Journal of Zoo and Wildlife Medicine 27(1): 61-70, 1996 Copyrighl 1996 by American Association of Zoo Veterinarians PAPILLOMAVIRUS-ASSOCIATED FOCAL ORAL HYPERPLASIA IN WILD AND CAPTIVE ASIAN LIONS iPANTHERA LEO PÉRSICA) John P. Sundberg, D.V.M., Ph.D., Richard J. Montali, D.V.M., Mitchell Bush, D.V.M., Lyndsay G. Phillips, Jr., D.V.M., Stephen J. O'Brien, Ph.D., A. Bennett Jenson, M.D., Robert D. Burk, M.D., and Marc Van Ranst, M.D. Abstract: Four Asian lions (Panthera leo pérsica), two wild and two captive, were diagnosed with focal oral hyperplasia affecting the ventral surface of their tongues. Focal, flat, sessile lesions consisted of hyperplastic, stratified squamous epithelium. Koilocytotic atypia was evident in the upper layers of cells, some of which contained characteristic intranuclear papillomavirus particles visible by electron microscopy. In addition, large amphophilic cytoplasmic inclusions were evident in the koilocytes and were considered to be a product of the viral E4 gene. Papillomavirus group- specific antigens were detected by immunohistochemistry in the atypical cell nuclei. Conserved papillomavirus antigenic epitopes differed from epitopes found in cutaneous papillomavirus-induced lesions from domestic cats. An 8,000-base pair DNA fragment, linearized by Bam HI digestion, was detected by Southern blot hybridization probed with a mixed human papillomavirus genomic probe. Limited restriction endonuclease studies of DNA prepared using an oral hyperplastic lesion from an Asian lion indicate that this is a novel feline papillomavirus different from the domestic cat cutaneous papillomavirus. This new virus has been designated "PIPV." Key words: Papillomavirus, Asian lion, Panthera leo pérsica, felidae, PIPV. INTRODUCTION or unsuspected diseases that could be dev- The success of captive breeding pro- astating for other species. grams for endangered species depends on The Asian lion {Panthera leo pérsica) is careful evaluation of wild-caught animals to an endangered species for which a captive prevent introduction of serious infectious breeding program was established in the diseases into the captive population. Fur- United States and India. During routine thermore, introduction of subclinical dis- physical examinations of seven wild-born, eases into taxonomic families can be dev- captive lions at the Szkkarbang Zoo in Gu- astating. Such has been the case in Felidae, jarat, India, and four wild lions at the Gir Forest Sanctuary in India, focal oral hyper- with outbreaks of feline infectious perito- plasia was observed on the ventral surfaces nitis'^ and rhinotracheitis*"* in captive chee- of the tongues of four animals. This lesion tahs. Careful physical examination together in felids and similar lesions in other verte- with sérologie testing during quarantine brate species are caused by papillomavirus- must be practiced in order to detect novel es,2' herpesviruses,^ poxviruses,'^ chemical carcinogens,^^ diet," and genetic defects.^*' From The Jackson Laboratory, Bar Harbor, Maine Several of these infectious agents can limit 04609, USA (Sundberg); The National Zoological the value of the animal in the breeding pro- Park, Smithsonian Institution, Washington, D.C. gram .^^ Furthermore, since poxviruses" and 20008, USA (Montali, Bush); School of Veterinary papillomaviruses""-" can infect related spe- Medicine, University of California, Davis, California cies, there is concern about possible spread 92112, USA (Phillips); The National Cancer Institute, National Institutes of Health, Frederick, Maryland of infection to other felids within the cap- 21702, USA (O'Brien); Georgetown University School tive collection and perhaps to domestic cats of Medicine, Washington, D.C. 20007, USA (Jenson); that enter zoo grounds. Rega Institute for Medical Research, University of Leuven, Leuven B-3000, Belgium (Ranst); and the Al- MATERIALS AND METHODS bert Einstein College of Medicine, Yeshiva University, Over a 10-day period, the eleven adult New York, New York 10461, USA (Burk, Ranst). Asian lions (seven males, four females) 61 62 JOURNAL OF ZOO AND WILDLIFE MEDICINE Figure 1. Ventral surface of tongue of Asian lion showing papillomaviras-induced focal epithelial hyperplasia. were anesthetized with tiletamine HCl and examined by light microscopy to select zolazepam HCl (Telazol, Aveco, Fort sites for electron microscopic evaluation. Dodge, Iowa 50501, USA) delivered by Ultrathin sections were stained with uranyl blow dart at a dose of 400-500 mg for a acetate and lead citrate and examined as male and 350-450 mg for a female. The previously described.