. The rash is usually unilateral.The commonest sites are areas innervatedby spinal cord segments T3 to L2 and trige-minal nerve mostly ophthalmic branch.The rash disappears in 2 weeks but painand paresthesia may persist for months

together. In some cases lower motor neu-ron paralysis with meningoencephalitisand generalized zoster may occur.

Laboratory diagnosis is like that of vari-cella infection. Varicella and zoster virusesappear identical antigenically. It appearsthat the same virus in children duringprimary infection produces varicella(generalized infection) and when adults areinvolved they develop zoster (localizedinfection).

4. Cytomegaloviruses: They are indistin-guishable from other viruses of herpesgroup. They may infect man, monkey,guinea pig, etc. Characteristically theycause enlargement of infected cell withacidophilic or basophilic intranuclearinclusion body. Human strains areantigenically heterogeneous. They can begrown on human fibroblast cultures. Sincecytopathic effects are slow in appearancethey require prolonged incubation.

It may cause subclinical localizedinfection of salivary glands, kidney andrarely generalized disease in infants. Theinfection can be transmitted by urine, salivadroplets via respiratory route and throughplacenta from infected mother to fetus. Thegeneralized infection is associated withhepatosplenomegaly, jaundice, thrombo-cytopenic purpura, hemolytic anemia andmicrocephaly. Apart from this there maybe chorioretinitis and cerebral calcification.Sometimes syndrome resemblinginfectious mononucleosis may occur. Itmay lead to insidious hepatitis orpneumonia.

Laboratory diagnosis is established byisolating the virus from throat swab, urineand various affected organs. This materialis inoculated on human fibroblast tissueculture and after 1 to 6 weeks characteristiccytopathic effects appear with presence ofinclusion body. Serological diagnosis likecomplement fixation and neutralizationmay be helpful. Histological study ofvarious organs may show large swollen cellwith inclusion bodies (intranuclear)suggesting cytomegalovirus infection.

5. Epstein Barr virus: It is indistinguishablefrom other viruses of herpes group. It hasthe affinity for lymphoblastoid cells. There

DNA Viruses 301

is now strong evidence that infectiousmononucleosis is caused by Epstein Barrvirus (EB virus).

To establish diagnosis bloodexamination shows leukopenia in earlystages followed by leukocytosis. Theabnormal mononuclear cell with basophilicvacuolated cytoplasm and kidney shapednucleus with fenestrated chromatin is seen.By electron microscope examination EBvirus may be identified. Serological testslike Paul Bunnel, complement fixation,immunofluorescence and gel diffusionmay be useful.

Human Herpes Virus Type 6

• First isolated in 1986 from peripheral bloodleukocytes from patients of lymphopro-liferative disorders. Reported to be widespread in UK, Japan, USA, etc.

• Although previously called B-lymphotro-pic virus, now identified as primarily T-lymphotropic.

• Two genetically distinct variants are reco-gnized and are HHV-6A and HHV-6B).

• HHV-6 frequently develops duringinfancy.

• HHV-6 can cause exanthem subitum, feb-rile seizures without rash during infancy,etc.

• In older age HHV-6B has been associatedwith mononucleosis syndromes, focalencephalitis, pneumonitis, disseminateddisease.

• HHV-6A has not been associated withdisease.

• Virus is transmitted through saliva andprobably by genital secretion.

• There is no established treatment or vaccineavailable for this virus.

Human Herpes Virus Type 7

• Isolated in 1990 from T-lymphocytes ofhealthy man from peripheral blood.

• The virus was subsequently isolated fromother persons too.

• Virus is acquired during childhood andpresent in saliva of healthy persons.

• Some cases of exanthem subitum may beassociated with this virus.

Human Herpes Virus Type 8

• Unique herpes virus like DNA sequencereported in 1994-95 in tissue derived fromKaposi’s sarcoma and body cavity basedon lymphoma in AIDS patients.

• HHV-8 isolation in cell culture may defineits role in disease.

• They are partially homologous to the DNAof Epstein Barr virus, herpes virus samiriof squirrel monkeys.

• These herpes virus like DNA sequenceshave also been reported from Kaposi’ssarcoma tissues sarcoma from non-AIDSpatients, in a subgroup of AIDS related B-cell body cavity based lymphoma, and inbrain tumor some proliferative skin lesionsof organ transplant recipients.

• These DNA sequences have also been seenin semen of both AIDS and non-AIDSpatients.Initially discovered by molecular biology

techniques in Kaposi’s sarcoma, this newherpes virus has been isolated in Kaposi’s sar-coma using cell cultures. This virus is asso-ciated with 3 conditions, i.e. Kaposi’s disease,B-cell lymphoma and Castleman’s disease.

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50 RNA Viruses

PICORNAVIRUSES A

They are very small, 20 to 30 nm in size, non-enveloped and resistant to ether. Picornavirusgroup of medical importance includes:A. EnterovirusesB. Rhinoviruses

Enteroviruses

From the medical point of view importantviruses of this group are polio, echo andcoxsackie viruses. Enteroviruses are stable,and resistant to bile and ether. They remainunharmed in water and sewage for quite along time. They are described as under:a. Poliovirus: They are 30 nm diameter with

capsomere arranged in icosahedral sym-metry and are spherical. They are resistantto ether, chloroform and bile. They survivein low pH and low temperature. They arekilled by formaldehyde, cholinination andlyophilization. By neutralization poliovirusstrains are classified into types I, II and III.Type I is the commonest and is responsiblefor epidemic. Natural infection occurs onlyin man.

The virus enters body by ingestion orinhalation. The virus multiplies in lym-phatic tissue of alimentary canal (fromtonsils to Peyer’s patches) enteringregional lymph nodes and then viruses arecarried to bloodstream. From here virusesare taken to spinal cord and brain. Theydestroy neurons with degeneration of Nisslbody. Lesions are mostly in anterior hornof spinal cord. Sometimes we may findextensive lesions like encephalitis. Theincubation period is about 10 days withrange from 4 days to 4 weeks.

Laboratory diagnosis is made by isolationof viruses from throat (early stage) andfeces (throughout the course of disease).After processing specimen is inoculatedinto tissue culture and virus growth isindicated by cytopathic effects in 2 to 3days. Identification of virus should beinterpreted along with clinical picture.Serodiagnosis is not of much use, stillcomplement fixation and neutralizationtest may be valuable.

Immunization is achieved by usingvaccine. Salk killed polio vaccine isformalin inactivated consisting of 3 typesof polioviruses. It gives 80 to 90 percentprotection against paralytic poliomyelitis.Killed vaccination is given by injection. Onthe other hand, Sabin live polio vaccine isalso available and is prepared by growingthe attenuated strain in monkey kidneycells. Live vaccine is easy to administer asit is given orally, much more economical,single dose gives lifelong immunity andgives local immunity in the intestine.

b. Coxsackieviruses: They are called coxsac-kievirus as first of all they were isolatedfrom patients coming from the village ofcoxsackie in New York. They are classifiedinto group A and B. By neutralizationmethod group A viruses are divided into24 types. Characteristically the viruses havethe ability to infect suckling mice and notthe adult mice. All group B viruses growon monkey kidney tissue culture and somegroup A viruses grow in HeLa cells.They may cause vesicular pharyngitis(group A), aseptic meningitis (groups Aand B), minor respiratory infections (A21),Bronholm disease manifested as stitch-like

RNA Viruses 303

pain in abdomen and chest (group B),myocarditis (group B) and pericarditis(group B).The laboratory diagnosis may be made byisolating the viruses from lesion or fecesby inoculation in suckling mice. Since thereare several antigenic types so serodiagnosisis not feasible.

c. Echoviruses: Their description designationis enteric cytopathogenic human orphanviruses (ECHO viruses). They are classifiedinto 33 serotypes. They infect man natu-rally. They are not pathogenic to labora-tory animals.

They may produce fever with rash andaseptic meningitis (types 4, 6, 9 and 16).Laboratory diagnosis is by inoculatingfeces, throat swab or CSF on monkeykidney tissue culture and virus growth isdetected by cytopathogenic changes.

Newer Enteroviruses (68 to 72)

Four types of viruses, i.e. 68, 70, 71, 72associated with diseases of man as under:

68 Pneumonia70 Acute hemorrhagic conjunctivitis71 Mumps72 Hepatitis A.

Rhinoviruses

They differ from enteroviruses in being moreacid labile and heat stable. They have beenclassified into over 100 types and immunity istype specific. Depending upon growth intissue culture, rhinoviruses are classified as Hstrains (grow only on human cells) and Mstrains (grow equally well on human as wellas monkey cells). Because of too manyserotypes (over 100) it is impossible to makeideal vaccine. However, antiviral chemo-therapy may be helpful in bringing specificcontrol.

ORTHOMYXOVIRUSES

It includes the enveloped RNA viruses capableof adsorbing on to mucoprotein receptor onerythrocytes. This results in hemagglutination.They are 80 to 120 nm in size and spherical inshape. Influenza virus represents this group.

INFLUENZA VIRUSES

They are responsible for infectious disease ofrespiratory tract occurring mostly in epidemicand pandemic forms. The classification ofinfluenza virus into 3 (A, B and C ) is based onthe antigenic nature of ribonucleoprotein.

Influenza virus is spherical with diameter80 to 120 nm. The virus has ribonucleoproteinin helical symmetry. Single stranded RNAgenome is segmented and nucleocapsid issurrounded by envelope having virus codedprotein layer and lipid layer derived from hostcell. Attached to lipid layer are hemagglutininspikes and neuraminidase peplomers(Fig. 50.1). The virus is inactivated at 50°C for30 minutes, ether, formaldehyde, phenol andsalts of heavy metals.

The characteristic feature of influenza virusis its ability to undergo antigenic variation.Depending on degree antigenic variation maybe classified as (Table 50.1):

i. Antigenic shift (abrupt, drastic, disconti-nuous variation in antigenic structurecausing major epidemic).

ii. Antigenic drift (gradual changes inantigenic structure regularly, resultingin periodical epidemic).

The virus grows in amniotic cavity andallantoic cavity of chick embryo. It is detectedby appearance of hemagglutinin in allantoicand amniotic fluid. They are also grown inmonkey kidney cells. Route of entry isrespiratory tract. The viral neuraminidase faci-litates infection by reducing the viscosity ofmucus lining and exposing the cell surfacereceptor for virus adsorption. These cells aredamaged and shed, laying bare the cells intrachea and branchi.

Fig. 50.1: Structure of influenza virus

304 Virology

The incubation period is 1 to 3 days. Theonset is abrupt with fever, headache, genera-lized myalgia and prominent respiratorysymptoms. If no complication follows thedisease resolves in 2 to 7 days. Complicationsinclude pneumonia due to bacterial super-infection, congestive heart failure and encep-halitis. Reye’s syndrome is associated withinfluenza B virus.

