0022-202X/ 8 1/ 7602-0097$02.00/ 0 THE J OU RNAL OF INV ESTIGATIV E DERMATOLOGY, 76:97- 102 1981 Vol. 76, No.2 Copyright © !98 1 by The Williams & Wilkins Co. Printed in U.S. A. Identification of Papillomaviruses in Butchers' Warts GERARD ORTH, D.V.M., STEFANIA JABLONSKA, MD., MICHEL FAVRE, PH.D., 0DILE CROISSANT, PH.D., SLAVOMIR 0BALEK, M .D., MARIA JARZABEK-CHORZELSKA, PH.D., AND NICOLE JIBARD G. R . [NSERM U.J90. ln~>titut Pa~>t e ur, Paris, France (GO, MF, OC, NJ) and Department of Dermatology, Warww School of Medicine, Warsaw, Poland (SJ, SO, MJ-C). We have studied the papillomaviruses found in the butchers' warts, and to look for a possible correlation between hand warts of 60 butchers, most of them from 2 distant the types of viruses and the histological patterns of the lesions. slaughterhouses. Warts differing in morphology and lo­ MATERIALS AND METHODS cation were studied separately. The viruses were iden­ tified by molecular hybridization, restriction enzyme Subjects analysis and immunofluorescence. Four known human Our studies have been p erformed in 60 butchers whose work was papillomaviruses (HPV-1, HPV-2, HPV-3, HPV-4) were either to slaughter cattle, sheep and pigs, or to cut meat and viscera. detected and one hitherto unknown papillomavirus was Most of the butchers were from 2 slaughterhouses located in different identified in 9 butchers. The DNA of the latter virus did cities. All the warts were located on the hands: in 21 butchers, they the not anneal with any of the RNAs complementary to were dorsal and palmar; in 8, they were dorsal, palmar and around ey were dorsal on ly. The clinical characteristics of either HPV-1 to HPV-5 or bovine papillomavirus type 1 nails; and in 14 th the warts and the epidemiological d ata will be reported elsewhere. • a Hind ll+ill restriction (BPV-1) DNAs, and showed The warts differed considerably in morphology, size, and growth pattern enzyme cleavage pattern distinct from those of known (endophytic or exophytic). When clinically different types of warts were HPV s and BPVs . This virus showed distinct antigenic present, each type was usua lly studied separately. The specimens used properties, as shown by immunofluorescence, using for the biochemical characterization of the viral DNA were obtained HPV-1, -2, -3, -5, and BVP-1 antisera. It may represent a from I to 40 lesions, either by removing the warts by c urretage or by new type of human papillomavirus (HPV-7) or a yet scraping their surfaces. The specimens were kept in Eagle medium unidentified animal papillomavirus. In addition, 6 butch­ before freezing at -70°C. The samples for immunofluorescence (IF) mples ers were found to be infected with a papillomavirus, and histological studies were obtained by punch biopsies. The sa froze n immediately, or placed in Eagle from the known skin HPVs and from BPV-1, for IF studies were either distinct medium before processing. which could not be characterized by restriction enzyme analysis. Eleven butchers were found to be infected by 2 Preparation of Viral DNA viruses. The extraction of viral DNA was performed according to a modified A characteristic histological pattern was found to be Hi.J·t's method (10,20,21 ). The DNA was precipitated with ethanol and associated with the different papillomaviruses. redissolved in 10 mM Tris-HCI, 1 mM EDTA, pH 7.9 (200 Ill). The concentration of ci.J·cular vi.J·al DNA molecules was evaluated b y elec­ tron microscopy (22], by comparison with a sample of the replicative It is now well established that the incidence of warts is form of bacteriophage fl X 174 DNA (fl X 174 RF DNA) of known significantly higher in butchers and meat-cutters than in the concentration. The formamid e technique described by Davi , Simon, general population [1-4]. Although some authors have raised and Davidson (23] was used for mounting undiluted or diluted samples t he question of the possible transmission of animal papilloma­ (5/ll). The grids were observed at a magnification of 8000. Supercoiled ules were scored by viruses to man [1,2,5-8], no identification of the viruses found (form I) and relaxed (form II) ci.J·cula.r DNA molec res per grid. One molecule per 5 squares corresponded in butchers' warts had yet been reported. The problem is of crossing 5 s qua to 0.06/lg/ 200 Ill. With this method, the concentration of vi.J·al DNA is special interest, since at least 5 types of cutaneous human underestimated, since li near vi.J·al DNA molecules and fragments pres­ types of bovine papillo­ papillomavi.ruses (HPV) [9-12], and 4 ent in the