Papillomavirus infection requires gamma secretase.

The mechanism by which papillomaviruses breach cellular membranes to deliver their genomic cargo to the nucleus is poorly understood. Here, we show that infection by a broad range of papillomavirus types requires the intramembrane protease γ secretase. The γ-secretase inhibitor (S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide (compound XXI) inhibits infection in vitro by all types of papillomavirus pseudovirions tested, with a 50% inhibitory concentration (IC(50)) of 130 to 1,000 pM, regardless of reporter construct and without impacting cellular viability. Conversely, XXI does not inhibit in vitro infection by adenovirus or pseudovirions derived from the BK or Merkel cell polyomaviruses. Vaginal application of XXI prevents infection of the mouse genital tract by human papillomavirus type 16 (HPV16) pseudovirions. Nicastrin and presenilin-1 are essential components of the γ-secretase complex, and mouse embryo fibroblasts deficient in any one of these components were not infected by HPV16, whereas wild-type and ß-secretase (BACE1)-deficient cells were susceptible. Neither the uptake of HPV16 into Lamp-1-positive perinuclear vesicles nor the disassembly of capsid to reveal both internal L1 and L2 epitopes and bromodeoxyuridine (BrdU)-labeled encapsidated DNA is dependent upon γ-secretase activity. However, blockade of γ-secretase activity by XXI prevents the BrdU-labeled DNA encapsidated by HPV16 from reaching the ND10 subnuclear domains. Since prior studies indicate that L2 is critical for endosomal escape and targeting of the viral DNA to ND10 and that γ secretase is located in endosomal membranes, our findings suggest that either L2 or an intracellular receptor are cleaved by γ secretase as papillomavirus escapes the endosome.

Increased immunity to cottontail rabbit papillomavirus infection in EIII/JC inbred rabbits after vaccination with a mutant E6 that correlates with spontaneous regression.

Our previous studies showed that a progressive cottontail rabbit papillomavirus (CRPV) strain containing a single amino acid change in E6 (E6G252E) induced papilloma regression in EIII/JC inbred rabbits. This finding implied that the point mutation might cause an increase in the antigenicity of the mutant versus the wild-type E6. To test this hypothesis, groups of four EIII/JC inbred rabbits were immunized with wild-type CRPVE6, CRPVE6G252E, CRPV E5, or with vector alone. A gene gun delivery system was used to deliver the DNA vaccines. Two of four rabbits from both E6G252E- and wild-type E6-vaccinated groups were free of papillomas at week 12 after viral challenge. Significantly smaller papillomas were found on E6G252E-vaccinated rabbits than on E6-, E5-, and control vector-vaccinated rabbits (p = 0.01, unpaired Student t test) and these small papillomas regressed at week 20 after viral challenge. E5 vaccination failed to provide protection against viral challenge, and the mean papilloma size was also comparable to that of the control vector-vaccinated rabbits (p > 0.05, unpaired Student t test). We conclude that a single amino acid change in the CRPV E6 protein (G252E) increased protection against wild-type infectious CRPV.

Papillomavirus E2 protein induces expression of the matrix metalloproteinase-9 via the extracellular signal-regulated kinase/activator protein-1 signaling pathway.

Papillomaviruses are involved in the development of cancers of the female cervix, head and neck, and skin. An excellent model to study papillomavirus-induced tumor induction and progression is the New Zealand White rabbit, where the skin is infected with the cottontail rabbit papillomavirus (CRPV). This leads to the formation of benign tumors that progress into invasive and metastasizing carcinomas without the need for cofactors. We have shown previously that specific mutations in the transactivation domain of the transcription/replication factor E2 cause a dramatic loss in the tumor induction efficiency of the viral genome and a major deficiency in tumor progression as we show now. By comparing wild-type (WT) and mutant E2-induced skin tumors, we found high levels of matrix metalloproteinase-9 (MMP-9) protein and transcripts in WT CRPV-E2-induced tumors in contrast to certain mutant CRPV-E2-induced papillomas and normal uninfected skin. Stable cell lines and reporter assays revealed that E2 from different papillomavirus types is able to transactivate the MMP-9 promoter via the promoter-proximal activator protein-1 (AP-1) site as shown in reporter gene assays with mutant MMP-9 promoter constructs. Furthermore, WT E2 but not mutant E2 strongly transactivated a minimal promoter reporter construct with multiple AP-1 sites. The MMP-9 protein induced in cells expressing E2 degrades collagen matrices as measured in Matrigel-based invasion/mobility assays. E2-induced MMP-9 expression can be blocked by a chemical inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (PD 098059), suggesting that E2 activates the MAPK/ERK signaling pathway, which is further supported by the induction of ERK1 in CRPV-E2-transfected cells.