Diagnosis in the laboratory is establishedby demonstration of virus antigen (immuno-fluorescence), isolation of virus (chick embryoor monkey kidney cell culture), serology(complement fixation test, hemagglutinationinhibition test) and radial immunodiffusiontests in agarose gel (screening test).

Influenza vaccine is in use. Vaccine may beprepared by growing virus in allantoic cavityand inactivating the virus with formalin.Because of presence of egg protein this vaccinemay cause allergic reactions. This difficulty isremoved by preparing subunit vaccines (virustreated with ether). The other vaccines in useare: (i) recombinant live vaccines obtained byhybridization between its mutants of estab-lished strain, (ii) new antigenic variant, a neura-minidase specific vaccine, and (iii) a live vaccineusing temperature sensitive (TS) mutant, etc.

Antiviral drug amantidine hydrochloridewhich inhibits adsorption of virus to cell isuseful in influenza infection. Combined yearlyvaccination of persons at high risk, usingthe best mix of important antigens and adminis-

tration of amantidine at time of stress, e.g.surgery or hospitalization, etc. is suggested.

PARAMYXOVIRUSES

They are larger and more pleomorphic thanorthomyxoviruses. They possesses hemagglu-tinins, neuraminidases and hemolysin. Theyare antigenically stable. This group includesviruses like mumps, parainfluenza, respiratorysyncytial and measles.

Mumps

It is responsible for acute infectious diseasecharacterized by parotitis. The name mumpsis derived from mumbling speech of patients.

The virus is spherical varying from 100 to250 nm. The envelope has hemagglutinins, aneuraminidase and hemolysin. The virus canbe grown on yolk sac or amniotic fluid of chickembryo, and human or monkey kidney cellculture. They are inactivated at room tempera-ture, ultraviolet light or by chemicals likeformaldehyde and ether. Two complementfixing antigens have been identified as soluble(S antigen) and viral (V antigen).

Infection may be by inhalation and throughconjunctiva. Incubation period is 18 to 21 days.Clinical symptoms start with sudden non-suppurative enlargement of parotid glands.Skin over the enlarged parotid glands may bestretched, red and hot. Viremia may be res-ponsible for the involvement of other organs.Orchitis and viral meningoencephalitis areimportant complications of mumps. The pan-creas, ovary, thyroid and breast may beinvolved. However, it is important and mostcommon cause of aseptic meningitis.

Diagnosis is confirmed by isolation of virusfrom saliva, CSF or urine. For this purposeamniotic cavity of chick embryo or monkeyor human kidney cell culture may be used.Serological test like complement fixation,hemagglutination inhibition and neutraliza-tion tests may be helpful. Skin test is not veryuseful but still it can be used to detectsusceptible patient.

Mumps infection confers life long immu-nity. Normal human gamma globulin prepa-red from mumps convalescent serum appearsuseful for prophylaxis.

TABLE 50.1: Emergence of antigen subtypes ofinfluenza A associated with pandemics or epidemicdiseases

1889-90 H2N8 Severe pandemic

1900-03 H3N8 Moderate epidemic1918-19 H1 N1 Severe pandemic

(formerly Hsw N1)1933-35 H1N1 Mild epidemic

(formerly H0N1)1946-47 H1N1 Mild epidemic1957-58 H2N2 Severe pandemic

1968-69 H3N2 Moderate pandemic1977-78 H1N1 Mild pandemic1988-1989 H1N1/H3N2 Have circulated

either in alternatingyears or concurrently

2009 H1N1(Swine flu) Pandemic April 2009

RNA Viruses 305

For active immunization killed vaccine(virus grown in allantoic cavity), Jeryl-Lynnstrain (live attenuated vaccine) and now livevaccine is available which can be sprayed intomouth without any side effect.

Respiratory Syncytial Virus

Although these viruses resemble paramyxovi-ruses structurally but they do not have eitherhemagglutinin or neuraminidase. They areantigenically stable and grow on HeLa cells,HEp-2 and in monkey kidney cells. They areresponsible for bronchiolitis and pneumonia.In adults, it may cause afebrile rhinitis and inaged persons it may cause exacerbation ofbronchitis.

For diagnosis, nasal and pharyngeal secre-tion are inoculated in human (He La, HEp2)or monkey kidney cell culture. It takes 5 to 14days’ time. Rapid diagnosis may be made byimmunofluorescent technique. Serologicaltechniques like complement fixation andneutralization test may be useful. No vaccineis available at present.

Parainfluenza Viruses

They may produce febrile respiratory infec-tions throughout the year. They possesshemagglutinin, neuraminidase and hemolysin.They grow well in human or monkey kidneycell culture. Growth in chick embryo is pooror absent. They are inactivated by heat and byether. They are classified into four groups:Parainfluenza 1, Parainfluenza 2, Para-influenza 3, Parainfluenza 4.

Parainfluenza viruses are responsible forabout 10 percent respiratory infection inchildren. Types 1 and 2 cause croup which isa serious clinical disease. Type 3 causes lowerrespiratory infections and type 4 causes minorrespiratory infections.

Measles

It is highly acute infectious disease character-ized for generalized maculopapular rashproceeded by fever, cough, nasal and conjunc-tival catarrh, etc. (Fig. 50.2).

The viruses possess hemagglutinin and noneuraminidase. They do not grow in eggs but

may grow on human embryonic kidney oramnion cell cultures. The virus’s core may beinactivated by heat, ultraviolet light, ether andformaldehyde. They are antigenically homo-geneous.

Incubation period is 10 to 12 days. Infectionmanifests as fever and respiratory tractinvolvement. At this stage Koplik spots maybe seen on buccal mucosa and 2 to 4 days laterrash appears. Uneventful recovery occurs inmost of the patients. In small number of casescomplications like croup or bronchitis, secon-dary bacterial infection, giant cell pneumoniaand meningoencephalitis may occur. Veryrarely we may have late complication likesubacute sclerosing panencephalitis (SSPE).

The diagnosis may be established by iso-lating the virus from nose, throat, conjunctiva,blood and urine. Primary human embryonickidney and amnion cells are quite useful.Rapid diagnosis of virus growth is possible byimmunofluorescence. However, smear can beprepared from nasal, pharyngeal and conjunc-tival secretion and examined microscopicallyafter staining with Giemsa’s method forpresence of giant cells and inclusion bodies(Cowdry type A). Serological techniques likecomplement fixation test, neutralization, andhemagglutination inhibition may be useful forestablishing diagnosis of measles.

Normal human gamma globulin if givenwithin 6 days of exposure can prevent disease.A formalin inactivated vaccine against mea-sles proved not of much use. Live attenuatedvaccine is developed using Edmonston Bstrain. This vaccine can be given in combina-

Fig. 50.2: Measles virus

306 Virology

tion with mumps and rubella vaccines (MMR).The other live attenuated vaccines areSchwartz and Mortin strain and Backham 31strain.

RUBELLA VIRUS

It is an enveloped RNA virus causing rash andlymphadenopathy (posterior and suboccipital)in children. In adults, there is involvement ofjoint and purpura. Infection in early pregnancymay lead to developmental defects in fetus(embryopathy).

The virus is pleomorphic, spherical, 50 to70 nm in diameter and enveloped. It has onetype of antigen (hemagglutinin). It is heat labileand inactivated by ether and chloroform.Rubella virus can be grown in primary Africangreen monkey kidney tissue cell lines (VERO,RK 13), human amnion and thyroid tissueculture. The presence of virus is detected byinterference test.

It enters the body by inhalation and replica-tion of virus occurs in cervical lymph nodes.After incubation period (2 to 8 weeks) viremiaoccurs which lasts till rash, fever and lymph-adenopathy appears. Arthritis is commoncomplication especially in females. If rubellaoccurs in early pregnancy the fetus may dieotherwise congenital malformation is commonin first trimester. The most common malfor-mation produced by rubella are cardiac defect,cataract and deafness. The other features inbabies of congenital rubella are hepatospleno-megaly, thrombocytopenic purpura, myocar-ditis and bone lesions.

Incidence of defects in rubella infection isclosely linked to stage of pregnancy when theinfection is acquired:

Stage of pregnancy Incidence of defects

1 month 50%2 months 25%

3 months 17%4 months 11%5 months 6%

6 months onwards Very low

Rubella virus is found in all excretions ofcongenitally infected infants. That is the reason

of infected babies constituting importantsource of infection to the staff in nurseries.

Diagnosis can be established by virusisolation from blood (early stage) and throatswabs. Growth on rabbit kidney or vero cellculture is detected by interference with Echo11 virus. However, serological diagnosis ismade by hemagglutination inhibition, neutra-lization, complement fixation, immunofluores-cent of platelets and aggregation tests. Four-fold or more rise in convalescent serums isdiagnostic. In congenital rubella diagnosis ismade by demonstrating lgM.

Rubella infection gives life-lasting immu-nity as there is one antigenic type of virus.Prophylaxis is relevant only to women ofchildbearing age. The vaccines (live atte-nuated) available are Cendehill and HPV 77(serial passage in tissue culture). They areadministered subcutaneously. Drawback isthat arthritis occurs after vaccination. To 336and HPV 77 DES are other vaccines. Newvaccine has come up (RA 27/3) which isadministered intranasally conferring localimmunity as well. The safe period of givingvaccine in young girls is 11 to 13 years and inwomen immediately after delivery.

RHABDOVIRUS

They are classified as Rhabdovirus with bullet-like shape. They are enveloped RNA whichmultiply in cytoplasm of host cell and matureby budding from plasma membrane. The mostimportant virus of this group is rabies virus.

Rabies Virus

They are bullet-shaped with one end blunt andother end pointed, 120 to 200 nm long withcylindrical diameter of 60 to 80 nm. The corecontains RNA in helical symmetry. The virionis surrounded by lipoprotein envelop fromwhich project hemagglutinin spikes (Fig. 50.3).They are killed by ultraviolet light, heating at56°C for one hour and 60°C for 5 minutes,ether, strong acid, strong alkalies and trypsin.All strains are antigenically similar. Theyinduce formation of fixing, neutralizing andhemagglutination inhibition antibodies.

RNA Viruses 307

Virus can grow almost in all warm bloodedanimals, suckling mice being better suited forvirus isolation. They can also be grown onchick embryo, duck embryo, tissue culturesprepared from mouse or chick embryo (fibro-blast), hamster kidney and human diploidtissue.

Man is infected by the bite of rabid animals,e.g. dog. Saliva of the infective animal containsrabies virus which are deposited in woundconferred by the bite of animal. The virusestravel through nerve fibers to the spinal cordand brain. From central nervous systemviruses spread to salivary glands and othertissues. Incubation period is from 1 to 3months. Prodromal phase usually lasts for 2to 4 days with manifestations as malaise, ano-rexia, nausea, vomiting, headache and fever.This stage is followed by sensory phase whenpatient feels peculiar sensation around woundand attempt to swallow results in painfulspasm of muscles of deglutition. This phase isfollowed by excitory phase in which patientgets generalized convulsions and coma. Manya time death is preceded by paralysis.