Hi.t·t' supernatants a re not taken into account. maviruses (BPV) [12-16] have recently been identified. Fur­ In preparations containing more than 2.5 llg of DNA, form I DNA t hermore, 2 of these viruses, HPV-5 and BPV-4, have been molecules were separated from form II and form III (linear) and from found associated with naturally occurring carcinomas [11,16- cellu lar DNA by sedimentation in sucrose gradients in the presence of 18], and 2 other viruses, BPV-1 and BPV-2, h ave been found ethidium bromide (less than 10 /lg) (18,24], or by CsCI-ethidium bro­ associated with natural cases of tumors of the connective tissue mide equil ibrium centrifugation (more than 10 llgl (10]. After dialysis occurring in an alien host, the horse [19]. against 10 mM Tris-HCl, 1 111M EDTA, pH 7.9, the DNA co ncentrations y electron microscopy, and the DNA preparations The aim of our work was to determine the types of viruses in were determined b were kept at -20°C. cRNA-DNA Hybridization Manuscript received May 19, 1980; accepted for publication July 28, 1980. Complementary HNAs were obtained by in vitro transcription of This work was supported by Grant PR6 26/76 for Cancer Research form I DNA molecules, as previously described [10,2 1). The viral DNAs fro m the Polish government and Grants ATP 67.78.99 and CRL were: HPY-1 DNA (subtype a) (12,18] obtained from a s ingle deep 1 79.4 .023.2 from Institut National de Ia Sante et de Ia Recherche plantar wart; HPV-2 DNA (a mixture of subtypes band c) obtained · Medicate. from the pooled common warts of a s ingle patient; HPV-3 DNA Reprint requests to: Gerard Orth, Unite des papillomavirus, G.R. (subtype a) t (18] isolated from pooled scrapings of fl at wart-like lesions INSERM U.190., Institut Pasteur, 28, rue du Docteur Roux 75724 Paris in a patient with EV (17,18]; HPV-4 DNA [9,18) obtained from the 1 Cedex 15, France. Jlooled palmar warts of a butcher; HPV-5 DNA (subtype a) (18) Abbreviations: B: bovine cRNA: complementary RNA • Jablonska S, Croissant 0, Obalek S, Favre M, Rzesa G, Orth G: EV: epidermodysplasia verruciformis Morphology and epidemiology of butchers' warts in relation to the type H: human of papillomavirus, in preparation. IF: immunofluorescence t Favre M, Jablonska S, Croissant 0, Obalek S, Orth G: On the MH: molecular h ybridization genetic heterogeneity of skin HPVs as analysed by restriction endonu­ PV: papillomavirus cleases, in preparation. 97 98 ORTH ET AL Vol. 76, No. 2 obtained from the pooled scrapings of pityriasis versicolor-like lesions TABLE III. Analysis of pa.pillomavirus DNAs by restriction enzym es of a patient with EV [17,18]; and BPV-1 DNA [13,14) isolated from cleavage skin fibropapillomas of cattle. The cRNA-DNA filter hybridizations Virus type as deLermined by under paraffin [25) were performed using amounts of form I and II viral No. of DNA varying from less than 5 ng to 50 ng per filter, under the conditions bu Lc hers Molecular Hind ll+III previously described [10,2 1]. h y bridi ~at i o n cleavage patterns 2 1 1a; Ib Restriction Enzyme Analysis 4 2 2b; 2b + Xa; 2b + 3f; 2x" + Xa The digestion of viral DNAs with a mixture of Hind II and Hind III 4 3 3e; 3f; 3f; 3f + Xa endonucleases (Boehringer, Mannheim) a nd the separation of the 1 2 + 3 2c + 3g cleavage products by vertical slab gel electrophoresis in agarose (1. 2%) 1 4 4 were performed as previously described [18). The molecular weights of 4 X Xa; Xa; Xa; Xa the fragments were evaluated from their electrophoretic mobilities, by 6 ND'' l a + lb; 3f; 3g + 4'; 4; Xa; X a'· comparison with A. Hind III DNA fragments [26) and Hae III 10 X 174 " An incomplete cleavage pattern did not all ow identification of the RF DNA fragments [27]. subtype. " ND, not determined. l mmunoflu orescence ,. Two DNA preparations from different locations were studied. Direct and indirect immunolluorescence (IF) studies were performed as previously described [17,28). Fluorescein-labeled anti-HPV-1 guinea pig IgG (G 121, G 122 ) and anti-HPV-3a (G 281) or HPV-5a (G 251) guinea pig sera were the same used in our previous studies [10,17,18]. Fluorescein-labeled anti-HPV-2 IgG were prepared from a guinea pig serum (G 314) obtained against full particles of a mixture of subtypes b and c. t This serum had a specifi city similar to that of the HPV -2a antiserum (G 206) used previously [10,11,17,28]. Histological Studies Sections prepared from specimens fixed in Bouin's solution or in 10% formalin and embedded in paraffin, or cryostat sections adjacent to sections studied by IF, were stained with hematoxylin and eosin.