Recent advances in preclinical model systems for papillomaviruses.

Preclinical model systems to study multiple features of the papillomavirus life cycle have greatly aided our understanding of Human Papillomavirus (HPV) biology, disease progression and treatments. The challenge to studying HPV in hosts is that HPV along with most PVs are both species and tissue restricted. Thus, fundamental properties of HPV viral proteins can be assessed in specialized cell culture systems but host responses that involve innate immunity and host restriction factors requires preclinical surrogate models. Fortunately, there are several well-characterized and new animal models of papillomavirus infections that are available to the PV research community. Old models that continue to have value include canine, bovine and rabbit PV models and new rodent models are in place to better assess host-virus interactions. Questions arise as to the strengths and weaknesses of animal PV models for HPV disease and how accurately these preclinical models predict malignant progression, vaccine efficacy and therapeutic control of HPV-associated disease. In this review, we examine current preclinical models and highlight the strengths and weaknesses of the various models as well as provide an update on new opportunities to study the numerous unknowns that persist in the HPV research field.

Protective cell-mediated immunity by DNA vaccination against Papillomavirus L1 capsid protein in the Cottontail Rabbit Papillomavirus model.

Papillomavirus major capsid protein L1 has successfully stimulated protective immunity against virus infection by induction of neutralizing antibodies in animal models and in clinical trials. However, the potential impact of L1-induced protective cell-mediated immune (CMI) responses is difficult to measure in vivo because of the coincidence of anti-L1 antibody. In this study, we tested the hypothesis that L1 could activate CMI, using the Cottontail Rabbit Papillomavirus (CRPV)-rabbit model. A unique property of this model is that infections can be initiated with viral DNA, thus bypassing all contributions to protection via neutralizing anti-L1 antibody. DNA vaccines containing either CRPV L1, or subfragments of L1 (amino-terminal two-thirds of L1 [L1N] and the carboxylterminal two-thirds of L1 [L1C]), were delivered intracutaneously into rabbits, using a gene gun. After three booster immunizations, the rabbits were challenged with several viral DNA constructs: wild-type CRPV, CRPV L1ATGko (an L1 ATG knockout mutation), and CRPV-ROPV hybrid (CRPV with a replacement L1 from Rabbit Oral Papillomavirus). Challenge of L1 DNA-vaccinated rabbits with wild-type CRPV resulted in significantly fewer papillomas when compared with challenge with CRPV L1ATGko DNA. Significantly smaller papillomas were found in CRPV L1-, L1N-, and L1C-vaccinated rabbits. In addition, rabbits vaccinated with either L1 or L1N grew significantly fewer and smaller papillomas when challenged with CRPV-ROPV hybrid DNA. Therefore, CRPV L1 DNA vaccination induced CMI responses to CRPV DNA infections that can contribute to protective immunity. Cross-protective immunity against CRPV L1 and ROPV L1 was elicited in these CRPV L1- and subfragment-vaccinated rabbits.

Shope papilloma virus. II. A comparison of several methods for purification of the virus.

