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! Textbook of HUMAN VIROLOGY EDI TED B Y ROBERT B. BELSHE PSG PUBLISHING COMPAtiY, INC. LfT7ZETON. MASSACHUSET75 HASTER-1:5PP

Library of Congress Cataloging in Pubitcatton Data 4tain entry under title: Textbook of human virology. Bibliography: p. Includes index. 1. ViroloQr. :. Virus diseases. 1. Belshe. Robert B. QR360.TsS 1984 616.0194 83-2S0I8 t5B` 0-d8416-438-6 Published by: PSG Publishing Company, Inc. 545 Great R;,ad Littleton. Massachusetts 01460. C.S.A. Medicine is an ever-chan8tn8 science. As new research and clinical experience broaden our knoWledQe. changes in treatment and drug therapy are required. The authors and the publisher of this work ha~,e made every effort to ensure that the treatment and drug dosage schedules herein are accurate and in accord with the standards accepted at the time of publication. Readers are ad- %+sed, howe.er, to check the product information sheet included in the package ot` each drug they plan to ad- minister to be certain that changes ha.e not been made in the recommended dose or in the indications and con- traindicanons for administration. This recommendation is of particular importance in regard to new or infre- quently used drugs. Copyright O 1964 by PSG Publishing Company, Inc. All ristts reserved. No part of this publication may be reproduced or transmitted in any form or by any tneans, electronic or mechanical, including photocopy, recording, or any information storage or retrieval system, without permission in writing from the publisher. Printed in the United States of America International Standard Book Number: as8416-4s8-6 Library of Congress Catalog Card Number: s3-2sa18

CONTENTS 1 !r 3 6 Foreword xi Maurice A. Mufson Introduction usi Robert B. Belshe Structure and C7assi6cation of Viruses 1 Joseph L. Melnick Introduction to Methods for Characteriation of Viruses and Virat Macromolecules 29 James M. Wilhelm Replication of DNA Virscses 49 Terry W. Fenger Replication of RNA Viruses 79 Terry W. Fenger Immunology of Viral Infections 103 Gerald V. Quinnan, Jr. RNA Tumor Viruses 139 1 Rudiger Hehlmann Hartmut Schetters Volker Erfie Diagnostic Virology 179 Marilyn A. Menegus Aativirai Chemotherapy 193 Lee P. Van Voris Nosocomial Viral infectioas 231 William M. Valenti InAuensa Viruses 267 Lee P. Van Voris James F. Young Jack M. Bernstein William C. Graham Edwin L. Anderson Geoffrey J. Gorse Robert B. Belshe ParaisAuenzr Vimses 299 Peter F. Wright Mumps Vir.f 311 Mark D. Toipin Victoria Schauf Messia Virus 333 13 John F. Modlin ix .

x 14 Respiratory Syncytia) Virus 361 Robert B. Belshe Jack M. Bernstein Karen N. Dansby 15 16 17 Coronaviru~es 395 Maurice A. Mufson Rhinoviruses 391 Roland A. Levandowski Enteroviruses, including Polioviruses 407 Melinda Moore David M. Morens 18 Rsbies Virus iaS Patrick A. Robinson 19 Atettavitvses 513 C. J. Peters 20 Bunyaviruses, Phieboviruses and Related Viruses 547 C. J. Peters James W. LeDuc 21 Topviruees 599 Robert B. Craven 22 other Vira! Hemorrhagic Fevers and Colorado Tkk Fever 649 Kelly T. McKee, Jr C. J. Peters Robert B. Craven D. Bruce Francy 23 Robens Vws em Cuherine L. Lamprecht 7A Hep.tltb A Visti 707 Gert Frosaer 25 Hepattds s Yient 729 Larry I. Lutwick 26 Nori-A. No" Hepadtb 757 Stephen M. Feinstone lr ! Adeuo.ietutet 779 Sharon R. Snavely C.ltien Liu f+S Rotaviruees and Other Viruses Causing Gastroenteritis 795 Robert H. Yolken 29 Hetpes Simpkx Vlruxs d11 Richard C. Reichman 30 VatkeUa.Zoster Virus in9 Victoria Schauf Mark Tolpin 31 Epstein•Barr Virns g33 Gary R. Fleisher 32 Cytomeiabvintses n7 Sirus Naraqi 33 Poxviruses m Derrick Baxby 34 Human Papillotnaviruses 951 A. Bennett Jenson Wayne D. Lancaster 35 Human Poiyomaviruses 969 Thomas F. Hogan Billie L. Padgett Duard L. Walker 36 Virus-induced Subacute Spongiofotln Encephaiopatbks (Kuru and Creutziddt,iakob Disease) 997 Colin L. Masters 37 Ro.eou i.ta.tnm (Euariem Sttbitttm) 1011 Prudence Krieger 38 En-*em i.t.ctio..m (Fiftk DNes.e) 10]S Prudence Kriefer 39 mwmw Syadro.e ><Q1g Marian E. Melish Nyven J. Muchetu 40 ,:,aa syadmee io3 Frederick L. Ruben Liwt liiS

Ir CHAPTER 34 !Sl HISTORY 9S2 THE STRUCTL7tE AND BIOLOGY OF HPV 9S3 CLASSIF7CATION OF HPV TYPES 9S4 EPIDEMIOLOGY fS4 PATHOGENFSIS 954 TRANSMISSION 955 tNCUBATiON PERIOD ns HOST RESPONSE 9% CLINICAL SYNDROII(ES (COMMON) 956 CUTANEOUS 956 MUCOSAL !SI CLINICAL SYNDROMES (UNCOMMON) 9SS IMMUNOLOGIC RESPONSES 93g CELL-MEDIATED IMMUNITY 958 HUMORAL IMMUNITY !S! LABORATORY DtAGNO6IS OF INFF.Cl7ON 959 GENUS-SPECIFIC TESTS 961 TYPE-SPECIFIC TESTS !f3 TRF'.AY'11lErri !i4 VACCINATION !64 SUMMARY Human Papillomaviruses A. Bennett Jenson Robert J. Kurman Wayne D. Lancaster 951 The human (H) papillomaviruses (PV) cause a variety of cutaneous (plantar, common, and fiat or juvenile) warts'•' and proliferative squamous lesions of mucosal surfaces such as the oral cavity; .• larynx,'.: and anogenital areas'-' Al. though most lesions associated with HPV infec- tion appear to be benign and many spontaneously disappear, some cutaneous and mucosal papillo- mas undergo malignant transformation into in situ and invasive squamous carcinoma.'•t8- " The inability to propagate HPV in tissue culture" or transmit it to other animal species" has been the major impediment in characterizing the virus and studying its oncogenic potential. Recent advances in molecular virology, however, have led to the recognition that HPV displays a remarkable plur- ality with at least 16 different types (HPV-1 to HPV.16) distinguished serologically and/or by molecular hybridization techniques (Table 34-1). From these studies evidence is emerging that the type of HPV determines, in part, the clinical and pathological appearance, and natural fate of HPV-associated lesions. HPV are members of the A (PV) genus of the family Papovaviridae; the polyomaviruses consti- tute the B genus." The genera do not share any antigenic detetTninants" or polynucleotide sequence homology." The PVs have a larger ico- sahedral capsid (55 nm vs 45 nm) and a larger supercoiled, double-stranded DNA genome (5.0 x 10• dahons vs 3.3 x 10' daltons)." The genera also differ in biological properties.'• PV are species specific, producing hyperplasias/neo- plasias in natural hosts. In vitro infection is relatively quiescent without expression of late viral genes.'•-" In contrast, the polyomaviruses give rise to both productive and nonproductive in- fections in vitro but do not cause tumors in the natural host." HLSTORY Warts have created a mystique dating from antiquity since many undergo spontaneous regres- sion frequently attributed to an assortment of HWSTER-S-PP002745

