Butcher's Wart Virus (HPV 7) Infections in Non-Butchers

Ethel-Michele de Villiers, Ph .D., Christine Neumann, M.D., Tilman Oltersdorf, M.D., Gerhard Fierlbeck, M.D., and Harald zur Hausen, M.D., D.Sc.(H.c.) Referenzzentrum fLir humanpathogene Papiliomviren, Deutsches Krebsforschungszentrum, Heidelberg (E-MdeV, HH), Institut fiir Virusforschung, Deutsches Krebsforschungszentrum, Heidelberg (TO), Universitats-Hautklinik, Munster (CN), and Universitats-Hautklinik, Tubingen (GF), F.R.G.

The analysis of a total of 654 benign and malignant lesions of the skin, genitalia, lungs and bronchi, intestine, kidneys, bladder, mammae, and of the head and neck region, re­sulted in the identification of human papilloma virus 7 (HPV 7) infections in 3 individuals. One of these was an ordinary "butcher's wart," whereas the other 2 patients have never been involved in meat-handling or farming . One of the latter revealed extensive verrucosis of hands,

Infections by different types of human papilloma viruses (HPV) reveal a remarkable preference for specific sites [1-6] and are frequently characterized by type-specific macroscopic and microscopic features [7,8]. One of these virus types, HPV 7, shows a unique pattern of infection. Thus far, it has been

identi fi ed only in warts of butchers and meathandlers [8,9). This has raised the suspicion that this virus may originate from slaugh­tered an imals or their products [8,9), although no evidence has yet been provided to support this claim [8-10].

Apart from the one case of a "butcher's wart" containing HPV 7 DN A, we describe here the identification of HPV 7 or closely related DNA sequences from 2 individuals, each with a long history of persisting verrucoses, neither of whom has been in­volved in meathandling or farming. In both patients, newly arising HPV 7-positive papillomas are characterized by their filiform morphology.

An additional 651 biopsies, originating from various localiza­tions in the body, were screened for the presence ofHPV 7 DNA sequences. These, however, all proved to be negative.

PATIENTS AND METHODS

Patient History Warts started developing on the hand of pa­tient WV -4274 (32 years) at 6 years of age. During the followin g yea rs, various methods were applied to remove these persistent warts, e.g., surgery, freezing, electrocoagulation , and various corrosive agents. At the age of20, warts were persisting on hands, neck, left axilla, and a few sma ll lesions were present in the face. Lesions resembling condylomata acuminata were found peri­anall y. All these were removed by electrocauterization. Papillo-

Manuscript received June 25, 1985; accepted for publication February 7, 1986.

Supported by the Bundesl11inisteriul11 fur Jugend, Familie und Gesund­hcit, Bonn (Referenzzentrul11 fUr hUl11anpathogene Papillol11viren , Deutsches Krebsforschungszentrul11, Heidelberg) .

Reprint requests to: Ethel~Michcle de Villiers, Ph.D., Referenzzentrul11 fiir humanpathogene Papillomviren, Deutsches Krebsforschungszen­trum, 1m Neuenheimcr Feld 280,6900 Heidelberg, F.R.G .

Abbreviation : HPV: human papilloma virus

feet, axilla, neck, and face, persisting for about 27 years, with new lesions arising in the neck region. Particularly the new lesions showed filiform morphology. The second patient has , for a period of more than 2 years, been showing filiform papillomas in the face which recurred after surgical removal. These 2 patients appear to represent the first cases ofHPV 7 infections in non-butchers or non-meathandlers . J Invest Dermatol 87:236-238, 1986

mas appeared again during the following years and 11 years later a massive recurrence was noted at all previous locations, as well as on the back of the leg, on the forearm, and toes . Additionally, condylomata-like proliferations developed on the scrotum and the groin .

Patient WV-4685 (43 years) had filiform warts in the face. During the 2 years of clinical manifestation these papillomas were removed surgically but they recurred and showed no sign of spontaneous regression.

Nucleic Acid Analysis Total cellular DNA was extracted from each biopsy as described by Gissmann et al [3]. Two different approaches were then followed. One approach entailed radiola_ beling of 1 J.tg of cellular DNA with [32P]thymidine triphosphate and hybridizing it to a nitrocellulose blot containing 100 ng of each of the human papillomaviral DNAs 1-19 and 21-26 [11]. The second approach was to digest a 10-J.tg sample of cellular DNA with a restriction endonuclease (for screening 204 of the biopsies Bam Hl or Eco Rl was used). The DNA was then separated on agarose gels and transferred onto nitrocellulose fil­ters. Hybridization was done under conditions of high stringency using cloned 32P-radiolabeled HPV 7 DNA [10] as probe. For more detailed analysis of the state of the viral DNA in the positive lesions, the enzymes A va II and Pst 1 were used.