Four methods for preparation of Shope papilloma virus have been compared. These comprise: (1) alternate centrifugation at low and high speed after grinding with alundum and saline, (2) purification with a fluorocarbon after similar extraction, (3) extraction with the aid of 2-mercaptoethanol and purification by alternate low- and high-speed centrifugation, and (4) extraction with 2-mercaptoethanol followed immediately by centrifugation to equilibrium in CsCI. It is necessary to remove CsCI before chemical analysis, and this has been accomplished by chromatography on Sephadex G-75. After banding, the virus from preparation (4) appears homogeneous in the electron microscope. The deoxyribonucleic acid (DNA) contents of the preparations were (1) 7.8, (2) 8.3, (3) 8.0, and (4) 11.9 percent of total nucleoprotein. At equal inputs of DNA, there was no significant difference in infectivity of any of the preparations. Method (4) is simpler than the others and results in a higher yield of a homogeneous preparation.

The cottontail rabbit papillomavirus model of high-risk HPV-induced disease.

Animal models are essential to study the pathogenesis of papillomavirus infection and develop strategies for treatment and prevention. This review details use of the cottontail rabbit papillomavirus (CRPV)-laboratory rabbit model. The protocols describe how to infect rabbits with CRPV DNA or CRPV virus to induce papillomas. They also describe the design and analysis of genetic pathogenesis experiments, prophylactic and therapeutic intervention experiments, and malignant progression experiments.

Delayed and Aberrant Presentation of VX2 Carcinoma in a Rabbit Model of Hepatic Neoplasia.

A socially-housed New Zealand white rabbit presented with a large subcutaneous mass on the ventral thorax approximately 11 mo after the intrahepatic delivery of a suspension of VX2 carcinoma cells to induce hepatocellular carcinoma as part of a nanoparticle study. The mass and closely associated axillary lymph node were removed en bloc. Immunohistochemical staining identified the mass as an undifferentiated carcinoma. The rabbit demonstrated no appreciable pathology at the study end point at 16 mo after VX2 inoculation. An additional rabbit from the same VX2 injection cohort was found at necropsy to have an unanticipated intraabdominal mass, also identified as an undifferentiated carcinoma. This case report summarizes the molecular analysis of both tumors through a novel PCR assay, which identified the delayed and aberrant onset of VX2 carcinoma in an extended timeframe not previously reported.

A Century of Shope Papillomavirus in Museum Rabbit Specimens.

Sylvilagus floridanus Papillomavirus (SfPV) causes growth of large horn-like tumors on rabbits. SfPV was described in cottontail rabbits (probably Sylvilagus floridanus) from Kansas and Iowa by Richard Shope in 1933, and detected in S. audubonii in 2011. It is known almost exclusively from the US Midwest. We explored the University of Kansas Natural History Museum for historical museum specimens infected with SfPV, using molecular techniques, to assess if additional wild species host SfPV, and whether SfPV occurs throughout the host range, or just in the Midwest. Secondary aims were to detect distinct strains, and evidence for strain spatio-temporal specificity. We found 20 of 1395 rabbits in the KU collection SfPV symptomatic. Three of 17 lagomorph species (S. nuttallii, and the two known hosts) were symptomatic, while Brachylagus, Lepus and eight additional Sylvilagus species were not. 13 symptomatic individuals were positive by molecular testing, including the first S. nuttallii detection. Prevalence of symptomatic individuals was significantly higher in Sylvilagus (1.8%) than Lepus. Half of these specimens came from Kansas, though new molecular detections were obtained from Jalisco-Mexico's first-and Nebraska, Nevada, New Mexico, and Texas, USA. We document the oldest lab-confirmed case (Kansas, 1915), pre-dating Shope's first case. SfPV amplification was possible from 63.2% of symptomatic museum specimens. Using multiple methodologies, rolling circle amplification and, multiple isothermal displacement amplification in addition to PCR, greatly improved detection rates. Short sequences were obtained from six individuals for two genes. L1 gene sequences were identical to all previously detected sequences; E7 gene sequences, were more variable, yielding five distinct SfPV1 strains that differing by less than 2% from strains circulating in the Midwest and Mexico, between 1915 and 2005. Our results do not clarify whether strains are host species specific, though they are consistent with SfPV specificity to genus Sylvilagus.