l I 952 Tabk 34-1 Classification of Human Papillomavirus Types and Subtypes and Lesion with which They are Most Often Associated Type Lesion Reference la,b,c Plantar warts Gissmann et al" Heilman et all' 2a.b Common warts Heilman et all' Orth et all' 3 f7at warts Orth et al" 4 Plantar warts Heilman et all' Gissmann et al" sa,b.c Epiderrnodysplasia verruciformis (EV) Kremsdorf et al "' Ostrow et all' H Pfister, personal communication 6a,b Anogenital warts Gissmann and zur Hausen" de Villiers et a!" 6c,d,e Laryngeal papilloma. anogenital warts Gissmann et al" 7 Common warts of butchers Orth et al "' S EV Pfister et al "s 9 EV Kremsdorf et al iu 10 EV G Orth, personal communication I i Laryngeal papilloma. cervical dysptasia Gissmann et al°' Gissmann et•a!" 12 EV G Orth, personal communication 13 EV H Pfister, personat communication 14 EV G Orth, personal communication is EV G Orth, personal communication 16 Cervical carcinoma H zur Hausen. personal communication unconventional forms of treatment,'"13 including genital Sact condyiomas to human volunteers hypnosis and psychotherapy (reviewed by Bun- who developed typical cutaneous warts at the sites ney'). HPV was the ftrst tumor virus to be tratu- of inoculation was interpreted as indicating that micced experimentally from one host to another. s there was JW one type of human wart virus and This was accomplished in 1894 by L3cht,h" who A~'that the site olAftdon d~ p~erhaps, the genetic transmi:tte~ warts from his=other to ltimaeK by mak~ of~~ Oatient d~e~ttitted the clinical inoculation of •crude watt &ateriat. .Cia~O" iA 5-tppaFattCt. tYf cfitme4ua warts and mucosal pa- 1907 mid Serra'& oftPear later derttonstrsted thak.- pilloeais. 1-4o+Mrva, recognition that there were ~._ warts taDUid be fiat*xW b3!- cell-free filtrates ~_..dia~: papilloata~tts types and subtypes"'" wart maieaal. in i919, Wile and Kingreyn sue4- stimul#ted a new-t intermt in the role of these cessfuIIlr traasmitfed waRS through a succtssion _.Mptaqj="n '?iyp501ssiaslneoplasias arising in of hutnan volunteers using sterile extracu. E3ec-- OwmFoWspithdiut>t. tron microscopic studies by Strauss et al: !~ Melnick A a1,'' Bntitit~," and Alteiidz -et aN' confuined- a viral tttiofo:y for cutaneoua-warts. z~ THE Melnick,a' in 1962, RottQed the papillotnaviruses7n_ 81006 together with the polyotrtttvitwea in the fat3dty Pa- _-: 4iPV. =tr* rii>~t- `' `t»tnvdoped icosahedral povaviridae beause bot>!~e DNA ptmorvirttses'- eapsi~ ~~ in dit~er) with 72 capso- with sittn'tar uitrawucttuat features. Repotts meres" Theq o aot contaitt~lipids and are inac- (reviewed by Rowson and Mahy')of transmission of tivated by treatment with 0.4% formalin for 72 filtrates of warts, larMeal papillomas, and hours at 4•C" Both complete and empty par-