It is more important to demonstrate virusin the rabid animal than in patients. However,in patients, demonstration of viral antigenfrom facial skin biopsy and in corneal smearmay be done. Immunofluorescent may beemployed for antemortem diagnosis whereaspostmortem demonstration of inclusion bodyor virus antigen in the brain or isolation ofvirus by mouse inoculation are the standardprocedures. For the diagnosis in animals sus-pected to die of rabies preferably severed headshould be sent to laboratory. If possible brainof the animal may be removed and dividedinto two portion, one in 50 percent glycerolsaline (biological test) and the other in Zenker’sfixative (microscopic examination). Hippo-campus major part of brain should be includedas it contains abundant inclusion bodies.Impression smears are stained by Sellertechnique. The inclusion bodies (Negri bodies)are seen as intracytoplasmic, round andpurplish pink structure (Fig. 50.4). By indirectimmunofluorescence test using antirabiesserum fluorescein conjugate, deposit of virusantigen may be demonstrated in infected cell

long before Negri bodies appear. Ten percentsuspension of brain is injected intracerebrallyinto mice. Impression smears of brain showmany Negri bodies. Isolation of virus fromsaliva of man or animal may be tried for thispurpose by intramuscular injection to Syrianhamster because of its susceptibility.

Prophylactic measures of rabies consists ofwashing the wound with water and soap andthen cauterizing it with carbolic acid or quarte-nary ammonium compounds. Currently, fol-lowing antirabic vaccines are available:a. Neural vaccines:

i. Fermi vaccines not in use now.ii. Semple vaccine is widely used.

iii. Sheep brain vaccine.iv. Inactivated vaccine is prepared from

suckling mouse or rabbit. It carries littlerisk of neurological complication.

b. Live attenuated chick embryo vaccine isavailable in two forms:i. Low egg passage (LEP)

ii. High egg passage (HEP)c. Duck egg vaccine.d. Tissue culture vaccines have low potency.

The recommended dosage schedule ofsemple vaccine for different classes is asunder:

Fig. 50.3: Rabies virus

Fig. 50.4: Negri bodies

308 Virology

CLASS SEMPLEVACCINE

CLASS ILicks on fresh cuts withsaliva all over the bodyexcept head, face andneck, licks on intact 2 ml for 7 daysmucosa of mouth, nose,and conjunctiva or bitesscratches without bleedingand handling raw flesh ofrabid animals.CLASS IILicks on fresh cut orabrasions on fingers,unlacerated bites orscratches on the fingers 5 ml for 14 days(½ cm long) and bites orscratches on all parts ofbody except head, face,neck or finger whichhave drawn blood.Number of bites shouldnot be more than five.CLASS IIILicks and bites or freshcut or abrasions on head,face and neck, bites,(lacerated) on finger(more than 1 cm), bitescausing laceration anddrawing blood, morethan 5 in number,all jackal and wolf bites 10 ml for 14 daysand class II patient whohas not received treatmentwithin 14 days of injury.

ARBOVIRUS

They are arthropod borne viruses (transmit-ted by blood sucking insects vectors). They areRNA viruses, spherical, 20 to 60 nm in dia-meter having lipid envelope and susceptibleto ether, bile salts, etc. Their ability to multiplyin arthropods is their special character.

Today 41 arboviruses have been isolatedin India. Of these 41 arboviruses, 12 are knownto cause human disease in India and anadditional 3 have been associated with human

disease in other countries but not in India(Sindbis, Wanowrie and Bhanja viruses).

These viruses are:Family/Genus Name

Togaviridae/Alphavirus 1. Chikungunya2. Sindbis3. Japanese encephalitis

Togaviridae/Flavivirus 4. West Nile5. Dengue types 1, 2, 3 and 46. Kyasanur forest disease

Bunyaviridae/ 7. Sandfly fever SicilianPhlebovirus 8. Sandfly fever NaplesBunyaviridae/Nairovirus 9. GanjamRhabdoviridae/ 10. ChandipuraVesiculovirusUngrouped 11. Wanowrie

12. Bhanja

Five of these viruses, i.e. Kyasanur forestdisease, Ganjam, Chandipura, Wanowrie andBhanja viruses were first discovered in India.

These viruses may be grown by intracere-bral inoculation in suckling mice, yolk sac,chorioallantoic membrane of chick embryo, cul-ture of insect tissue and tissue culture primarycell like Vero, HeLa. Three antigens areidentified and they are complement fixing,hemagglutinin and neutralizing antigens. Basedon antigenic relationship in hemagglutinationinhibition and complement fixation test 300arboviruses are placed into over 20 groups.Medically important groups are A (20 mosquitoborne viruses), B (26 mosquito borne and 8 tickborne), C (11 mosquito borne) and so on.

Arbovirus infection in man occurs by thebite of an arthropod and through micropunc-ture virus is introduced. Viral multiplicationoccurs in capillaries and lymphatics and fromhere the virus goes to blood (viremia) ofpatient. As such virus may be present inmacrophages or lymphocytes or adsorbed tothe surface of platelets or RBC and thuswidespread viral dissemination occurs all overthe body. Encephalitis producing virus causelesions all over the brain. In yellow fever, virallocalization may occur in the liver resulting inmidzonal necrosis.

Blood collected during acute phase ofdisease may be used to isolate and identify theviruses for establishing diagnosis. Isolationmay also be made from CSF but brain is thebest specimen for this purpose. Specimens are

RNA Viruses 309

inoculated in suckling mice intracerebrally andanimal develops encephalitis after some time.Tissue culture and eggs may be used forculture of virus. Serological techniques likehemagglutination inhibition, complement fixa-tion, gel precipitation and neutralization withantisera are useful for establishing thediagnosis. However, virus isolation frominsect vector also helps in identification ofarbovirus activity in the area.Group A viruses:1. Encephalitis viruses

a. Western equine encephalitisb. Eastern equine encephalitisc. Venezuelan equine encephalitis.

2. Febrile illness causing virusesa. Chikungunya virus (in India it was

demonstrated in 1963)b. O’nyong nyong virusc. Sindbis virus (also recovered from

India).Group B viruses:1. Encephalitis virus

a. St. Louis encephalitis virusb. Ilheus virusc. West Nile virusd. Murry Valley encephalitise. Japanese B encephalitis virus occurs in

Korea, Japan, India and Malaysia. Theyare called Japanese “B” encephalitis todistinguish it from encephalitis Agroup. Severe epidemics have occurredsince its isolation in 1935 in Japan. Culextritaeniorhynchus (mosquito breeding inricefields) is principal vector. Birds andpigs are reservoir hosts.

This was recognized in India in 1955when virus was isolated from Culexvishnui mosquito. Epidemics have beenreported in West Bengal, Bihar andAssam since 1973 latest being in 1978.

A formalin inactivated vaccine isused. Vaccination of pig is suggestedfor checking of epidemics.

2. Yellow fever does not exist in India becauseof strict vigilance on vaccination andquarantine for travel from endemic area.The other reason may be that stray virus

introduced may not be able to get estab-lished in vector due to prevalence in localAedes aegypti of dengue fever virus. 17Dvaccine is safe for prevention.

3. Dengue: This virus is distributed all overtropics and subtropics. It occurs inter-mittently in large epidemics. It is trans-mitted from man to man by Aedes aegyptimosquito. It is common in India. All fourtypes are identified in India. Control ofdisease may be achieved by controlling thevector. No vaccine against dengue isavailable so far.Four serotypes of dengue viruses are

identified abbreviated as DEN-1, DEN-2,DEN-3 and DEN-4.

Laboratory diagnosis includes detectionof virus (Immunofluorescence techniques,monoclonal antibodies, radiolabeled RNAprobes, etc.), detection of antibodies hemag-glutination inhibiton test, IgM capture test, dotenzyme immunoassay) and rapid diagnostictests (reverse transcriptase PCR, fluorogenicELISA).

Dengue vaccine is a live tetravalent con-taining all 4 serotypes. It has been developedin Thailand and in currently undergoingphase II trials in children. Molecular biologytechniques used for preparing vaccines eitherexclude the “enhancing epitopes” in the Ecomponents or use other protein like NS-1.More recently, it has been shown that denguepre-M can also confer protective immunity.

Tick Borne Group

i. Russian spring summer encephalitisvirus.

ii. Kyasanur Forest disease viruses causehemorrhagic fever and occur in India(Karnataka state). Forest birds and ani-mals are believed to be the reservoirhosts.

iii. Omsk hemorrhagic fever virus.iv. Louping ill: This is only arthropod borne

togavirus infection occurring in Britain.In sheep it causes cerebellar damage andcharacteristic ataxic movements. It maybe transmitted by tick (Isodex ricinus) toman who may develop mild encephalitis.

310 Virology

Bunyaviridae

The term Bunyaviridae is derived from alocality in Africa Bunyamwera. This group ofviruses is the largest arboviruses.

Morphology

They are enveloped, single stranded, RNAviruses about 60 to 100 nm in diameter, havinghelical symmetry. They multiply in thecytoplasm of host cell and attain maturationby budding into vacuoles.

Classification

There are about 100 species in this group.Recent classification is as under:1. Bunyavirus.2. Phlebovirus.3. Nairovirus.4. Unkovirus.

These are further divided into strains andserological types based on reservoir animals/vectors, geographical distribution and sero-logy.

Pathogenesis

They produce infection in man rarely.The vector (mosquito, etc.) infects the virus

directly into the bloodstream through saliva.Multiplication of virus occurs in vascularendothelium and in the reticuloendothelialcells of lymph nodes, spleen, liver, etc. After 4to 7 days viremia occurs with systemicsymptoms.

Clinical Picture

It may start with fever, chills, aches, arthritis,myositis, hemorrhagic skin rash, nephritis orencephalitis.

Laboratory Diagnosis

Culture on cell lines like, BHK-21, Vero maybe used. With repeated passage there arevery little cytopathogenic effects. However,granular cytoplasmic inclusion may bedemonstrated.

Serological methods like complementfixation test, hemagglutination inhibition testand neutralization test are useful.

Severe Acute Respiratory Syndrome (SARS) 311

51 Severe Acute RespiratorySyndrome (SARS)

Towards the tail end of 2002 a new syndromeemerged in southern China. It was named asSevere Acute Respiratory Syndrome (SARS).The initial outbreak was in peak in April 2003.By June 2003 there had been 8,000 casesworldwide and 775 deaths.