Immortalization of inbred rabbit keratinocytes from a Shope papilloma and tumorigenic transformation of the cells by EJ-ras.

An immortalized cell line of keratinocytes, named SPG1-3, was established from a papilloma induced from cottontail rabbit papillomavirus (CRPV)-infected inbred rabbit skin. The cells have reached 60 passages in culture and are still growing well, but they are not tumorigenic in athymic mice. Although CRPV DNA was present as extrachromosomal episomes in the papilloma from which the cell line was derived from a single colony of keratinocytes, there was no CRPV DNA detectable in the cells. Three sub-cell lines of SPG1-3EJ, SPG1-3EJ1 and SPG1-3EJ2 were then established from the EJ-ras transfected SPG1-3 cells. All of the three sub-lines contained both EJ-ras DNA and a 1.2 kb transcript of EJ-ras, and they are malignantly tumorigenic in athymic mice. These data indicate that CRPV genome and its expression might be essential for the initiation and maintenance of neoplasia, but not for the maintenance of immortalization of the tumor-derived cells. In addition, some oncogenes such as EJ-ras may play an essential role in tumorigenic and malignant conversion of the immortalized cells. These cell lines derived from inbred rabbit skin may provide a useful in vitro system for better understanding of the oncogenic processes of papillomavirus-involved neoplastic progression by transfecting the cells with CRPV genes and serial transplantation to the inbred rabbits for studying host immune responses to the viral oncogenic potential.

In vivo anti-papillomavirus activity of nucleoside analogues including cidofovir on CRPV-induced rabbit papillomas.

A series of nucleoside analogues were tested for in vivo anti-papillomavirus activity using the cottontail rabbit papillomavirus (CRPV) domestic rabbit model. Compounds were delivered either topically, injected into growing papillomas, or delivered subcutaneously at a site remote from the papillomas. Compounds tested included cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (HPMPC); cyclic HPMPC (cHPMPC); cyclopentenylcytosine (CPE-C); lobucavir [1R(1alpha,2beta,3alpha)]-9-[2, 3-bis(hydroxymethyl)cyclobutyl]guanine; 9-((2-phosphonylmethoxy)propyl)adenine (PMPA); adefovir 9-((2-phosphonylmethoxy)ethyl)adenine(PMEA) and cyclopropyl 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (cyclopropylPMEDAP). Dose response curves and time-course treatments were included for most compounds tested. Strong anti-viral activity was detected using cidofovir and cHPMPC when delivered either topically or by the intralesional route. Complete cures were obtained using 1% (w/v) topical cidofovir at dosing schedules of twice daily for 8 weeks beginning at 4 weeks after CRPV infection, which represents a time when papillomas were clearly visible. Complete cures of large established papillomas were obtained by intralesional injection of 1% cidofovir three times per week for 8 weeks. Topical treatments with adefovir had strong anti-viral activity, cyclopropyl PMEDAP had moderate anti-viral activity, and CPE-C, PMPA and lobucavir showed no effects. These data indicate that certain nucleoside analogues have strong in vivo anti-papillomavirus activity and that the CRPV/rabbit model is a good model for assessing clinical responses of anti-viral treatments for patients with HPV disease.

Optimization of cottontail rabbit papilloma virus challenge technique.

Disease induced by Cottontail Rabbit Papilloma Virus (CRPV) scarification in domestic rabbits shares many attributes with disease induced by human papilloma virus (HPV). CRPV induces squamous papillomas in domestic rabbits, of which approximately 70% transform into invasive carcinomas. In advanced tumors, virus is often undetectable, and occasionally, some rabbits undergo spontaneous regression of papillomas. Techniques utilized to scarify rabbit skin are diverse, often labor intensive and time consuming with the possibility for significant variability. Using four unique infection techniques, resultant papilloma incidence, time to onset, and total papilloma volumes were compared to determine an optimal challenge method. Five rabbits were each infected with CRPV via a tattoo gun with and without ink, an intradermal injection, manual use of a tattoo needle, or a sterile blade followed by manual use of a tattoo needle. Papilloma formation was monitored weekly after inoculation for 6 weeks. CRPV papillomas began as pinpoint foci at 3 weeks post challenge and grew exponentially throughout the course of measurement. Individual foci coalesced rapidly to form larger papilloma aggregates. Although intradermal injection was well tolerated and easily performed, it was the worst method of papilloma production (2.2 mm(3) at 6 weeks). The best method, a sterile blade followed by manual use of a tattoo needle, produced significantly larger papillomas over all time periods (>1100 mm(3) at 6 weeks, P<0.01). Inoculation of CRPV using this method produces highly repeatable papillomas beginning 3 weeks post-infection.