953 icles may be present.,' I The complete particles nave a sedimentation coefficient of 296 to 300 S and a buoyant density in CsC1 of 1.34 g/mL; empty particles have sedimentation coefficients of 168 to 172 S and a bouyant density of 1.29 g/mL (tubular capsids are frequently observed at this density). Molecular analysis of PV DNA was first deter- mined by Crawford and Crawford'0 and Craw- ford." " Using stringent hybridization techniques without the aid of various restriction endonucle- ases, it was concluded that although PV from the various species had similar structures and molecu- lar weights, there were no polynucleotide se- quences shared among the different genomes analyzed. The PV genome exists in virions and in infected cells in three forms (Fo); FoI is a cova- lently closed, circular, supercoiled molecule with a sedimentation coefficient of 23 S; FoII is an open, circular molecule with a sedimentation coefficient of 17 S and, Fo1II is a linear molecule with a sedi- mentation coefficient of 16 S u," AU forms appear to exist as nonintegrated, replicating plas- mids in both benign and malignant lesions."~" The molecular size of the genome based on agarose gel electrophoresis and contour length measure- ment of DNA molecules by electron microscopy reveals a molecular weight of approximately 5 x 10• daltons, corresponding to 8000 base pairs of genetic information, which is sufficient to code for 300,000 daltons of protein?'-" The guanine-plus- cytosine content of HPV DNA is 41 moi°1o,•' and nearest-neighbor analysis indicates a similarity to host species DNA" Two pairs of inverted repeti- tive (palindromic) sequences have been detected in the genome of HPV-I" the significance is unknown. Structural viral proteins constitute approxi- mately 88% of the mass of the PV virion," and up to 10 polypeptides have been resolved by sodium dodecyl sulfate polyacrylamide gel dectrophoresis. Each PV has a major capsid protein with a molec- ular weight of 54,000 to 63,000 daltons" "-" A variety of other polypeptides in the molecular weight range of 30,000 to 53,000 daltons are con- sistently resolved in most virus preparations. Al- though some may represent degradation products of the major capsid protein, others are most likely minor capsid proteins. Four low-molecular- weight polypeptides closely associated with viral DNA have been resolved and are similar to cellu- lar histones." At present nonstructural viral pro- teins and/or PV tumor antigens have not been identified in HPV-associated lesions using a variety of techniques. CLASSMCATION OF HPV TYPES The papillomaviruses are classified into types or subtypes on the basis of polynucleotide sequence homology." To be classified as a new PV type, a maximum of 5007o polynucleotide sequence ho- mology with other classified viruses should exist in conjunction with significant serological deviations in reciprocal assays. Those viruses with greater than 50°to but less than 100eto deoxyribonucleic acid (DNA) homology are subtypes. The closely related subtypes usually differ by having one or more distinct restriction endonuclease cleavage patteras. For example, HPV-6 and HPV-11 share about 2507o DNA sequence homology"•" and are thus considered different types; however, HPV types la and lb are subtypes since they are indis- tinguishable by hybridization and differ only with respect to a limited number of restriction sites." HPV DNA studies are dependent on extraction of nonintegrated supercoiled viral DNA from either purified virions or directly from HPV-asso- ciated lesions'-" The recoverability of virions appears to be dependent on both the type of HPV and the extent of differentiation of squamous epi- thelium as characterized by the degree of keratini- zation.30 PV structural antigens (and therefore virions) have not been found in lesions composed primarily of proliferating squamous epithelial cells with little or no differentiation or in squamous carcinomas regardless of the degree of differentia- tion (Jenson, unpublished data). Cutaneous warts, particularly plantar and common warts caused by HPV-1, contain the largest quantity of virions" whereas mucosal papillomas appear to contain the fewest viral particles. Recovery of viral DNA for purposes of classification can be accomplished by a variety of different approaches. One approach requires an abundance of HPV virions that are ex- tracted from the wart/papilloma and purified in CsCI equilibrium density gradients. The virions are ruptured by treatment with sodium dodecyl sulfate, deproteinized by phenol extraction, and supercoiled DNA subsequently isolated in CsCI-ethidium bromide equilibrium density gra- dients"•" Another method, particularly suitable if there are only small amounts of HPV particles, is extraction of supercoiled HPV DNA as a plas- mid from infected celLs"•"•"''° usually using modifications of the Hirt" differential salt extrac- tion technique. Early studies of endonuclease restriction pu- terns and physical maps of HPV DNA were limited to those viruses that produced large amounts of virions in cutaneous warts"•" How- - --tlmr- n_ P

954 ~s. HPV-S iid i~V~t have been frequently :w_ d ~idtffiobyca! studier, case reports, and = detected in pityriasis rosea-tike l,esions oa patients fe+r traaswlstion sttidies a¢wns humans.' For W year=:hgr-ieea known that cutaneous with epiderttiodysptisia vesrirciformis, a rare fa- .a& ,~,:_ milia~e#isorder, and in-immunosuppressed pa- warts ¢ould be traasmitted between individuals, tients. HPV-6 is the predominant virus associated either directly by skin contact, or indirectly from occasional mosaic plaatac warts and atftc '_al ~-- xlM[oa of_tlte,svailabk endeace that HPVs are warts. HPV-3 isdoiM aftnon exclusively ia 'lfat --tr~rt~u'3hibk, .- infectiotss Wnts comes from in common watts..*ii it.3tas al:o been found in "M11am1MM reassociation kiaetics" _ w*-nOw know t!~'most naturally occurring anti- Using molecularrb3+tfidizatioa techniques and - bodies are t7ps specific, reqtming the appropriate restriction easyme, diRst~on aaalysis. at least ii ~'.._ HPY type-speci8c anti,st for a positive reaction different types~of HPV bavar been clauified (Table : regii•dleia-of ,,,~e test. Ia- the past, most HPV an- 34-I): Tlwa vinues ace peefetentially, but not ex- ftea grep.is~ons have been obtained from pooled te%* speciSc clinical lesions. _erat4, which, in r+etrospecttPreclude interpreta- clusively, atsociad HP1%rpe 1 and 4_(HPV_.i_aad HPV.4) are asso- tioa_of some of the results. ciued mith pWttar waeta: I#PV-1 has also been ` ideati5ad..ia a small pe+oportioa of common and !A?HOGENFSIS anaseslital warts. HPV-2 is pfeCasttinantly found ever, with the development of molecular cloning with anogenital warts. HPV-7 is only associated technology, many new HPV types have been iden- with warts of meat handlers such as butchers, etc. tified from tissues containing small amounts of HPV-10, 12. 13, 14, and 15 have been detected viral DNA."'* Viral sequences are cleaved with a only in patients with epidermodysplasia verruci- battery of restriction enzymes of which one may formis. HPV-li is associated most commonly yield a single cleavage site. facilitating cloning of with largyngeal papillomas but, along with the entire viral genome. The vector systems cur- HPV-16, has been reported in cervical dysplasias rently in use are bacterial plasmids, particularly and cervical carcinoma. pBR3,1-1, and derivatives of bacteriophage lambda. The viral sequences to be cloned are ligated to the EPIDE!rtIOLOGY %ector and then used to transform competent bac- Accurate information on the prevalence of terial cells. Most vectors have selective markers HPV-associated lesions is not available because tt such as antibiotic resistance or metabolic pathway is based primarily on clinical observations and his- inactivation. Once a bacterial colony that contains tories of patients.'.'-" Detailed epidemiolog,c the proper insert of viral DNA is selected, vir- studies have not been performed. Laryngeal papil- tua4ly unlimited amounts of viral sequences can lomas occur predominantly in preschool children" be prepared from mass culture for detailed but exhibit a second lower peak in later life.'0 Ver- characterization."" rucae plana (flat or juvenile warts) occur primarily Preliminary identification andior classification in preschool and grade-school children. Common of DNA sequences extracted from biopsy material warts appear to occur most frequently in school- are accomplished by probing for HPV sequences age children and adolescents with the frequency using blotting methodologies such as described by of current HPV infection varying from S% to Southern." With this method the low-molecular- 13?7.. Piantar warts appear to be more common in weight DNA sequences are extracted from virions adolescents and young adults, probably as a result or tissues and cleaved using restriction enzyme en- of a high exposure in communal activities such as donucleases.43 The DNA fragments are separated bathing and swimming, where a combination of by gel electrophoresis and then transferred to a moisture and minor trauma to the feet play a role. nitrocellulose membrane and hybridization with a Anogenital warts are most often seen in young, radiolabeled PV probe can be performed under sexually promiscuous adults.' stringent (2S°C below the melting temperature of Epidemiologic studies of HPV-associated the DNA or Tm -2S°C) or relaxed (43°C below lesions must take into account that the spontan- the melting temperature of DNA or Tm -43°C) eous regression of warts, and probably mucosal conditions."-°'-" Under relaxed conditions. papillomas, is high, approaching 25074 to 35°7o in regions of HPV DNA homology with as much as the first six months and 359% to 70% within two 337% base mismatch can be detected. The percent years.?~v_ Assessment of antibody prevalence is -homology among the various HPVs, however, pebalWft most reliable measure for evaluating can only be accutatdy measured by DNA-DNA tlW,epideteti0W of HPV infections." However, I 0