In both cases, after washing, the filters were exposed to all x- ray film for 1 day or 1 week.

RESULTS

DNAs from 654 lesions were screened for the presence of HPV 7 sequences. Radiolabeled DNA of 445 of these biopsies were tested against HPV DNA of types 1-19 and 21-26 blotted Onto

nitrocellulose filters. This test reveals papilloma virus DNA of the respective type if present in 10 or more genome copies per cell [11]. Cellular DNA of the remaining lesions were blotted onto nitrocellulose filters and hybridized with 32P-radiolabeled HPV 7 DNA. As little as 0.1 HPV genome copies per cell could be detected by applying this procedure. Table I summarizes the results with both testing methods.

Interestingly, the DNA from papillomas of 3 patients hybrid-

0022-202X/86/$03.50 Copyrigh t © 1986 by The Society for Investigative Dermatology, Inc.

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VOL. 87, N O.2 AUGUST 1986

Table I. Skin Specimen Analyzed for the Presence of HPV 7 DNA

Epidermodysplasia ve rru ciformis

Verruca vulga ri s Verru ca p lantaris Verruca sebo rrhoica Verruca fi lifo rmis Butcher's wa rt Keracosis Poikiloderma congenitale Lichen sci eros us et atropicus Porokeratosis Mibelli Nevus cell nevus Epiderma l nevus Systemic nevus Keracoacanthoma Melanoma Basal cell carcino ma Squamous cell ca rcinoma Normal skin

Number of Biopsies T es ted

21 19 2 3 2 1 5 2 1 1 2 I 1 2 I 1 6 1

Number of HPV 7-Positive

Samples

2 1

Additionally, specimens (rom the fo llowing biopsies we re all nega tive (number o( biopsies tes ted): no rmal cervical ti ssue (14), genital warts (142), cervical intra­e pithelial neoplasias and genital bowenoid papu los is (15 1), an ogenital cancer (9 1). noncancerous endo metrium (14). endo metrial and ova ri an ca ncer (12) , mamilla fibrode nomas and ca rcino mas (7), normal lymph ocytes ( I) , bladder and kidney ca rcinomas (17), normal ora l mucosa (2). beni gn o ro pharyngea l and lar yngea l tu­mors (31), oropharyngea l and laryngea l carcinomas (53), lun g ca rcin omas (40) and eso phagus, stomach, and colon ca ncer (7).

ized exclusively with HPV 7 DNA. One representative result is shown in Fig 1. Since one of these papillomas originated from a butcher (WV-3983) it was not analyzed subsequently . The 2 other patients, however, from w hom HPV 7-positive hybridization signals were obta ined, were not professionally active as butchers o r mea thandlers. Therefore these materials were selected for a more detailed anal ysis .

Patient WV-4274 presented himself with particularly large hand warts, some exceeding 2- 3 cm in diameter (Fig 2). Warts of long duration had been removed from this region and were subj ected to the HPV ana lysis. Similarly, recently developing filifo rm pro­liferations had been obtained from the neck region (WV-4S96) and were investigated accordingly. Unfortunately, no histologic preparations were avai lable from this patient.

From the second patient (WV -4685) filiform papillomas had been obtained after surg ical removal. The histology of these le­sions is shown in Fig 3. The pattern revea ls structures described as characteristi c for HPV 7 infections, such as papillomatosis and acanthosis, heavily stained cells containing medium-sized kera­tohyaline gra nules, and a pronounced hyperkeratosis with para­keratosis [8]. Although this patient revealed persisting warts for more than 2 years, he is presentl y receiving interferon therapy resulting in regression of these papillomas.

HPV 1 2 3 4 5 6 7 8 9 '10 11 12 13 14 15 1~ 11 18 19 21 22 23 24 25 26 30 31 LPV

Figure 1. Radiolabcled cellular DNA (WV-4274) was hybridized to types HPV 1-19 and 21-25. Onl y HPV-sequences with cross-h ybridiza tion to HPV 7 DNA were found.

HPV 7 INFECT IONS IN NON-BUTCHER 237

Figure 2. Patient WV-4274 showed ex tensive les ions on the hands .

Figure 3. Histo logic section of patient WV-4685. N ote the acanthotic epidermis. Some clustered heav il y stained cells contain medium-sized keratohya line g ranules. A pronounced hyperkeratos is is evident . Mag­nifica tion X 60.

Res triction enzyme analysis of the D NA from these papillomas with Ava II and/or Pst 1, blotting of this DNA onto nitrocel­lulose filters , and subsequent hybridization w ith 32P-labeled HPV 7 DNA resulted in the typical cleavage patterns as described for HPV 7 DNA [8-10] (Fig 4). N o evidence was obtained for in­tegrated HPV 7 sequences; thus the vira l DNA appears to persist in an episomal state, w hich seems to be the prevalent state of the HPV DNA in benign tumors [12].