Synonymous codon changes in the oncogenes of the cottontail rabbit papillomavirus lead to increased oncogenicity and immunogenicity of the virus.

Papillomaviruses use rare codons with respect to the host. The reasons for this are incompletely understood but among the hypotheses is the concept that rare codons result in low protein production and this allows the virus to escape immune surveillance. We changed rare codons in the oncogenes E6 and E7 of the cottontail rabbit papillomavirus to make them more mammalian-like and tested the mutant genomes in our in vivo animal model. While the amino acid sequences of the proteins remained unchanged, the oncogenic potential of some of the altered genomes increased dramatically. In addition, increased immunogenicity, as measured by spontaneous regression, was observed as the numbers of codon changes increased. This work suggests that codon usage may modify protein production in ways that influence disease outcome and that evaluation of synonymous codons should be included in the analysis of genetic variants of infectious agents and their association with disease.

Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits.

The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.

Differences in methodology, but not differences in viral strain, account for variable experimental outcomes in laboratories utilizing the cottontail rabbit papillomavirus model.

The cottontail rabbit papillomavirus (CRPV) animal model is used in several laboratories worldwide to investigate immunogenicity, carcinogenicity and life cycle aspects of papillomaviruses. It is the only animal model in which the full life cycle of the virus from initiation of infection to malignant progression can be studied. A major strength of the model is that the viral DNA is infectious. This feature allows for the study of mutant genomes without the need to create infectious mutant virus. Results from laboratory to laboratory have not always been consistent. Different laboratories use different methods for creating infections from DNA and it was postulated that the different challenge methods could play a role in the differential outcomes. Because different laboratories use different strains of CRPV, it was also desirable to test if the difference in CRPV genomes contributed to the differential outcomes. In this study, three of the CRPV strains used most widely (Washington B, Orth CRPV and Hershey CRPV) were cloned into PUC19; the E8 ATG ko mutants for each strain were also generated. We employed the infection technique reported previously in which scarification is done first and is followed with delivery of DNA by pipette 3 days later. The papilloma outgrowth generated by these three wild type constructs and their E8 ATG ko mutants was compared. No significant difference was found among the three strains or their E8 ATG ko mutants. E8 ATG ko mutants induced significantly smaller but persistent papillomas when compared to their respective wild type CRPVs. The gene gun was also used to create infections with both Hershey CRPV DNA and the corresponding E8 ATG ko and was found to lead to less vigorous growth as well as some regressions. Further studies suggested that gene gun delivery might have induced an immune response which then resulted in compromised growth of papillomas. It was concluded that the E8 gene is not required for infection. We suggest that standardized infection methods should be used in laboratories so that inconsistencies in conclusions will be minimized.

Impact of genetic changes to the CRPV genome and their application to the study of pathogenesis in vivo.

The cottontail rabbit papillomavirus (CRPV)/rabbit model has been used to study oncogenicity and immunogenicity of different antigens from the papillomavirus genome and has therefore served as a preclinical model for the development of preventive and therapeutic vaccines against papillomavirus infections. One unique property of the CRPV model is that infection can be initiated using viral DNA. This property allows for the functional testing of viral mutants in vivo. We have introduced point mutations, insertions and deletions into all of the different coding and non-coding regions of the CRPV genome and have tested their infectivity in this model. We found that the majority of the mutant genomes retained viability and could induce papillomas in domestic rabbits. These data indicated that the CRPV genome is tolerant of many modifications without compromising its ability to initiate skin papillomas. In combination with our recently established HLA-A2.1 transgenic rabbit model, this plasticity allows us to extend the utility of the CRPV/rabbit model to the screening of HLA-A2.1 restricted epitopes from other human viral and tumor antigens.