955 clothing, jewelry, bathroom floors, etc. Warts and mucosal papillomas/condylomata, particu- larly those occurring at pressure points such as the sole of the foot or in loose traumatized tissue like the laryrtx, are likely to be multiple and recur after therapy. This is probably due either to auto- inoculation or seeding of HPV at the site of iatro- genic trauma. There is good evidence that infants may contract the HPV that causes juvenile laryn- geal papilloma as they pass through the birth canal of mothers who have genital condylomata, and, perhaps, cervical dysplasia.'•' Some cases of _#rai condylomata occur concurrently in individ- uals with genital condylomata.•" All of these ob- servations suggest that HPVs are the tra8stz>Wle agents that cause cutaneous and mucosal paj5il- lomZs in man. Two populations appear to be partictelarftore- disposed to HPV infections: 1) institunonalized individuals, and 2) immunosuppressed individ- uals. In one study over one quarter of patients in a mental institution had warts." This probably only reflects increased contact among individuals in close quarters, since the spontaneous regression rate was as high as other populations. The increased incidence and severity of warts in patients with naturally acquired and iatrogenic immunosup- pression undoubtedly reflects the role of the im- mune system in HPV infections.= '13-76 Although generalized warts have been seen in patients with agarnmaglobinemia, it appears to be more fre- quent in patients with decreased cell-mediated responses. Incubation Period The incubation period of warts is best assessed by reviewing data from experiments on the trans- mission of human warts.' The incubation period for most of the experiments was one to three months, but some incubation periods were as long as six to 20 months. The incubation period for genital warts has been estimated at two to three months with an infectivity rate of approximately bS47o." Host Response All three types of squamous epithelium, 1) cu- tane.ous (keratinized), 2) mucosa! (nonkeratinized), and 3) metaplastic, are susceptible to infection by HPV.' Generally, the various types of human warts and mucosal-.,papillomas are well-circum- scribed papillomatous growths that vary in size and color and tend to have a roughened horny surface but some HPV-associated lesions such as juvenile warts, cervical dysplasia, and pityriasis rosea-like lesions are flat and difficult to delineate precisely on visual examination.=•'• It is thought that the HPV genome is present in a stable form in the basal layer of the wart," and that early gene expression is associated with acan- thosis (increase in stratum spinosum or prickle cell layer). Vegetative viral DNA synthesis begins in the suprabasal cells of the prickle layer" and is followed by late gene expreuion (structural poly- peptides) and virion assembly occurring almost ex- clusively within nuclei of the granular layer of cells." The phenotypic expression (histopathologic Nwe«~ rsor~om. - - hoi+wn~ow ~ ° tlie4. o1n" Figure 34-1 This illustration depicts the hi:tolopc features of normal skin and the histaoat6biogic features of an adjacent papilloma (wan). in the normal itiitt, there is a basal layer of od(i,which under" divisdon to fotut a multipk cell layer of prickle cells. The number ofprictk cells determines the thiftness of the skin, which varies aecorditt; to location. As the prickle cells are pushed toward the surface, they flatten and develop cytoplauaic keratohyaline =ranules (Ranular cells). The Rattular cells then lose nudei, and the cytoplasm and keratohyatitte giranuks coalesce into a flat, acetlular keratinized layer. In the papilloma. there is a sinlle layer of basal celis but there is hyperplasia of prickle cells (acanthosis) and production of excess keratin (hyperkeratosis) which is associated with the formation of epithelial spikes (papillomatosis). Virus inclusions andlor expression of HPV structural and{ens usually appear in granular cells with degenerative cytoplasmic vacuolization (koilocytotic cells). Reprtnted by permission from Jenson et al.'4 ~~~~ ~~ -S, 0 Aw

956 features) of most cutaneous warts and mucosal papillomas are similar' (Figure 34-1). Productive HPV infection is manifested by proliferation of prickle cells (acanthosis), degenerative cytoplas- mic vacuolization (koilocytosis), nuclear altera- tions (wrinkling and pyknosis) of granular cells. and the production of excess keratin. The acan- thosis and hyperkeratosis are associated with the formation of epithAal spikes referred to as papillomatosis. The epithelial proliferation may also extend downward, resulting in accentuated rete pegs referred to as inverted papillomatosis. Inverted papillomatosis is a feature of all but the flat lesions such as juvenile warts, pityriasis-like lesions, and cervical dysplasia. Inverted papillo- matosis is frequently encountered in vulvar lesions such as bowenoid papulosis (dysplasia), where it may be°mistaken for stromal invasion. Another characteristic feature of PV-induced lesions is their vascularity. As the epithelial proliferation becomes more exuberant, capillaries from the underlying -1dermis or stroma are drawn into the lesion. This accounts for the vascularity of warts and the abnormal vascular patterns seen in cervical dysplasia when viewed through the colposcope. CLINICAL SYNDROMES (COMMON) Cutaneous' " (Table 34I ) Verruca vulgaris (HPV-2; occasionally HPV-1) is the most common (vulgar) of all the warts. It is sessile and exophytic with a crusty appearing pa- pillomatous surface. Common warts can occur singly but are frequently multiple. They are usu- ally located on the hand. Verruca also occur in the oral cavity but much less frequently than on the skin. They are most frequently seen in young chil- dren. Some verruca disappear, but many are treated. Nonetheless a small but significant per- centage will recur regardless of the form of thera- peutic intervention. Verrua platttarfs or plantar warts (HPV-1: HPV-4) art seen mainly in adolescents and young adults. They usually occur as a single lesion on the sole of the foot. Although clinically aggressive, these warts usually respond well to treatment. They ars not elevated above the surface of the skin but extend deep into the sole where they are frequently painful. Although many forms of ther- apy, including irradiation. have been used on these lesions, there are no reports of malignant transformation. Verrnca plana (HPV-3) are flat warts which usually appear over the face and those parts of the extremities other than the hands and feet. Thev are multiple. Since these warts usually appear in children, they are frequently called juvenile wans. Juvenile warts are usually asymptomatic. cause few cosmetic problems, and may persist for a number of years, only to spontaneously disappear within a few weeks of each other.'°-'1 Mucosal Oral cavity and larynx Focal epithelial hyper- plasia (HPV type unknown) occurs only in the oral cavity, appearing clinically to be flat or slightly elevated and either white or the color of the surrounding mucosa.' These lesions have been reported to occur primarily in natives of Green- land and North and South America. suggesting the influence of environmental andtor genetic fac- tors. In Greenland Eskimos, it has been observed primarily in adults, whereas in the southwest In- dians, it has been reported to occur primarily in children." These lesions are usually multiple and spontaneously disappear. Single oral papiIlomas (HPV type unknown) are the most common benign epithelial tumors of the oral cav%ty!' They are pedunculated with a fibrovascular stalk and usually have a rough papil- lary appearance to their surface. They can occur in any age group, are usually solitary, and rarely recur after surgical excision. Multiple papillomas (HPV type unknown) are clinically and pathologically similar to condylo- mata, which occur infrequently in the oral cavity.4 Laryngeal papillomas are associated with HPV-11 "•" and are the most common benign epithelial tumors of the larynx." The clinical pic- ture is that of small exophytic papillomas which can coalesce and appear very exuberant.' They occur more frequently in children than adults, are typically multiple, and frequently recur after surgical removal. Laryngeal papillomatosis is usu- ally considered a life-threatening condition in children because of the danger of airway obstruc- tion and suffocation; they frequently disappear after puberty. Occasionally papillomas may ez- tend down the trachea and into the bronchi. Ma- lignant transformation may occur after radiation therapy.,."-" Aaogtaftal Condyloma acuminata, so-called anogenital or venereal warts, are caused primarily by HPV-6u•"•" but also HPV-li 1!' -1, and -2!' and occur almost exclusively on mucosd surfaces of external genitalia and perianal areas.".« They are occasionally found in the oral cavity where MRSTER-S3PP