SARS is caused by a novel coronavirus(CoV). It does not appear to be related with 3known classes of coronavirus. It is hypo-thesized on the basis of available data thatanimal virus recently mutated and developedthe ability to productively infect man. Groups1 and 2 contain mammalian virus while Group3 contains avian virus. SARS CoV defines4th class of coronavirus and it exhibitsfollowing features:

1. It has 29,727 nucleotides in length.2. It has 9 open reading frames that are not

found in other coronavirus and may codefor proteins that are unique to SARS virus.

3. It is large, enveloped having positivestranded (27 to 30 kb) and may causerespiratory and enteric diseases in man andanimal.

4. Its genome is largest found in any RNAvirus.

5. Human coronaviruses are found both inGroup I (H Cov -229 E) and Group II (HCoV-OC 43) and are responsible for 30percent mild respiratory tract infection(Figs 51.1 and 51.2).

Fig. 51.1: Severe acute respiratory syndrome (SARS)

312 Virology

Fig. 51.2: SARS-Associated coronavirus

7. Mortality and morbidity resembles 1918influenza epidemic.

8. Death may occur from progressive res-piratory failure in 3 to 30 percent of cases.

LABORATORY DIAGNOSIS

1. Isolation of SARS CoV in monkey Vero E 6cells in tissue culture.

2. Reduction in lymphocyte count.3. Rise in aminotransferase activity which

indicates damage to the liver.4. Reverse transcription PCR using respira-

tory secretions.5. Indirect immunofluorescent to demon-

strate rising antibodies.6. ELISA to detect rising titer of antibodies.7. Chest radiograph.

MANAGEMENTThere is no consensus so far treatment forSARS is concerned. Only symptomatic treat-ment and quarantine of the patient seems tobe feasible for the time being. The drug ofparticular interest and promising is the onewhich blocks the protease function. No vaccinehas been developed. A major hurdle towardsdevelopment of vaccine is antigenic shift.

CLINICAL PICTURE

SARS is transmitted by inhalation because thevirus may be present in droplets aerosol ofrespiratory tract secretions of the patients.Incubation period of SARS is 5 to 7 days.Manifestations of SARS are as under:1. Fever 38°C or more.2. Dry non-productive cough.3. Myalgia.4. Sore throat.5. Shortness of breath.6. Atypical pneumonia.

Avian Influenza (Bird Flu) 313

52 Avian Influenza(Bird Flu)

Birds are especially important species becauseall known subtypes of influenza A virusescirculate among wild birds, which are consi-dered the natural hosts for influenza A viruses.Influenza viruses that infect birds are calledavian influenza viruses.

The causative agent is H5 N1 a subtype ofinfluenza A virus. It generally affects birdpopulation all over Asia. Outbreaks of avianinfluenza A (H5 N1) have been confirmedamong poultry in Cambodia, China, HongKong, Indonesia, Japan, Laos, South Korea,Thailand and Vietnam. The virus H5 N1 wasfirst isolated from birds in South Africa in1961.

Influenza A viruses may be divided intosubtypes on the basis of their surface proteins,i.e. hemagglutinin (HA) and neuraminidase(NA). There are 15 known H subtypes.Whereas all subtypes are found in birds, only3 subtypes of HA (H1, H2 and H3) and twosubtypes of NA (N1 and N2) are known tohave circulated widely in man. This virusmakes wild birds sick, but may make domes-ticated birds very sick and may kill them. Sev-eral instances are on records where humaninfections and outbreaks have occurred in last8 years. There is possibility of different varia-tions of H5 N1. Following information hasbeen gathered about H5 N1:1. All genes are of bird origin. Since virus has

not acquired genes from human influenzavirus so possibility of person to personspread is more likely.

2. There is possibility of different variationsof H5 N1 virus which is in circulation atthis time. Genetic sequencing of virussamples from South Korea and Vietnam

suggest that viruses of these countries aredifferent.

SPREAD OF INFECTION

Infected birds shed viruses in saliva, nasalsecretions and feces. Bird to birds transmissionis by contact with contaminated excretions. H5N1 infection in man may be because of contactwith infected poultry or contaminated surfaces.Of late human to human transmission has beenreported.

SYMPTOMS

The symptoms in man include fever, cough,sore throat, muscle aches. Additionally theremay be eye infection, pneumonia, acuterespiratory distress and other severe lifethreatening conditions.

PREVENTION AND TREATMENT

Prevention measures include killing of sick andexposed birds, isolation and treating thepatients. Travellers to countries in Asia withH5 N1 outbreaks must avoid poultry farms andany surface contaminated with feces frompoultry. Other preventive measures includewearing of mask and gloves, cleaning kitchensurfaces, cooking chicken till boiling tempera-ture, controlling human traffic into poultriesand reporting to authorities any unusual deathor illness of chicken or other birds as well asillness of workers in poultry farms.

Antivirals drugs like oscmltamvir andzanamavir are quite effective. Some strainshowever do show resistance to amantadine andrimantadine.

314 Virology

53 Acquired ImmuneDeficiency Syndrome

(AIDS)

IMPORTANT EVENTS IN AIDS

1981 The first case of AIDS reported in USA.1983 LAV, i.e. HIV isolated.1984 Serological tests developed to identify

infected persons.1985 Report of anti-HIV activity of suramin,

ribavirus published.1985 Report of in vitro anti-HIV activity of

HPA 23, interferon alpha, foscamet andzidovudine published.

1986 Clinical trials of zidovudine showefficacy in AIDS and advanced AIDS-related complex.

1987 Zidovudine (AZT)—licensed for clini-cal use in many countries. Large scaleclinical trials of zidovudine and otheragents began.

1991 Didanosine is licensed for use byUSFDA in selected AIDS patients.

AIDS is a immunoregulatory disorderthat is often fatal because it predisposes theperson to severe opportunistic infections orpossibly to neoplasms. It happens so becauseof depletion of helper T cells owing toinfection by HIV (human immunodeficiencyvirus).

History

AIDS was first recognized in USA in July 1981.In August 1981, AIDS was reported in intra-venous users. In June 1982, clusters of AIDSpatients appeared among homosexuals andlater in hemophiliacs and blood transfusionassociated patients. In January 1983, it wasreported in heterosexual cases in female.

Isolation of etiological agent of AIDS wasfirst reported in May 1983 by Luc Montagnier

from Pasteur Institute, Paris. They isolateda retrovirus from a West African patientwith generalized lymphadenopathy and theynamed it lymphadenopathy associated virus(LAV). In March 1984, Robert Gallo fromNational Institute of Health, Bethesda (USA)reported isolation of retrovirus and named itHTLV-3. In March 1985, ELISA test kit wasapproved by FDA. In May 1985, blood bankscreening for HIV was introduced. In 1986,the virus was named HIV.

India started a serosurveillance amonghigh risk groups in 1985 to know the magni-tude of HIV infection. First case of HIVinfection in India was reported in 1986 andthat of HIV-2 in 1991.

Structure and Properties of HIV

It belongs to the Lentivirus subgroup ofRetroviridae family. HIV is an RNA retrovirus(see also page no. 328). The unique morpho-logic feature of HIV is its cylindrical nucleoidin the mature virion. The diagnostic bar-shaped nucleoid may be seen in electronmicrographs. Under electron microscope itexhibits the characteristic exotic flower appear-ance (Fig. 53.1). Dr. Rober C. Gallo discoveredHIV in 1984.

The virus contains the 3 genes requiredfor a replicating retrovirus—gag, pol and env(Fig. 53.2). The virus has outermost enveloperich in glycoproteins (gp41, gp120, gp160) andinner core with two component proteins (p18,p24) while the enzyme reverse transcriptasecapable of the retrograde transcription ofviral RNA to viral DNA marks its specialfeature. The core proteins, the surfaceproteins and the regulatory proteins (p31, p66)

Acquired Immune Deficiency Syndrome (AIDS) 315

are under the genetic control by the gag, envand the pol loci respectively. Amongst thethree inbuilt control mechanisms the env locusis under frequent genetic alteration, leadingto modification in the antigenic structure ofthe virus.

In addition following 5 genes code forpolypeptides are identified as under:

TAT (transactivationgene)REF (regulator of May be involvedvirus gene) in regulation ofNEF (negative factor HIV expression.gene)VIF (viral infectivityfactor gene)VPU in HIV-I—May weaken transcrip-

tional activator.VPX in HIV-II—May be required for

efficient budding.HIV is T lymphotropic especially for helper

T cells identified by monoclonal antibody OKT4 (Leu-3). Virus infection causes cytopathic

effect including the formation of multinuc-leated giant cells followed by cell death. Thisexplains the quantitative and functionaldepletion of T4 lymphocyte subset that is thehallmark of AIDS lymphodepletive terminalevent. In the end stage, there is reduced T4count (less than 60%), nonspecific prolifera-tion of B lymphocytes producing functionallyincompetent wasteful serum immunoglobu-lins (IG and IgA) and nonreactivity (energy)to recall antigens. The final outcome is totaldepletion of lymph node, impairment of theCMI making the patient susceptible to vastspectrum of opportunistic infection (bacterial,viral, fungal and parasitic).

Etiopathogenesis: HIV effects T4 lympho-cytes through infected semen, the contami-nated blood and blood products and rarelythrough saliva, urine and the fecal material.

Once the virus binds with CD4 receptor,the outermost cover is lost at the site of entry.The inner protein coat is being subsequentlycast off and the bare enzyme RT transcribesviral RNA and DNA (provirus). Subsequentto its entry into host nucleus the viral DNAgenome (provirus) integrates with host DNAgenome. In the absence of immunological acti-vation the T4 lymphocyte continues to survivewith provirus and the subsequent integrationin host DNA is halted leading to the latentHIV infection. What influences the viralreplication or dormancy is unpredictable atthis stage. The viral messenger RNA (mRNA)subsequent to its transcription redirects thehost cytoplasmic machinery to synthesize thenewer viral particles. The cell eventually diesand newly generated viruses bud out fromdying cell, leading to lymphodepletiveterminal events.

The end stage is characterized by reducedT4 count (60%), nonspecific unrestrainedproliferation of B lymphocytes producingexcessive functional incompetent wastefulimmunoglobulins (IgG, IgA) and nonreactivityto recall antigens. The lymph node in theterminal stage may show a total depletion withthe characteristic “burnt-out” picture. The finaloutcome is long-standing impairment of CMImaking the affected person unusually suscep-tible to a vast spectrum of life-threateningopportunistic infection and malignancies ofvarying types.

Fig. 53.1: Face of AIDS virus

Fig. 53.2: Structure of HIV

316 Virology

Clinical picture: The incubation period seemsto be long, ranging from 6 months to morethan 2 years. AIDS occurs in homosexuals(75%) but bisexual males, heterosexuals, intra-venous drug users and hemo-philiacs treatedwith blood products or factor VIII are also atrisk to get infected.