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Figure 4. To verify the presence of HPV 7 DNA sequences in biopsies WV-4274, WV-4685, and WV -4596, restriction enzyme ana lysis of the DNA was carried out. Using cnzy mes A va II and/o r Pst 1 the typical pat­tern for HPV 7 DNA was obtained.

DISCUSSION REFERENCES

Human papilloma virus type 7 has been described as being as­sociated thus far excl usively with butchers or meathandlcrs [8,9). The lesions were located almost exclusively on the hand of such individuals. * The hi stology revea ls a typical pattern of papillom­atous hyperkeratosis.

In this report we describe the presence of HPV 7 DNA in 2 patients, neither of w hom had any history of mea thandling o r farm ing. T he long-persisting les ions as w ell as, in particular, newly arising proliferations had a multifaceted filiform appear­ance. They were found on hands, arms, legs and feet, face, and neck in one patient and on the foreh ead in the other. In this series we were able to investi ga te papillomas from the hands, the face, and the neck and all of them contained HPV 7 DNA.

It is noteworthy that in accordance with other reports on HPV 7 infections* [8,9] the proliferations in our 2 patients were oflong duration (in one patient 27 years) without any signs of sponta­neous regressions. In one patient the infecti ons proved to be re­sistant to various different forms of treatment whereas, in the other patient, interferon therapy has been, at least partially, ef­fectiv e.

The origin of HPV 7 infections still rem ains obscure. Earlier suspicions that HPV 7 may originate from anima l sources, par­ticul arl y from bovine papilloma vi rus infections , have not yet been substantiated by experimental studies [8-10J. Although the 2 patients described in this report appear to represent the first HPV 7-positive individuals not linked to meathandling o r farm­ing, an accidental exposure to viruses o ri ginating from meat prod­ucts cannot be excluded.

This report indicates , however, that persisting H PV 7 infections do not require long-time exposure to meathandlin g or to con­ditions of handling refrigerated products.

T"e skilljid lec/Illieni ass iSlml ce oj Ulc f.) lI salko i5 gralejidly ackll olll l e~ged . We are .~ ralejid 10 C. 01"1" , 1-1 . I>jiSIC/" , ali!I R . 05/1'0111 Jor p l"O lIidillg 1/1(' c/olles oj I-IP V 9, 12, 14-17, 19- 25, mId 26.

*Jablonska et ai, m anuscript in preparation.

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2. Gissmann L, zur Hauscn H: Partial characteriza tion of viral DNA from human genital warts (condylomata acuminata). Int] Cancer 25:605-609. 1980

3. Giss mann L, Diehl V, Schultz-Coulon H-], zur Hausen H : Molecular cloning of human papilloma virus DNA derived from a laryngeal papilloma. ] Virol 44:393-400, 1982

4. Durst M, Gissmann L, Ikenberg H, zur Hausen H : A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy sa mples from different geographic regions . Proc N at! Acad Sci USA 80:3812-3815, 1983

5. Boshart M , Gissmann L, Ikenberg 1-1, Kleinhcinz A, Scheurlen W , zur Hausen H: A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cel l lines derived from cervical cancer. E MB O ] 3: 1151 - 1157, 1984

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7. Gross G, Pfister H, Hagedorn H , Gissmann L: Correlation between hum an papilloma virus (HPV) type and histology of warts. J Invest Dermatol 78:160- 164, 1982

8. Orch G, ]ablonska S, Favre M, Croissant 0 , Obalek S, Jarzabek­C horzclska M, ]ibard N : Identification of papillomaviruses in butchers' warts. Jlnvest Dcrmatol 76:97-102, 1981

9. Ostrow RS, Krzyzek R, Pass F, Faras AJ: Identification of a novel human papilloma virus in cutaneous warts of meat-handlers . Vi­rology 108:21-27. 1981

10. O ltersdorf T , Campo MS, Favre M , Dartmann K, Gissmann L: Molecular cloning and characterization of human papillomavirus type 7 DNA. Virology 149:247-250, 1986

11 . de Villiers E-M, Schneider A. Gross G , zur Hausen H: Analysis of benign and malignant urogenital tumours for human papilloma­virus infection by labellin g cellular DNA. Med Microbiol Im­munoI174:281-286, 1986

12. Dtirst M, Kleinheinz A, Hotz M , Gissmann L: The physical state of hum an papillomavirus type 16 DNA in benign and malignant gen ital tumours . ] Gen Virol 66:1515- 1522, 1985