957 they are indistinguishable from multiple papil- lomas.• " In females, condylomata may regress spontaneously, persist for years, or undergo a ra- pid increase in size during pregnancy. Genital warts of long duration appear to have a low risk for undergoing malignant transformation.' Most show evidence of mild dysplasia but greater de- grees of dysplasia are extremely rare. Approxi- mately 5074 of squamous carcinomas of the vulva have been reported to develop in association with condylomata.' •' Perhaps the best clinical evi- dence of maGgnant potential of genital warts occurred in two rare cases in which carcinoma of the vulva in young teenage girls was preceded by condylomata (reviewed by zur Hausen'). In males, 15074 of squamous carcinomas of the penis have been reported to develop in association with preexisting condylomata.' " Many penile carci- nomas are of the verrucous type with features of giant condylomata acuminatum of Buschke and Lowenstein" Of interest is the observation that circumcision reduces the incidence of penile carci- nomas but not the appearance of penile condylo- mata.' Perineal and perianal condylomata may also undergo malignant transition to squamous cell carcinomas, particularly in homosexual maies."O Of particular importance is the recent observation that papillomas/condylomas of the distal male urethra may be flat or exophytic with some showing atypia.^• •' these lesions are most likely transmitted by sexual contact to the cervix where a productive infection gives rise to cervical dysplasia. Cervical dysplasia (cervical intraepithelial neo- plasia, CIN) has been associated with HPV-6,1 -11!' and -16 (personal communication, zur Hau- sen), and other unknown types, and is the precur- sor of squamous carcinoma." The development of cervical cancer is thought to proceed through a continuum of progressive cellular changes from mild (CIN 1) to moderate (CIN 11) (Figure 34-2) to severe dysplasia and/or carcinoma in situ (CIN III)"" This sequence of events has been docu- mented by a number of studies with transit times from mild dysplasia to carcinoma in situ ranging from 84 to 96 months." Approximately 40074 to 70074 of the dysplasias undergo spontaneous re- gression. Although the etiology of cervical cancer is still unknown, epidemiolojW-studies suggest that both precursor lesions and carcinoma are linked to venereally transmitted agents." For many years, herpesvirus type II was thought to be the most likely etiologic agent based on seroepi- demiologic evidence.1O01Ot However, since 1980, morphologic, immunologic, and molecular viro- logic studies support the view that HPV plays an important role in the development of cervical neo- piasia."" i42"O' HPV is one of the most com- monly sexually transmitted agents, having been identified by the presence of koilocytotic cells in cervical smears of 1076 to 2°70 of all women and S% to 10% of young women screened.'o1.uo-1u E'ig.ee 34-2 Moderate cervical dysplasia showing zone of papillomavirua-auodated hyperplasia occupying the lower half of the thickness of the epitheliutn. The upper half of the epithelium shows cells with vacuoiated cytoplasm and mild nuclear uypia. Papillomavicus an- tigens and DNA were present in this case (hematoxylin and eosin, x I0o). k G