TABLE 53.1: Break-up of HIV infectedpersons in India

Category % of total

• Heterosexually promiscuous 45.30• Homosexuals 0.12• Blood donors 19.33• Patients on dialysis 0.38• Antenatal mothers 0.42• Recipient of blood/blood products 1.98• Relatives of HIV patients 0.90• Suspected AIDS related cases or

AIDS cases 3.68• Drug addicts (I/V) 18.03• Miscellaneous 9.86

AIDS is characterized by pronounced sup-pression of immune system, the developmentof unusual neoplasms especially Kaposi’s sar-coma or wide variety of severe opportunisticinfections. Other symptoms include fatigue,malaise, unexplained weight loss, fever, short-ness of breath, chronic diarrhea, white patcheson tongue (hairy leukoplakia or oral candi-diasis) and lymphadenopathy.

The most common complication of AIDSmay be (protozoal Toxoplasma gondii, etc.),fungal, (Pneumocystis carinii, Candida albicans,Cryptococcus neoformans, Histoplasma capsula-tum, etc.), bacterial (Mycobacterium avium,Mycobacterium tuberculosis, Listeria monocyto-genes, salmonella, Nocardia asteroides, etc.) andviruses (cytomegalovirus, Herpes simplex,adenovirus, Hepatitis-B, etc.).

HIV can infect other cell types includingB lymphocytes and monocytes in vivo and avariety of human cell lines in vitro. Virusescan spread throughout the body, and aredemonstrated in lymphoid cells, brain,thymus, spleen and testes. No animal modelshave been developed for AIDS.

Inactivation of HIV: 10 minutes’ treatmentat 37°C can inactivate this virus, with:

i. 10% household bleachii. 50% ethanol

iii. 35% isopropanol

iv. 1% NP40

v. 0.5% lysolvi. 0.5% paraformaldehyde

vii. 0.3% H2O2

Besides extreme pH (pH 1.0 and 13.0),heating at 56°C for 10 minutes can inactivateHIV.

Global situation of AIDS: 23 million peopleall over the globe are now infected with HIV.8500 people are infected with HIV each dayand as per calculation it is ample clear that by2000 AD every 6th or 7th person in worldwill be a victim of this infection. WHO datasuggested 1.3 million adult AIDS cases in 193countries with epicentre of epidemic shiftingfrom Africa to Asian subcontinent. India hasemerged quickly as the country with largenumber of people infected with AIDS virus.More than 3 million of India’s 950 millionpeople are estimated to be infected with HIV.Trailing India in the number of people infectedare South Africa 1.8 million, Uganda 1.4million, Nigeria 1.2 million, Kenya 1.1 million.

The first case of AIDS in India was regis-tered in 1986. Since then, HIV prevalence hasbeen reported in all states and unionterritories of India. By October 31, 1997, of atotal of 3.20 million individual practising riskbehaviors and suspected AIDS case who werescreened for HIV infection, 67,311 were foundto be seropositive. Up to May 31, 1998, thenumber of AIDS cases in India is reported as6052, the largest number of cases are fromMaharashtra (2,955) followed by Tamil Nadu(1,424) and Manipur (301). The break-up ofHIV cases in India is depicted in Table 53.1.

Laboratory Diagnosis

Direct Methods

i. Cultivation of T lymphocytes: T lympho-cytes with normal lymphocytes arecultured on special cell lines (Hg, HUT78

U937) with interleukin II. Stimulation ofT cell and demonstration of multi-nucleated giant cells is possible by 2 to20 weeks. It is the classical method toconfirm the HIV infection.

ii. Detection of reverse transcriptase: It is pos-sible on virus grown cells by radiolabel-ing.

Acquired Immune Deficiency Syndrome (AIDS) 317

iii. Solid phase ELISA: It is useful to detectHIV antigen.

iv. Phase contrast microscopy: It is helpful inassaying the prevention of reclusteringof MT-4 cells.

v. Electron microscopy: Demonstration offuzzy envelope of infected T4.

vi. Animal study: Reproduction of humanlesions in nonhuman primates have alimited diagnostic importance.

Indirect Methods

Antibodies to HIV usually appear in 6 to 8weeks after the exposure to virus. Followingmethods are simple, easy and quite useful todiagnose AIDS patients.

i. ELISAii. Western Blot Assay: The antigene frac-

tions specific for HIV, initially resolvedby SDS polyacrylamide gel are trans-ferred on nitrosocellulose membraneby electroblotting. The antigenic profileon membrane contains P17, P24, P31, gp41,P55, P56, gp160 in the increasing order ofmolecular size. The later steps involvereaction with HRP labelled anti-IgGantibody and chromogen substratereaction. The development of pinkcolored bands indicate the site ofspecific antigen antibody complex onthe membrane.

However, IgM-Western Blot Assaytest is slightly different from Western

Blot Assay. In this test basic techniqueis identical, diaminobenzedine as asubstrate and P24 as a positive controlare two deviations. Besides resolutionof brown components indicate the siteof immune complex. Appearance of allthe three gene products (env, gag andpol) are consistent with a classical HIVinfection. Otherwise two env bands(gp41, gp120) show a positive pattern too.

The ELISA at times, particularly soin multiparous women gives rise tofalse-positive results because of anti-bodies crossreacting with T4 lym-phocytes. This is rarely seen in WesternBlot technique (Fig. 53.3).

iii. Radio-immunoprecipitation assay: Thistechnique involves radioactive antigen.

iv. Polymerase chain reaction: The poly-merase chain reaction (PCR) is a newand exciting technology. It is gainingwidespread use in the diagnosis andmanagement of genetic, oncologic,hematologic and infectious diseases. Inthis method, there is direct detectionof disease specific sequences of eitherRNA or DNA from the tissue or bodyfluid of patient. It is quite sensitive.

This technique is widely used inHIV infection. In addition to quanti-tative PCR technique is useful inmonitoring the efficacy of antiviralchemotherapy.

Fig. 53.3: Western blot technique

318 Virology

v. Dot blot hybridization.vi. Agglutination tests using RBC/latex

gelatin, colloidal gold, immuno dot/stripand PAT (particle agglutination tests).

vii. Detection of antibody to nef gene pro-duct (27 KD protein) along with p20 andgp120.

viii. Fujerbio agglutination test: In this test,antigen coated gelatin particles areagglutinated by antibody present in theserum of patient. It is quite simple andconvenient test. However, false-positive reactions do occur.

ix. Karpas test: In this test, HIV infected cellsare fixed on teflon coated slide wellsto which serum of the patient is added.After some time horse radish pero-xidase labeled antihuman immuno-globulin is added. Later appropriateand corresponding substrate is added.Color develops if test is positive. Nocolor develop if test is negative. It iseasy to do and not expensive as slideimmunoperoxidase test.

Surrogative markers: They are directpredictors of HIV infection and are:1. Low CD 4 cells2. Low B2 microglobulin3. Increased neopterin

Nobel Prize for Medicine 2002

Sydney Brenner and John Sulston of Britain and H.Harvitz of US were awarded Nobel Prize for Medicine2002. Their pioneer work on gene research shednew light on killer diseases, e.g. AIDS and cancerfetched them Nobel Prize. They discovered abouthow gene regulate organ growth and a process ofprogammed cell suicide.

Working with tiny worms, these workers identifiedkey genes regulating organ development andprogrammed cell death, a necessary process forprinting excel cell.

Brenner broke new ground by linking specificmutation to particular effects on organ development.Sulston discovered that certain cells in the developingworm are destined to die, through programmed celldeath. He demonstrated the first mutation of genesthat participate in that process. Harvitz identified thefirst two “death genes” in the worm and showed thathumans have a gene similar to one of them. Nowscientists know that most genes controlling cell deathin worms have counterparts in humans. Informationabout programmed cell death has helped scientistsunderstand how some viruses and bacteria invadehuman cells.

Other Investigations

Reduced T4 lymphocytes, reversal T4: T8 ratio,defective T cell plus NK cell cytotoxicity andanergy to tuberculosis indicate a basic T celldefect which possesses a definite role in thediagnosis of HIV infection.

WHO and national HIV testing policyrecommends HIV testing for following pur-poses:• Screening blood, organ and tissue for

transplantation.• Epidemiological surveillance.• Diagnosis of symptomatic infection

(AIDS).• Early diagnosis of HIV infection among

asymptomatic persons with only informedconsent.Laboratory tests for diagnosis of HIV

infection are:1. Screening test

a. ELISAb. Rapid tests like

• Latex agglutination• Dot blot test

c. Simple tests, i.e.• Particle agglutination test.

2. Supplement tests: These tests are requiredfor validation of the positive results ofscreening tests. They include:• Western blot tests• Immunofluorescence test

3. Confirmatory tests• Virus isolation• Detection of p24 antigen by ELISA• Detection of viral nucleic acid may be

detected by in situ hybridization andpolymerase chain reaction.

Treatment: In a nutshell, thymus and bonemarrow transplantation, interferon andinterleukin therapy is useful in reconstitutingbasic T cell dysfunction. The antibiotics havea key role in taking care of opportunisticinfection. Many HIV drugs are found usefullike AZI and suramine (block the action ofreverse transcriptase). Inactivation of thevirus with monoclonal antibodies is anotherapproach to combat against HIV infection.Phosphonoformate, ‘Posearnet’, are otheranti-HIV drugs reported recently.

Acquired Immune Deficiency Syndrome (AIDS) 319

Now it is beyond doubt that zidovudinbenefits disappear within a year because HIVmutates into new forms that are resistant tothis drug. Hence, a new AIDS therapy devisedby Yung Kang Chaw is advocated. It consistsof administration of 3 drugs, i.e. zidovudine(AZT), dideoxyinosine (DDI) and eithernevirapine or pyridinone.

Vaccine against HIV infection: DNA recom-binant vaccine with yeast and vaccinia virus,the synthetic peptides are few candidates.Unfortunately, due to genetic alterationsvaccine trials are ineffective.

Recent report on the trial of fusioninhibitor Enfuvirtide is encouraging. This newdrug prevents the entry of HIV 1 into thetarget cells of the host. It also prevents fusionof HIV transmembrane (gp41) glycoproteinwith the CD 4 receptor of the host cell.

Currently treatment for HIV comprisedof HAART (Highly active antiretroviraltherapy). Here two nucleoside analogue

reverse transcriptase inhibitors or NRTI pluseither protease inhibitor or a nonnucleosidereverse transcriptase inhibitor (NNRTI).

STRATEGIES OF HIV TESTING IN INDIA

Strategy I: (Blood Donation)

Serum is subjected once to ELISA, Rapid,Simple tests for HIV. If negative, serum isconsidered free of HIV. In case it is positive,sample is taken as HIV infected.

Strategy II: (Surveillance and Diagnosis)

Test serum sample is taken as negative forHIV if first ELISA test report is negative, andsubjected to a second ELISA which utilizes asystem different from first one.

Strategy III: (Diagnosis)

Here 3 ELISA kits are used. First ELISA isdone with highest sensitivity, and second andthird ELISA with highest specificity.