^' lions were clinically evaluated for repro- Serial sections of paraffin-embedded tis- ductive and genetic studies. Two of the wild sues were stained for papillomavirus group- and two of the captive lions (three males, specific antigens using a rabbit polyclonal one female) had small, soft, light pink, oval, antibody (DAKO Corp., Carpenteria, Cali- slighdy raised, flat sessile lesions ranging fornia 93013, USA) that broadly cross-re- from 4-8 mm in diameter on the ventral acts with mammalian and avian papillo- surfaces of their tongues (Fig. 1). Repre- maviruses.'"" A panel of mouse monoclo- sentative tongue lesions from a wild and a nal antibodies directed against linear epi- captive lion were bisected, fixed in 10% topes of phylogenetically conserved bovine neutral buffered formalin and cacodylate- papillomavirus type 1 (BPV-1) LI and L2 buffered glutaraldehyde, and frozen in tis- gene products was also used.'" The reaction sue culture media in liquid nitrogen for fur- was developed using a modification of the ther testing. Formalin-fixed tissues were avidin-biotin complex technique.'" embedded routinely in paraffin, serially sec- Frozen hyperplastic lesions were finely tioned at 6 |i,m, and stained with hematox- minced, and genomic DNA was prepared ylin and eosin. Thin sections of the glutar- as previously described." Briefly, the tis- aldehyde-fixed specimens were prepared sue was digested with proteinase K (Boeh- for electron microscopy utilizing standard ringer Mannheim, Indianapolis, Indiana embedding techniques. One-micron-thick 46250, USA), treated with DNase-free sections, stained with toluidine blue, were RNase A (Boehringer Mannheim), extract- SUNDBERG ET AL.•LION ORAL PAPILLOMATOSIS 63 _f,-^p^Sr^-lK^» \ .^^m^'^ '-^"^'^m Figure 2. Focal sessile plaque of proliferating stratified squamous epithelium is supported by thin fibrovas- cular stalks. H&E, X40. ed with phenol and chloroform, and pre- RESULTS cipitated with 70% ethanol. Total cellular Microscopically, the sessile tongue le- DNA (5 |jLg) was digested with Bam HI sions consisted of marked squamous cell restriction endonuclease, and electropho- proliferation with short, broad, rete ridge retically separated on a 1% agarose gel. formation. Thin dermal papillae containing The DNA on the gel was depurinated, de- capillaries separated the short rete ridges natured, and transferred under alkaline (Fig. 2). Foci of degenerating cells were ev- conditions to a charge-modified nylon ident in the upper stratum spinosum and membrane (Genescreen Plus, DuPont NEN stratum granulosum (Fig. 3). These cells Research Products, Boston, Massachusetts displayed atypical nuclei surrounded by 02118, USA).^'' The membrane was hy- clear cytoplasm. Single large, amphophilic, bridized under low-stringency conditions inclusion-like structures were eccentrically with a radiolabeled cocktail probe consist- located in the cytoplasm adjacent to the nu- ing of equal amounts (50 ng each) of hu- cleus; a prominent round, vesiculated nu- man papillomavirus (HPV) DNA (HPV-1, cleus was centrally or eccentrically located -11, -16, and -18). After posthybridization within affected cells (Fig. 3). These degen- washing of the membranes under low- erating cells had features consistent with stringency conditions, the membrane was those described for koilocytes, cells exhib- exposed to Kodak XAR-5 film (Eastman iting the cytopathic effects of papillomavi- Kodak, Rochester, New York 14650, USA) rus infection.^' By electron microscopy the for 72 hr at -70° C using intensifying inclusions in the koilocytes appeared as screens (Cronex Lightning-Plus, DuPont finely granular to fibrillar, electron-dense, de Nemours, Wilmington, Delaware cytoplasmic aggregates that often molded 19801, USA). around nuclei (Fig. 4). Within the nucleus 64 JOURNAL OF ZOO AND WILDLIFE MEDICINE • Figure 3. Higher magnification of a field from Figure 2 sliowing a cluster of degenerated cells (koilocytes) in the upper stratum spinosum with swollen, clear cytoplasm, cytoplasmic inclusion bodies, and vesicular nuclei. H&E, X500. of some of these cells were 50-52-nm, vi- ization of a Bam HI restriction endonucle- ruslike particles that were ultrastructurally ase digest on the DNA extract of the oral compatible with papillomaviruses (Fig. 5). lesion with a cocktail probe of HPV-1, -11, Papillomavirus cross-reactive antigens, -16, and -18 DNA revealed a single, linear, several of which are recognized by careful- 8,000-bp hybridizing band (Fig. 7). ly characterized monoclonal antibodies, This novel papillomavirus was abbrevi- were identified within the nuclei of koilo- ated "PIPV" following the nomenclature cytotic cells (Table 1, Fig. 6). The cyto- guidelines for nonhuman papilloma- plasmic, inclusion-like structures did not viruses.^'* stain for structural viral proteins. The BPV- 1 epitopes conserved by the Asian lion oral DISCUSSION papillomavirus were different from those of Until recently,