958 CLINICAL SYNDROMES (UNCOMMON) Epidermodysplasia verruciformis (HPV-3, -S, -8, -9, -10, -12, -13, and -I4) or verrucosis gener- alisata"3 is a rare, autosomal recessive, disease characterized by varying degrees of decreased cell- mediated immunity and increased susceptibility to HPV infection manifested clinically by poly- morphogic skin lesions resembling either flat warts or macules indistinguishable from pityriasis rosea." f2 114-11' In approximately 25070 of Cauca- sian patients malignant transformation occurs within the pityriasis rosea-like lesions in sun- exposed areas such as the face and hands. Only lesions containing HPV-S appear to progress to premalignant lesions, histopathologically similar to cervical dysplasia before developing into carci- noma in situ (Bowen's disease) and, eventually invasive squamous carcinoma. HPV-5 can be identified in primary or metastatic malignant le- sions as a replicating, nonintegrated plasmid!' Immunosuppressed patients, particularly those undergoing renal transplantation, also develop pityriasis rosea-like lesions associated with HPV-S, which may undergo malignant transfor- mation."• Although warts that are present at the beginning of therapy become aggressive,3 newly acquired warts are usually seen only after the first year of immunosuppression." Squamous cell carcinomas usually do not develop in patients with immunosuppression of short duration, sug- gesting that HP V-S is acquired from some reser- voir which is as yet unknown. Flat warts asso- ciated with HPV-3 have also been reported in renal transplant patients."' IMMUyOLOGIC RESPONSES Available evidence suggests that spontaneous regression or persistence of warts is related to the immunologic responsiveness of the individual (re- viewed by von Krogh"). Studies of HPV infec- tion suggest that stimulation of cellular immunity is associated with regression of lesions and such immunity is HPV type specific. Humoral antibody responses to HPV are almost always type specific and can apparently be used to determine evidence of previous infection." The antibody response can also be used as a prognostic indicator of spon- taneous regression and/or successful therapy. Many of the previous studies of immune re- sponses to HPV infection are, however, difficult to assess in view of our current recognition of the plurality of immunologically distinct HPV. Cell-Mediated Immunity Patients with clinical evidence of altered cell- mediated immunity have an increased incidence of warts that are usually flat." This has been seen in immunosuppressed patients,"3-7" patients with cellular immunodeficiency,'• and patients with weak cutaneous hypersensitivity responses.i° aIl Specific immunologic defects may be involved, however, since not all patients with persistent warts have evidence of nonspecific abnormalities in cell-mediated immunity. There is pathologic evidence of cell-mediated regression of warts, particularly flat warts." A perivascular infiltration of mononuclear leukocytes is usually seen in the upper dermis with epidermal invasion localized to the flat wart. Patients with regressing flat warts will maintain plantar or common warts, suggest- ing that HPV type-specific structural or non- structural proteins are recognized at the level of the cellular immune response.'0 Noteworthy is the observation that spontaneous regression or cure following treatment is frequently associated with enhanced specific cellular immunity as tested by cutaneous hypersensitivity to wart virus-asso- ciated antigens 1::.1u The role of substances associated with cell-mediated responses such as lymphokines is unknown. Humoral Immunity Serologic evidence of wart virus antibodies ha~e been detected by a variety of techniques in various population studies (reviewed by von #Crogh"). The significance of these sudies is unknown because the tests have been performed on dif- ferent preparations of wan virus material. Pa- tients without wart virus antibodies or with 1gM alone are less likely to undergo spontaneous regression or be cured than those with IgM and (gG virus-specific antibodies'•'••i" Furthermore, after spontaneous or therapeutic cure, IgG anti- bodies will remain elevated for up to six months and as long as nine years." IgG antibodies are known to be circulating in half of patients who give no history of having warts."'-"' Patients with chronic andlor multiple warts are much less likely to have IgG against HPV." Of particular interest is that patients with warts that do not con- tain viriotts are less likely to have antibodies."' Regardless, the role of serologic tests in evaluating HPV infections must be performed using HPV type-specific polypeptides as antigens. In such a study PSster and zur Hausen,:' using purified HPV-I polypeptides, demonstrated by radioim- munoassay that 40% of an unselected group of individuals had circulating antibody to HPV-I.

959 LABORATORY DIAGtiOSIS OF iNFECTION The multiplicity of HPV types and the relative lack of cross-reactivity of type-specific reagents has impeded an assessment of the role of HPV types in lesions suspected of a HPV etiology. In the past, evidence for the presence of HPV in a particular lesion was mainty inferential, based on the morphologic similarity of the putative HPV-induced lesion to warts or condylorna acu- minata, lesions known to be caused by HPV. '. Identification of PV particles by electron mi- croscopy" " 103 ` I=s us offered a direct method (Figure 34-3). However, because of the possibility of sampling error, particularly with the mucosal lesions, and the fact that it is a time-consuming, ex- pensive technique, its value is diminished as a prac- tical screening technique.' u Recently, application of immunological and molecular hybridization methods to assay tissues for the presence of PV genus- and HPV type-specific structural antigens and DNA sequences have become possible and are routinely used for diagnostic purposes.'. 54 #9 Genus-Specific Tests Human and animal papillomaviruses contain regions of conserved polynucleotide sequence homology that can be detected only under non- stringent hybridization conditions such that heteroduplexes with 25076 to 350/o base mismatches remain thermally stable." In addition, papil- lomaviruses from diverse species have cross-reac- tive internal capsid antigens that can be detected using antisera prepared from disrupted papillo- mavirus."" Using these genus-specific reagents, it is now possible to test for the presence of HPV structural antigens and DNA sequences regardless of the HPV type associated with a particular lesion. Productive HPV infections, identified by the presence of genus-specific structural proteins using hyperimmune sera prepared against common anti- gens derived from either HPV or bovine papilloma- virus, can be demonstrated in acetone-fixed frozen sections by immunofluorescence (Figure 34-4) or fotinalin-fixed paraffin-embedded sections rou- tinely processed for pathology using imrnunocyto- chemical techniques (Figure Since 1980, a variety of cutaneous and mucosal lesions have been screened for the presence of the PV com- mon antigen. The frequency of HPV antigenic ex- pression using these techniques was 5007s to 7001o in plantar and common warts•" 40e/o to 60% in ver- rucae (Figure 34-6), multiple papillomas, and con- Finrs 34-3 F.lectron miaoaaph of Fanular layer-keratin junction in same piantar wart as Figures 34-4 and 34-5. PapilLonuvirus particles (50 to $5 nm) in crystalline array fill nucleus of cell immediately prior to undergoing keratiniution. Reprinted by permission from Jenson et al'= ( x 27,000) © ®

1% 960 Flfure 3i-i Acetone-fixed frozen section of plantar wart containing HPV.1. Type- specific structural antigens (arrows) are seen in many nuclei of cells in upper prickle and granular layer of epithelium. Reprinted by permission from Jenson et al.`s (Fluorescent antibody stain, x 400.) E'is.es 344 Formaiin-(lmd paraalnembedded section of same piancar .raet ai in Figure 34-3. Papillomaviru: senus-specific (comaion) struaucal antigens (acrows) stia seen in nudd of koilocytotic Qanular cells. Reprinteri by permission from Jeasoa a al. `s (Peroxidise-aaaperoaidase stain and hernatoxytin, x a00.) I ~~ ~ ~~~~ PP 7a~'~ 5'~

dylomata of the oral cavity;'-" 30474 to 45'1% of single papiAomas;' u approximately 5047s in laryn- geal papillomas;•-"° approximately S0e/i of vulvar condyiomas;10f,10' 4S%a to 6S41a of male urethral papillomas/condylomas^"' and 4047% to 30% of mild dysplasias of the cervix (Figure 34-7).••'-1" leis currently unknown why viral antigens are detected in only 40014 to 70479 of these lesions. Although lack of antigen detection may be due to sampling error, limited sensitivity of the test, or denaturing of viral antigens during tissue processing, it probably is related to a cyclic or periodic expression of struc- tural viral antigens by the viral genome, perhaps modulated by the immune response. In a study of 102 laryngeal papillomas, 48% were positivt for viral antigens.' However, when four or more con- secutive recurrences of the same lesion were ex- amined, at least one was always stained positively. Thus, although a positive immunologic reaction for HPV means that the lesion is associated with the virus, a negative reaction does not necessarily ex- dude an HPV etiology. 961 Figure 3a-6 Verruca from hard palate of oral cavity. Papiilomavirus genus-specific structural antigens are identified by posi- tive PAP reaction (arrows), mainly in koilocytotic cells of granular layer of a single papillary frond. Reprinted by per- mission from Jenson et al.' (Peroxidase- antiperoxidase stain and hematoxylin, x 197.) Nonstringent hybridization techniques have been used to demonstrate HPV sequences in two of four laryngeal papillomas"' and 10 of 16 cer- vical dysplasias (Figures 348A and B)."-10' When correlated with immunocytochemical staining for genus-specific antigens, it was concluded that at least 75% of laryngeal papillomas and 93+ti of cervical dysplasias, respectively, were associated with HPV infections. Type-Specific Tests HPV-1, -2. -3, and -5 type-specific viral anti- gens have been identified by immunofluorescence in frozen sections of cutaneous warts"•"'•"• Although no cross-reactivity was observed among these HPVs using heterologous antisera, the im- munofluorescence technique is of little practical use for diagnostic purposes since tissues for most retrospective studies are preserved in formalin. Recently, HPV-1 type-specific antigens have been identified in formalin-fixed tissue using hyperim- mune rabbit serum and the PAP technique" MASTEER ~ :avp Z. # C'