Preventive Measure in Biosafety

Steps Preventive Measures1. Pre-use screening • Avoid avoidable risky procedures like mixing, grinding etc.

• Avoid the use of sharp objects• Keep away person with ulcerating or weeping skin lesions

2. Barrier precautions • Use of gloves contact with blood, body fluids, mucous membrane,broken skin etc,

• Use of mask protective eye wear face shield to prevent dropletinfections

• Use of gowns, aprons, long shoes to prevent splash of blood, bodyfluids

3. In use precaution • Prevention of injuries by sharp objects, i.e. needles, scalpel etc.• Needles need not to be recapped.

4. After use precaution • Drop all used instrument in disinfectant jar.• Placing the jar with disinfectant as close as possible to the working

place.

320 Virology

54 Miscellaneous Viruses

Hepatitis Viruses

Viruses responsible for hepatitis are hepatitisA, hepatitis B, hepatitis C, hepatitis D, hepatitisE, Epstein Barr (E B viruses), yellow fever,cytomegalovirus, rubella, Herpes simplex andcoxsackievirus. Recently, some more virusesare included in this list, i.e. Marburg virus,Lassa virus and Ebola virus.

The term viral hepatitis refers to infectionwith hepatitis type A, hepatitis type Band non-A non-B virus like hepatitis-C,hepatitis-D and hepatitis-E virus (Table 54.1).Difference between Hepatitis A and HepatitisB type is tabulated in Table 54.2.

a. Hepatitis A Virus

Type A hepatitis occurs endemically in allparts of the world with frequent minor andmajor outbreaks. The older the patient moreare the chances of this infection. Lowersocioeconomic status class and females havehigh prevalence. Infection shows peak in themonth of March and April. Incubation periodis 2 to 6 weeks. Routes of spread may be stools,urine and nasopharyngeal secretions. Modesof transmission can be fecal-oral. The virusenters the body by ingestion, starts multiply-

ing in intestinal epithelium and then throughblood it reaches liver.

Hepatitis A virus (Figs 54.1 and 54.2) is aspherical RNA virus which belongs topicornavirus group (26 to 30 nm in diameter).Lipid is not an integral component and so thisvirus is stable to treatment with ether, acid andsurvives heating at 56°C for 30 minutes, and1 ppm chlorine for 30 minutes.

It is inactivated by ultraviolet irradiationheating at 100°C for 5 minutes, autoclaving121°C for 20 minutes, 1:4000 formalin at 37°Cfor 3 days and by RNase. This virus was firstidentified in 1937 by Feinstone, Kapikian andPurcell using immune electron microscope.Only one serotype is known and there is noantigenic cross-reactivity with hepatitis B virus.

Culture of this virus in the laboratory hasbeen possible and so provision of vaccine isnow there. Provost and Hillermann in 1979claimed cultivation of this virus in liver cellcultures of marmosets and in fetal rhesuskidney cell lines.

Laboratory diagnosis is possible by thedemonstration of virus by immunoelectronmicroscopy. The other tests are complementfixation, immune adherence, hemagglutina-tion, radioimmunoassay and ELISA (enzyme

TABLE 54.1: Properties of viral hepatitis (various types)

Hepatitis type virus Route of Mortality Risk of chronic Incubation Virustransmission liver diseases period classification

A. Unenveloped SS RNA Feco oral Low None 2 to 6 weeks PicornavirusB. Enveloped DS DNA Parenteral High High 1 to 6 weeks HepadnavirusC. Enveloped SS RNA Parenteral Moderate High 1 to 5 months Flavivirus likeD. Enveloped circular With hepatitis B High High 2 to 6 weeks Animal satellite

SS RNA virusE. Unenveloped SS RNA Feco oral High in None 3 to 6 weeks Calcivirus like

pregnancy

Miscellaneous Viruses 321

linked immunosorbent assay). More sensitiveserologic assay, e.g. the microtiter solid phaseimmunoradiometric assay and immune

adherence have made it possible to detect thisvirus in stool, liver homogenates, bile and alsoto measure specific antigen in serum.

Hepatitis A vaccine which is live attenua-ted procured from H-2 strain is reported safeand highly immunogenic at a subcutaneousdose of 106 TCID50 (1 ml).

Another hepatitis A vaccine comprises offormalin inactivated virions grown in humanfibroblasts or monkey kidney cell line foractive vaccination. Two doses are injected onemonth apart. Booster dose may be given after6 months giving adequate immune responsein 99 percent cases lasting for years together.It is expensive because of low yield of virusesfrom cultured cell. It is required in sewageworkers, homosexuals and intravenous drugusers and for visitors visiting HAV endemicregions.

b. Hepatitis B Virus

23.6 percent cases of endemic hepatitis in Indiaare HBs Ag positive with majority of patientsbeyond third decade. Incidence of thisinfection is more in males. Incubation periodis 28 to 160 days. The common subtypesreported from India are adr, adw. InTamil Nadu, adr is reported in 64 percent ofcases studied by Thyagarajan et al. In northIndia, the peak incidence occurs in the monthof July and August.

TABLE 54.2: Differences between hepatitis-A type and hepatitis-B type

Characters Hepatitis type A Hepatitis type B

1. Diameter of virus 27 nm 43 nm2. Symmetry of virus Icosahedral Icosahedral3. Nucleic acid RNA DNA (double stranded)4. Primary route Feco-oral Parenteral and also feces,

saliva, transplacental5. Incubation period 2 to 6 weeks 2 to 6 months6. Mortality Low High7. Chronic active hepatitis Rare Common8. Onset Acute Insidious9. Fever Present Rare

10. Age incidence Children All age group11. Extrahepatic lesion Absent Common12. Australia antigen in serum Absent Present13. Viremia Brief Prolonged14. Viruses in stool Present Mostly absent15. Human gamma globulin Effective Not effective

prophylaxis

Fig. 54.2: Hepatitis A virus

Fig. 54.1: Hepatitis A virus (27 nm)

322 Virology

Routes of spread are serum, saliva, tears,nasopharyngeal secretion, urine, feces, semi-nal fluid, breast milk, gastric fluid, synovialfluid and intestinal juice. Mode of transmis-sion may be blood transfusion, intravenousdrug abuse, tattooing, ear piercing, sharing ofrazors, sexual transmission, insect vectors andhuman bites, etc.

They are spherical DNA viruses. Electronicmicroscope shows three types of particles insera from type B hepatitis patients (Fig. 54.3).

The most abundant form is spherical(20 nm in diameter). The second type ofparticles are tubular with diameter of 20 nmand varying length. These two types ofparticles represent Australia antigen. The thirdtype of particles are double shelled spherical(42 nm diameter) and also called Dane’sparticles. Dane’s particles are consideredcomplete type B hepatitis virus (Fig. 54.4).

The terms in current use for virus, viralantigen and antibodies are:

HBs Ag: It is hepatitis B surface antigen foundon 20 nm spheres tubules and outer envelopeof Dane particle also called Australia antigen.

HBc Ag: It is hepatitis B core antigen presentin the core of Dane‘s particles.

HBV is hepatitis B virus identical with Dane’sparticles.

Hepatitis B—”e” antigen was described in1972 by Magnius et al. It is smaller than HBsAg and HBah Ag. Takashi, 1979 demonstratedthat HBe Ag exists in the core of Dane’sparticles in cryptic form.

Delta antigen is the latest to the addition inlist of antigens. It was reported for the first timeby Rizzetto in 1977. The antigen is distinct fromknown antigenic determinants of hepatitis Bvirus. It is localized in hepatocyte nuclei thatdo not contain HBc Ag.

Subtypes of HBs Ag: In addition to thecommon antigen “a” HBs Ag contain 2 pairsof subdeterminants b/y and w/r. These fourantigen types are: a d w, a b r, a y w, and a y r.Subtypes are useful as epidemiologicalmarkers.

A variety of serological techniques areavailable for detection of hepatitis B antigensand antibodies. HBs Ag becomes detectableabout a month after exposure to infection withpeak levels in preicteric phase of disease. Itdisappears with recovery of disease. Anti-bodies to HBs Ag appear within week afterdisappearance of HBs Ag and they may persistfor years. These antibodies are rarelydetectable in chronic cases. HBs Ab is notdetectable in the serum of patient. Antibodiesof HBc Ag are detectable in preicteric phase.However, HBc Ag may be demonstrated ininfected liver cells by immunofluorescence.Viral DNA-polymerase may be detectable inpreicteric phase of illness.

Serological tests are immunodiffusion,counter immune electrophoresis, complementfixation, indirect hemagglutination inhibition,immuneadherence, hemagglutination, reversepassive latex agglutination, radioimmuno-assay and ELISA. Nowadays, new monoclonalEnzygnost kit is available which is more spe-cific, simpler to perform and needs a shortertotal incubation period than polyclonal kit.

Hepatitis B carriers: Carrier form may bedefined when there are persistence of HBsantigen in circulation for more than 6 months.Carriers may be of following types:1. Super carriers: Here HBe antigen, high titre

HBs antigen and DNA polymerase remainpresent in blood. Needless to say that veryminute amount of serum or blood fromcarriers may transmit this infection.Fig. 54.4: Structure of hepatitis B (3 types)

Fig. 54.3: Hepatitis B virus

Miscellaneous Viruses 323

2. Simple carriers: Here HBs antigen is foundin blood in low titre. However, HBe antigenand DNA polymerase are absent in blood.Transmission of infection is possible whenlarge amount of blood is transferred, e.g.blood transfusion. By the way simplecarriers are more common.

Prophylaxis of hepatitis B infections:Hepatitis B virus infection may be checked byobserving the followings:1. Screening of blood donors.2. Use of sterile disposable syringes or

needles.3. Reduction in the number of sexual

partners.4. Use of condoms.5. Blood spills should be cleaned with 2%

glutaraldehyde or 0.5% sodium hypochlo-rite.

Passive immunization: Hepatitis B globulin(HBIG) should be given as early as possiblewithin 48 hours of exposure. A second doseshould be given after 30 days in conjunctionwith active immunization. HBIG is preparedfrom donors with high titre of anti-HBs. It maybe administered in the doses of 300 to 500 IUintramuscularly.

Indications of passive immunizationinclude needle stick injury, splash of bloodfrom HBs antigen positive patient, to new-borne infants born to HBs positive mothers.

Active immunization: Indications of activeimmunization are:1. Healthy personnels in direct contact with

blood and sharp instruments.2. Patients in need of repeated blood trans-

fusion and blood products.3. Patients on dialysis.4. Parenteral drug users.5. Prostitutes and sexually promiscuous.6. Spouses of known HIV positive.

The vaccines available against Hepatitis Bare:1. Plasma derived vaccine containing 22 nm

particles of HBs antigen. It is cheap,immunogenic and safe. It is administeredintramuscularly in deltoid region in threedoses at 0, 1 and 6 months.

2. Recombinant yeast Hepatitis B vaccine issafe, immunogenic and free from side

effects. It is administered intramuscularlyinto deltoid region in three doses 0, 1 and6 months.

3. Synthetic vaccine are yet under evaluation.4. Hybrid virus vaccines are prepared by

incorporating HBs antigen into vacciniavirus DNA. This vaccine is cheap havinglong self life.The virus shows considerable genetic and

antigenic diversity. There are different geno-types and many subtypes thus, indicating highmutability. Hence, there is little heterologousor even homologous postinfection immunityin infection due to this virus.

It is noteworthy that hepatitis virus has notbeen grown in culture. However, it has beencloned in Escherichia.

Patient may be treated by administeringinterferon in large doses. It has reduced thelevel of HBV and related antigens in the bloodwith improvement of health in some patients.Vidarabine (arc-A) and acyclovir have beensuccessful in reducing HBV levels.

Hepatitis C Virus

It belongs to family Flavivirdae. It is 50 to60 nm virus with linear single stranded RNAof positive polarity enclosed within a core andsurrounded by an envelope carrying glyco-protein spikes. It has multiple genotypeswhich has made vaccine preparation difficult.It is difficult to culture this virus. Recently, ithas been included in a separate genus calledHepacivirus under family flavivirdae.

Hepatitis D Virus

Morphology

It is an RNA virus which has the outer shell ofHBs antigen and the core is the delta antigen.It is smaller than HBV and measures around37 nm (Fig. 54.5). The RNA is small chainedand codes for 2 to 3 proteins. Animal modeluseful for its study is chimpanzee. It requiresthe helper function of HBV to produce disease.It is an incomplete virus. This virus is uniqueamongst the satellite viruses as it can also infecta second animal species Marmota Monax inassociation with woodchuck hepatitis virus.

324 Virology

Patients having repeated blood transfu-sions and drug abusers are likely to be infectedwith this virus.

Pathogenesis: Coinfection of HBV and HDVwill usually cause an acute disease. In case aperson becomes a chronic HBs Ag carrier,chronic delta hepatitis occurs.

HBV replication rate is evidenced byHBe Ag. When HDV infection occurs it willcompete with HBV for replication resulting inlow rate of HBV replication and hence, HBe Agis absent. Thus, in patients with high HBs Agand absent HBe Ag, HDV infection will causechronic delta hepatitis.

Clinical features: Both acute and chronic HDVinfection is more severe than acute and chronic(uncomplicated) HBV infection.

About 39 percent cases of fulminatinghepatitis are HDV positive. About 16 percentof all HDV infections are fulminating.

Laboratory diagnosis: Radioimmunoassay, atpresent, carries some diagnostic importance.Even electron microscope is not of much use.

Treatment: Alfa steroid therapy is considereduseful in both acute and chronic hepatitis Dinfection.

Hepatitis E Virus

This virus causes enterically transmittednon-A non-B hepatitis. This virus is nownamed hepatitis E virus. Hepatitis caused byE virus is found in epidemic form as well assporadic form. Large epidemics of this infec-tion has occurred in many countries includingIndia with very high mortality in pregnant

ladies. The higher mortality in pregnancy hasbeen attributed to reversal host factors likeheightened immune response, malnutritionand altered harmonal status. Disseminatedintravascular coagulation, which is the mostcommon cause of mortality in hepatitis Eassociated pregnancy, seems to be a severemanifestation of a Schwartzman like reaction.

Morphology: The virus is spherical, unenve-loped measuring 34 to 37 nm with spikes andindentations seen on the surface of virusparticle. These features resemble with calci-virus (Norwalk virus).

Resistance: This virus is heat labile, sensitiveto freezing-thawing, cesium chloride andpelleting by ultracentrifugation.

Successful cloning of viral genome ofthis virus is now possible. Development ofdiagnostic tests and preparation of vaccine,now seems to be feasible. The diagnosis ofacute hepatitis E can be conveniently esta-blished by demonstrating virus specify IgM/IgG antibodies against recombinant proteinsfrom open reading frame (ORF-2,3) by enzymeimmunoassay. In this assay, polystyrene beadscoated with HEV proteins representingsequence from ORF-2 (SF-3 antigen ORF) andORF-3 (8.5 antigen) of Burmese strain of HEV.

Hepatitis F Virus

• It is putative virus causing non-B, non-C,non-D hepatitis.

• May cause hepatitis in persons havingblood or blood product transfusion (nega-tive of hepatitis B virus and hepatitis Cvirus).

• These viruses have not been studied andcharacterized.

Hepatitis G Virus

It was discovered independently by 2 differentgroups, one naming it hepatitis G, other callingit GB virus. Like hepatitis C, hepatitis G virusis a blood borne RNA virus sequence analysisof 10 kilobase genome shows that Hepatitis Gand three recently identified viruses HepatitisGBV-A, GBV-B and GBV-C have genomestructure characteristic of flaviviridae family.It is transmitted by transfusion. Infection may

Fig. 54.5: Hepatitis D virus

Miscellaneous Viruses 325

occur in patients coinfected with hepatitis C,and that hepatitis G virus does not changeseverity of hepatitis C virus. Hepatitis G virusmay be the major infection associated withbone marrow failure. Hepatitis producingbone marrow failure (aplastic anemia) is notusually severe.

Reoviruses

It is 70 nm in diameter enclosing secondprotein shell containing genome of doublestranded RNA. The virion is icosahedron. Itmay be cultured on monkey kidney, humanamnion and HeLa cells. Type 3 reovirus hasbeen isolated from Burkitt lymphoma show-ing oncogenic potential. This role in disease isnot proved. They have been isolated frompatients of respiratory infection and gastro-enteritis.

Coronavirus

It is pleomorphic, 80 to 160 nm in diameters,enveloped with wedge-shaped projection onthe surface. The core contains RNA. Theseviruses have been isolated from cases ofcommon cold. When inoculated in humanembryonic trachea it may cause loss of ciliarymovements in ciliated epithelial cells. It hasmany serotypes.

Lymphocytic Choriomeningitis

Man may acquire this infection from excretaof rodents. It may cause influenza like illnessor meningitis. It is enveloped RNA virus.Particle inside virus shows electron densegranules resembling sand. It is a naturalparasite of mice.

Persistent Virus Infections

Persistent viral infection are kept intofollowing three categories:1. Latent infection is intermittent acute epi-

sodes of disease between which viruses arenot demonstrable, e.g. smallpox, measles,varicella, etc.

2. Chronic infection is persistent infection inwhich virus is always demonstrable butdisease is either absent or is associated with

immunopathological disturbance, e.g.leukemia, Aleution disease, etc.

3. Slow infection is a persistent infection witha long incubation period followed byslowly progressive disease that is lethal,e.g. scrapie. Discussion on slow virusinfection follows:

Slow virus diseases: These diseases arecharacterized by very long incubation, slowcourse, fatal termination, predilection forinvolvement of central nervous tissue, absenceof immune response and genetic predis-position.

Slow virus infection are grouped as under:I. Group A consists of slowly progressive

infection of sheep, e.g. leukoviruses cau-sing Visna and Maedi.a. Visna is a demyelinating disease of

sheep with incubation period of 2years. Onset of disease is insidious withpareses, total paralysis and death.Virus can be isolated and grown insheep choroid plexus tissue culturefrom CSF, saliva or blood of infectedsheep.

b. Maedi is slow progressive fatal hemor-rhagic pneumonia of sheep. Incubationperiod is 2 to 3 years.

II. Group B consists of four infections (1)scrapie (2) kuru (3) mink encephalopathy(4) Creutzfeldt-Jakob disease.

These four conditions are collectivelycalled subacute spongiform viral ence-phalopathy.1. Scrapie is a slow virus disease of sheep.

Incubation period is about 2 years. Theanimal becomes irritable, developspruritus, and later on emaciation andparalysis occur leading to death.

2. Kuru is an endemic disease of NewGuinea. Incubation period is 5 to10 years. Symptoms are cerebellarataxia and tremors. The disease endsfatally in 3 to 6 months. It occurs inman. The disease is transmitted bytribal practice of eating the dead bodiesof their relatives.

3. Mink encephalopathy is a disease ofmink and resembles scrapie of sheep.

326 Virology

4. Creutzfeldt-Jakob disease is subacuteencephalopathy of man with pro-gressive incoordination and dementiabecause of degeneration of brain.Death occurs about a year after onset.

III. Group C consists of two unrelated CNSdiseases of man (1) subacute sclerosingpanencephalitis, and (2) progressive leuko-encephalopathy.1. Subacute sclerosing panencephalitis

(SSPE): The disease sets in many yearsafter initial infection characterized byprogressive deterioration of mentaland motor functions. Brain cells doshow serological and microscopic evi-dence of measles virus infection butvirus cannot be cultured in routinecultural techniques. Patient shows highlevels of measles virus antibodies inserum. Subacute sclerosing panence-phalitis may develop as a very late butrare complication of live measlesvirus vaccination. Rubella infectionmay present similar picture as a rarecomplication.

2. Progressive multifocal leukoencephalopa-thy: It is a rare subacute demyelinatingdisease of old persons whose immunestatus is quite low because of malig-nancy or immunosuppression. Motorfunctions, speech and vision deteriora-tion do occur resulting in death after 3to 4 months. Papova virus has beendemonstrated from the brain of thesepatients.

Other possible examples of slow virusdisease of man are: Echo virus in Guillian-Barré syndrome and paramyxovirus in multi-ple sclerosis.

Mechanism of Slow Virus Infection

Following possibilities are proposed so far asmechanism of slow virus infection is con-cerned:a. Spread of virus in vivo is restricted.b. Infected cells spontaneously produce virus

at low rate in vivo. Hence, destructive effectis slow.

c. Virus acts for a series of host mediatedpathologic processes that destroy tissue. It

is the evolution of tissue damage that isslow.

Marburg Virus

It is an RNA virus resembling rhabdovirusstructure. It is larger and more pleomorphic.Natural asymptomatic infection does persistin monkeys in Uganda and Kenya. A fatalnatural death was reported in 1975 in man.Originally the virus was isolated fromhemorrhagic fever that occur simultaneouslyin laboratory workers in Marburg, Frankfurt(Germany) and Belgrade (Yugoslavia) in 1967.The mortality rate is over 20 percent. It causesfever, headache, muscle pain, diarrhea, vomi-ting and maculopapular rash.

Ebola Virus

They are 80 nm in diameter and 800 to1400 nm in length. They are RNA virus. Theenvelope comprises of lipid bilayer coveredwith peplomers. It is SS RNA having helicallywound tubular nucleocapsid.

Culture: Vero cells may be used for the cultureof Ebola virus. Virus multiplies in the cyto-plasm forming large inclusion bodies. BGMcell line is also useful to study cytopatho-genesis.