1 962 Figure 34-7 Mild cervical dysplasia with prominent epithelial spikes containing numerous cells with intranuclear genus-specific papiilomavirus antigens (arrows). Papillomavirus DNA was also recovered from this case. (Peroxidase-antiperoxidase stain without counterstain, x 197.) Reprinted by permission from Lancaster et al.s' HPV-1 antigens were found in S407o of plantar warts and 11 07e of common warts. Moreover, the presence of HPV-1 viral antigens corresponded to the presence of HPV-I specific nucleotide se- quences determined by molecular hybridization studies. Preliminary classifications of HPV DNA in biopsy specimens are made on the basis of the hybridization spectrum of viral DNA fragments generated by appropriate restriction endonu- cleases and by assessing the relatedness of viral se- quences as judged by hybridization efficiency under relaxed and stringent conditions.'••" When warts and/or mucosal papillomas undergo malig- nant transformation, late gene expression (struc- tural viral proteins) is no longer detected, but the PV genome continues to replicate as a plasmid, al- though at a lower copy number. Detection of HPV DNA associated with the transformed state is best accomplished under stringent conditions that are at least ten times more sensitive then non- stringent conditions, a difference which appears to be particularly critical in PV systems. At least 16 different HPV types have been char- acterized by molecular hybridization studies (Table 34-1). Besides being preferentially asso- ciated with specific clinical lesions, subsets of HPV appear to be primarily cutaneotropic or mu- cosotropic (HPV-6, -1 l, and -16) and noncarcino- genic or potentially carcinogenic (HPV-S, -6, -11. -16). Therefore, most studies on squamous cell carcinoma of ttie skin have been probed with HPV-S DNAs, whereas most studies of squatnous cell carcinoma of the laryngeal and anogenical areas have been probed with HPV-6,"'6 -11,'• and, more recently, HPV-16 (zur Hausen, per- sonal communication). HPV-S has been detected in both primary and metastatic squamous carcinomas of patients with EV•= and in the primary carcinomas of patients undergoing immunosuppressive therapy."• Dem- onstration of HPV in metastatic lesions is of par- ticular importance since HPV DNA sequences found in the primary lesions could possibly be a contaminant from an adjacent benign skin lesion. HPV-Il has been found in half of laryngeal papillomas, approximately one fourth of genital warts, cervical dysplasias, and four of 24 cervical cancers (two in situ; two invasive)!' HPV-6 se- quences have been found in the majority of typical condylomata acuminata (venereal warts) and condylomata acamittata of Lowenstein and Bushke," an ag=ressive infiltrating squsmous lesion that usually does not metastasize." Other evidence that HPVs are associated with malignant tumors has been the finding of an untyped DNA isolated from a puient with EV in four squamous cell carcinomas of the vulva and cervix." Re- cently, HPV-16 has been found in 609e of squa- mous cell carcinomas of the cervix from German

A --IWE #LttMBER - ..v -C=- 13 Kr 19 23 30 Figures 344A&B Aucoradiograms representing hybrid- ization of in vitro 'lP-labeled bovine paptllomavirus type 1(BPV-1) DNA to human papillomavirus type I (HPV-1) DNA and DNAs isolated from five cervical biopsies. HPV-1 DNA (10 ng) (lane C) and tissue DNAs were digested with either BamHl (panel A) or Hindlil (panel B) and electrophoresed in 0.8m+ agarose gels. The separated DNA fragments were transferred to nitrocel- lulose membranes and subsequently hybridized to 3jP-BPV-1 DNA. Bands I and 2 represent the migration of circular and linearized HPV.I DNA. respectively. In both panels A and B. four of the five cases tested con- tained DNA sequences to which the BPV-i probe hy- bridized. These sequences comigrated with either open circular and: or linear forms of HPV-1 DNA. Arrows indicate migration of low molecular weight DNA. pre- sent in case 19 (seen in Figure 34-7), that specifically hybridized to the BPV- I DNA probe. (Performed under relaxed or nonstringent conditions to detect PV genus- specific polynucleotide sequences). Reprinted by permis- sion from Lancaster et al." patients but in only 10*l0 of cervical dysplasias (zur Hausen, personal communication). TREATMENT Bunney,' in her assessment of a 10-year pro- gram evaluating wart cures in approximately 14,000 patients, concluded that 709s to 8044 of paiients could have been cured of warts by simple methods applied either at home or in the clinic. Whether warts are treated or left alone is usually a prerogative of the individual patient. Although most warts are usually treated for cosrttetic tYSsons, others are treated because they cause discomfort or are truly disabling. Since warts frequently undergo spontaneous regtession, veatmett should not result in permanent damage such as excessive scarring or disfigurement. 8 C 963 CASE NUMBER ~r 13 15 19 23 30 In general, warts that respond most favorably to treatment are usually single, of short duration, and found in children.' Home treatment of warts includes topical application of salicylic acid and formalin or glutaraldehyde; podophyllin or strong acids or bases should be used under supervision. Cryotherapy is the most universally popular treat- ment for warts, although surgical intervention may be indicated in some cases. Perhaps the best biological reason for not routinely recommending surgical intervention is that surgical extirpation of the wart is not associated with enhancement of the immunologic response such as is seen following multiple topical or freeze-thawing treatments that appear to facilitate recognition of HPV-asso- ciated antigens by the immunologic system.'='-"_ Quick removal of the bulk of wart virus antigens without concomitant exposure to the immune sys- tem is probably the main reason for the high re- currence rates of warts following surgery. Other treatments, such as interferon and retinoic acid, appear promising but are experimental at this time. The duration of treatment and criteria for cure of warts are important factors for clinical man- agement of these lesions. Bunney' suggests that 12 weeks after beginning treatment is an adequate length of time to expect warts to disappear. Since failure to completely eradicate the initial lesion is the main reason for recurrence, a valid criterion for cure is the appearance of a normal skin after disappearance of the wart(s). Similar criteria have not been developed for treatment and cure of HPV- associated iesions arising in mucosal epithelium. HITRS T E Ik ip