Pathogenesis: It is thought that Ebola virusenters the host through broken skin andmucous membrane. Incubation period is 4 to10 days.

Norwalk Agent

They are small (27 nm in diameter) RNA virusresembling picornaviruses. They are capableof inducing antibody formation. They havebeen found to cause gastroenteritis. They havenot been successfully cultured so far.

Rotavirus

They are 62 to 66 nm in diameter characterizedby well-defined outline like rim of a wheel(Fig. 54.6). They are RNA double strandedvirus. They can be grown on monolayer andorgan culture of human fetal intestine.Etiological role is established and may havepathogenic role in infantile diarrhea.

Miscellaneous Viruses 327

Rotaviruses infect cells in the villi of smallintestine. They multiply in the cytoplasm ofthese cells and damage their transportmechanisms. Damaged cells may slough intothe lumen of the intestine and release largequantities of virus which appear in the stool.Diarrhea may be due to impaired sodium andglucose absorption.

Laboratory Diagnosis

Laboratory diagnosis of rotavirus is based ondetection of virus and viral antigen. Followinglaboratory procedures are useful for thepurpose:1. Electron microscopy is quite helpful but it

does not specify serogroup.2. ELISA.3. Latex agglutination test.4. PAGE (Polyacrylamide gel electrophore-

sis). It is more useful for epidemiologicalpurposes as serogrouping is possible withit.

Rotavirus Vaccines

Bovine Rotavirus Vaccine

i. RIT 4237 vaccine.a. It is safe and provides partial

protection against rotavirus illness.b. This is greater for severe illness than

for all rotavirus diarrhea.c. Protection appears to be induced more

readily in infants over 6 months, fordisease due to serotype 1 rather thanserotype 2 rotavirus.

Because of low efficacy in young infantsand variable protection observed in deve-loping countries, RIT 4237 vaccine has beenwithdrawn by its manufacturers.ii. WC-3 vaccine.

A single efficacy treatment has been carriedout in USA with bovine vaccine WC-3.Marked protection against rotavirus illnesswas observed among infants 3 to 12 monthsgiven a single dose. Use of vaccine indeveloping countries is being planned.Dose: 107–108 PFU with little or no excre-

tion of virus and there were no side effects.

Rhesus Rotavirus Vaccine

• Dose in 104 PFU• Mild and brief fever reaction were obser-

ved in 20 to 30 percent in 2 to 4-month-oldinfants who received 104 PFU dose.

• Potential advantage over bovine strain isthat its VP7 neutralizing specific surfaceantigen is identical to that of rotavirusserotype 3.

• Protection by RRV-1 (formerly MMU-18006) is largely serotype specific (i.e. forserotype 3).

• Protection is substantial against severedisease and can be achieved with singledose, even in very young infants.

Rhesus Human Reassortment Vaccine

This vaccine is developed which incorporatesin RRV-1 strain, the gene segments of humanrotavirus that encode for the synthesis ofsurface protein (VP7) specific for each humanrotavirus serotype 1, 2 and 4. Combining thesereassortment strains with the parent RRV-1(serotype 3) would yield tetravalent vaccinethat might be useful to induce immunityagainst each of the 4 major human rotavirusserotypes. Its evaluation is being conductedin Burma, Brazil, India, Israel and Thailand.

Other Rotavirus Vaccine

i. Cloned rotavirus gene segments in pro-karyotic and eukaryotic cells.a. To produce large quantity of rotavirus

antigen for use in non-living vaccine.

Fig. 54.6: Rotavirus

328 Virology

b. When cloned, E. coli is used to developa potential live vaccine based upon E.coli that elaborate protective antigensof rotavirus.

c. Expression of VP7 is achieved ineukaryotic cells using recombinantvaccines virus as vector.

d. In USA, expression of rotavirus antigenis achieved successfully in culturedinsect cells using baculovirus vector.

ii. When cold adapted mutant of humanrotavirus is used several such mutants aredeveloped.

Lassa Virus

It was described for the first time in Lassa in1969 and subsequently seen as outbreaks inother corners of Africa. Rodents may be thesource of virus. The virus may be demonstra-ted in sera by complement fixation test. Theirisolation can be done by tissue culture. It hascaused mortality up to 45 percent in someoutbreaks. The disease manifests with fever,sore throat, ulceration of mouth and pharynx,vomiting, abdominal and chest pain and diar-rhea. It does cause leukopenia and proteinuria.

The virus is a member of Arenaviruses andcontain RNA with projection form surface. Itis 70 to 150 nm in size.

Retrovirus (Table 54.3)

It is single stranded RNA virus which istranscribed from RNA to DNA by reversetranscriptase. The name retro is derived fromReverse transcriptase. It is 100 nm, round,enveloped and covered by surface projection.The nucleoprotein is helical and surroundedby icosahedral capsid protein to form nucleoid.

The replication of the virus is unique andis initiated with the transcription of positiveSS RNA of virus to negative SS DNA by

viral reverse transcriptase (enzyme releasedduring uncoating from capsid). The negativeDNA becomes double stranded circular DNAwhich gets integrated into host cell chromo-some by viral enzyme DNA ligase. Now viralDNA genes code for virus component.

Parvoviruses

They belong to family parvoviridae and aresmall about 20 nm, nonenveloped, icosahedralviruses having single stranded DNA. It hasthree genera: Parvovirus, Dependovirus(defective virus always requiring helper virusfor growth and replication) and Erythrovirus.Parvovirus B 19 has been included inErythrovirus.

Parvovirus B 19: They may be associated withaplastic crisis in sickle cell disease and otherhemolytic anemias as they have affinity forimmature red blood cell precursors. Inimmunodeficient cases it may cause persistentanemia, erythema infectiosum of cheek(slapped cheek appearance) further spreadingto trunk and limbs followed by lymphadeno-pathy and arthralgia. It may involve childrenfever with rash sometimes referred as fifthdisease as this condition was placed 5th inthe old list of six exanthematous fever ofchildren. If pregnant woman happen todevelop primary infection, the result may beerythroblastosis fetalis with hydrops.

Nipah virus: It was discovered in 1999 and isrecognized zoonotic virus. The virus is namedafter the location where it was first detectedin Malaysia. Nipah is closely related to anotherzoonotic virus (1994) called Hendra virus.Nipah and Hendra are members ofParamyxoviridae.

Certain species of fruit bats are naturalhosts of both Nipah and Hendra viruses. Thebats appear to be susceptible to infection withthese viruses. Bats do not themselves becomeill. The role of species other than pigs intransmitting infection to other animals has notbeen determined. Human to humantransmission of Nipah virus has not beenreported.

The incubation period is 4 to 18 days.Patient develops high fever muscle pains,inflammation of brain with drowsiness,

TABLE 54.3: Classification of retrovirus (3 groups)

Group Human pathogens

I. Oncovirinae 1. Human tumor leukemiavirus

2. HIVII. Lentivirinae 1. Human tumor leukemia

virus2. HIV

III. Spumavirinae 1. Human foamy virus (HFV)

Miscellaneous Viruses 329

disorientation, convulsions and coma. Fiftypercent cases die.

Early treatment with ribavirin can reduceboth duration of feverish illness and severityof disease.

Bacteriophage

They are viruses that infect bacteria. They arealso called phage and are 10 to 100 µ (Fig. 54.7).Recently, more concentration is being givento phages of E. coli especially T even phage. Tphages are easy to purify because of their largesize and high frequency of recombinationwhich is quite useful for genetic studies.

Bacteriophage each exists in structurallydifferent states, e.g. extracellular form, vege-tative form or prophage strain of bacteriawhich are themselves not susceptible to lysisbut carry phage (prophage) are known aslysogenic strains.

Even numbered T phages made up of headand tail are tadpole in shape. Head containsDNA and has the shape of two halves oficosahedron connected by short hexagonalprism. Tail varies in dimension and structure.It is composed of tube through which DNApasses during cell infection. Tube is sur-rounded by parallel rows of protein manomer,are connected to this disc at head end and tothe plate at tip end. This sheath is capable ofcontraction. The plate tip end is hexagonal, haspin at every corner and is connected with 6very long thin tail fibers.

Lifecycle of phage starts with adsorptionand penetration. Receptors of different phagesare located in different layers of cell wall of

say E. coli. Nucleic acid (DNA) is injected intothe cells.

NONBACTERIAL GASTROENTERITIS(VIRAL GASTROENTERITIS)

This is the second most common viral disease.Sign and symptoms last only for 24 to 48 hours.Viruses responsible are:1. Rotavirus2. Norwalk agent Proved etiological3. Hawaii virus agents4. Coronavirus5. Adenovirus6. Echovirus7. Coxsackie virus8. Reovirus9. Asteroid virus10. Poliovirus11. Hepatitis virus

Diagnosis is made by isolating the virusfrom fecal samples. Viruses like adeno andecho are excreted for a week after onset ofdisease. Rectal swab (fecal sample) in TC 199with albumin, streptomycin, penicillin andpolymyxin is taken. It may be inoculated inchick embryo, tissue culture and laboratoryanimals. Diagnosis may be confirmed byelectron microscopy, immunofluorescence,hemadsorption, hemagglutination, inhibitionand neutralization with specific antisera.

ACUTE ASEPTIC MENINGITIS(VIRAL MENINGITIS)

The term acute aseptic meningitis syndromeincludes number of disorders which have incommon an acute onset usually self-limiting

Fig. 54.7: Bacteriophage

330 Virology

course with meningeal manifestations ofvarying degree, an increase in cells of spinalcord and absence of organisms on directsmear.

Viruses responsible are:1. Mumps2. Echo 4, 6, 8, 11, 14, 18, 303. Coxsackie B16

4. Polio5. Measles6. Herpes simplex7. Lymphocytomegalovirus8. Arboviruses9. E.B. virus

Laboratory diagnosis may be establishedby examining cerebrospinal fluid and inocu-lating an egg proceeded by animal patho-genecity. In case of Echo and Coxsackie, miceis the animal of choice and route of injection isintracerebrally. Serological procedure likecomplement fixation test, neutralization andhemagglutination test may be undertaken forthe diagnosis.

PERINATALLY ACQUIRED VIRALINFECTION

Infants may come in contact with vaginalsecretion and get viral infection. Virusesresponsible are:1. Herpes virus2. Cytomegalo virus3. Varicella zoster virus4. Coxsackie B virus5. Hepatitis B virus.

INTRAUTERINE INFECTION

1. Rubella virus2. Cytomegalo virus3. Herpes simplex (cerebral abnormality)4. Coxsackie B (congenital heart defect)5. Influenza (teratogenic effect?).

POSTNATAL VIRAL INFECTION

1. Respiratory syncytial virus2. Influenza virus3. Varicella virus4. Adenovirus5. Measles virus.