964 VACCINATION Vaccination offers potential prevention and/or cure for cutaneous warts and HPV-associated pa- pillomas of the tnucosal surfaces. It is apparent that effective vaccination will utilize either type- specific structural or nonstructural antigens and that the lesions to be targeted will be those of the anogenital area that are sexually transmitted and are at risk of malignant transformation.' Future vaccines will depend on the progress made in the molecular virology of HPV and the development of bacterial expression vectors capable of synthe- sizing highly purified, immunogenic structural and nonstructural HPV proteins. Otherwise, the use of an autogenous vaccine derived from tlte pa- tient's own lesion is the only other alternative for vaccination because of the possibility of poten- tially harmful biological contaminants associated with heterologous vaccines. Most cutaneous warts either spontaneously regress or can be cured by simple treatment.' Ap- proximately 20074 to 3Q07o of these lesions will be recalcitrant to therapy. Aithough presumably associated with general or specific immunologic defects, half of these recalcitrant warts may dis- appear after vaccination of patients with formalitt- inactivated HPV'• all patients cured developed a positive IgG response and delayed hypersensitivity reaction to the HPV preparation. Vaccination of persons at high risk for develop- ing or transmitting anogenital HPV-associated lesions appears to be indicated for several reasons. First, many of these are sexually transmitted and reinfection by consorts must occur frequently. Second. S°1o to 154% of squamous carcinomas of the vulva and penis appear to arise in preexisting condylomata, usually of long duration. Third, successful autogenous vaccination appears to be higher for venereal warts (up to 80410) than cutan- eous wans.+" Fourth, recombinant DNA tech- nology should be able to provide the necessary biological materials in the near future for vaccina- tion. Evaluation of the efficacy of vaccination for lesions of the aaogenital area, however, will de- pend on well-controlled epidemioiogic studies and clinical ttiaLt.rith evaluation of humoral (possibly secretory ISA) and cell-mediated responses to type-specific antigens. Regardless, preventive measures to avoid transmission of these viruses is much preferable to the best cure. SiJMIri.+1RY HPVs are a beterotenous group of oncogenic viruses that replicate as true plasmids in selected benign (wares/papillomas). dysplastic (cervical dysplasias/?bowenoid papulosis), and malignant (anogenital carcinomas) lesions of squamous epi- thelium. Spontaneous regression or successful topicallcryosurgical treatment of the benign lesions are largely dependent on either naturally acquired or iatrogenically-related stimulation of HPV type-specific cellular immunity. Malignant transformation of warts and papillomas appears to relate to a variety of factors: 1) infection by certain HPV types (HPV-S, -6, -11, and -16), 2) decreased cellular immunity to HPV-associated antigens, and 3) interaction with physical carcin- ogens such as sunlight or x-rays. Research on HPV has benefited more from re- combinant DNA technology than any other area in virology, mainly because HPV cannot be grown in culture or transmitted to other animal species. Future use of recombinant DNA technology in HPV research should: 1) allow many more HPVs to be identified and characterized; 2) permit the products of early and late gene regions to be syn- thesized in bacterial expression vectors and used as immunogens for diagnostic and vaccination purposes, and 3) provide a substantial amount of information about mechanisms involved in carcinogenesis. REFERENCES i. Rowson ICEK, Mahy BWJ: Human papova ( wart) virus. Bacterrol Rev 1967;31:114-131. 2. Bunney MH: Viral Wans: Their Biology and Treatmettt. New York, Oxford University Press. 1982. 3. Praetorius-Clausen F: Rare oral viral disorders (moUuscum contaposurn, localized keratoacan- thotna, verrucae, condyioma actuninatum and focal epithelial hyperplasia). Ora/ Surs Oral Med O.al Pathol 1972;34:60~b1n. 4. Jenson AB, Lancaster WD, Hartman DP. Shaffer EL Jr: Frequency and distribution of papillomavi- rus structural antigens in verrucae, multiple papillomas, and condylomsta of the oral cavity. ,4 m I Pathol 1962a:147:212-2I a. S. Cook TA, Cohn AM, &unschrvit JP, et at: Laryngeal papilloma: Etiolopc and therapeutic considerations. Am I Otol 1973;32:649-653. 6. Lack EE, Jenson AB, Smith HG, a al: Itttmuno- peroxida>ee loaliation of human pzpillomavirus in laryngeal papillomas. tnnrvirolotr 196Q:11: 148-154. 7. zur Hausen H: Human pspillomaviruses and their possible role in squamous cell carcinomas. Cttrs Top MteroDicl hnmuttol 2977t78:I-30. . i. Kurtnaa RJ. Jenson AB, Lmeasta WD: Papillo- mavirus infection of the cavitt.lI. Rdadonship to irttraepitlsdial neoplasia based on the preseace of speci6c viral structural proteia:. Am JSrrj Aathol 1963a:7:39-52. 9. Kutman RJ, Jmsac A8. Sinelair C, et al: Detec- tion of human pepillomaviruses by immuno- cytochemisay, in Detsiis RA (.d): Advasica in €

AL , Immunocyrochemi.rtry. New York, Masson, 1983b. 10. Orth G, Breitburd F, Favre M, et ai: papillomavi- ruses: Possible role in human cancer. Cold Sprtnt Harbor Conf Cell Prollf 1977a;4:1043-1068. 11. Orth G. Jablonska S, Breitburd F, et a1: The human papiliomavtruses. Bull Cancer 1978a;6S: 151-164. 12. Butel J: Studies with human papillomavirus modeled after known papovavirus systems. J Natl Cancer Inst 1972:48:285-299. 13. Koller LD, Olson C: Attempted transmission of warts from man, cattle and horses and of deer fibroma to selected hosts. J invest Derrrtatol 1972:58:366-368. 14. Melnick JL, Allison AC, Butel JS, et ai: Papova- viridae Intervirol 1974;3:106-120. 15. Jenson AB, Rosenthal JD, Olson C. et al: Irttmu- nologic relatedness of papillomaviruses from different species. I Nat/ Canrer Inst 1980;64: 495-500. 16. Law M-F, Lancaster WD, Howley PM: Conserved nucleotide sequences among the;enomes of papiI- lomavirttses. I Viro! 1979;32:199-207. 17. 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