Cottontail rabbit papillomavirus E8 protein is essential for wart formation and provides new insights into viral pathogenesis.

The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma growth but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin. The scarce warts induced showed very slow growth, despite sustained expression of E6 and E7 oncogenes. This points to an essential role of E8 in disturbing epidermal homeostasis. Using a yeast two-hybrid screen, we found that E8 interacted with the zinc transporter ZnT1, protocadherin 1 (PCDH1), and AHNAK/desmoyokin, three proteins as yet unrelated to viral pathogenesis or cell transformation. HPV16 E5 also interacted with these proteins in two-hybrid assay. CRPV E8 mainly localized to the Golgi apparatus and the early endosomes of transfected keratinocytes and colocalized with ZnT1, PCDH1, and AHNAK. We showed that ZnT1 and PCDH1 formed a complex and that E8 disrupted this complex. CRPV E8, like HPV16 E5, increased epidermal growth factor (EGF)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and both the EGF-dependent and the EGF-independent activity of activating protein-1 (AP-1). Competition experiments with a nonfunctional truncated ZnT1 protein showed that E8-ZnT1 interaction was required for AP-1 activation. Our data identify CRPV E8 as a key player in papilloma induction and unravel novel cellular targets for inducing the proliferation of keratinocytes.

Large cutaneous rabbit papillomas that persist during cyclosporin A treatment can regress spontaneously after cessation of immunosuppression.

Cottontail rabbit papillomavirus (CRPV)-induced papillomas can progress into malignant carcinomas, remain persistent or regress. Both host immunity and virus genetic background play critical roles in these events. To test how host immunity influences CRPV-induced papilloma evolution, both EIII/JC (inbred) and New Zealand White (outbred) rabbits were treated with an immunosuppressive drug, cyclosporin A (CsA), for 80 days and the regression of three regressive constructs, H.CRPVr (a CRPV regressive strain), H.CRPVp-E6r (a progressive strain with regressive E6) and H.CRPVp-CE6rm (H.CRPVp with the carboxyl terminal of regressive E6, containing mutations at amino acid residues E252G, G258D and S259P) was checked. Papillomas induced by H.CRPVr and H.CRPVp-E6r on control inbred and outbred rabbits regressed totally around week 8, whereas papillomas on all CsA-treated rabbits grew progressively. After cessation of CsA treatment, papillomas began to regress in six outbred rabbits: 14 of 18 papillomas induced by CRPVr, 11 of 18 papillomas induced by H.CRPVp-E6r and eight of 10 papillomas induced by H.CRPVp-CE6rm regressed around week 21. In four CsA-treated inbred rabbits, two of 17 papillomas induced by H.CRPVr and one of 17 papillomas induced by H.CRPVp-E6r regressed. These data indicate that papillomas induced by a regressive CRPV strain can become persistent in the transiently immunosuppressed host. However, returning immunity can lead to regression and clearance of large papillomas (with increased antigenicity) in an outbred population, whilst these same antigenic papillomas persist in inbred rabbits.

Rabbit genital tissue is susceptible to infection by rabbit oral papillomavirus: an animal model for a genital tissue-targeting papillomavirus.

Rabbit oral papillomavirus (ROPV) is a mucosatropic papillomavirus which naturally infects oral mucosal sites of domestic rabbits. In this study, we tested the hypothesis that rabbit genital mucosa is also susceptible to ROPV infection by using the athymic mouse xenograft system and adult immunocompetent rabbits. Subrenal xenografts of ROPV-infected rabbit vulvar and penile sheath tissues were strongly positive for ROPV infection by histologic, in situ hybridization, and Southern analyses. Direct inoculation of adult rabbit penises with infectious ROPV produced small raised lesions of approximately 1 by 1 by 1 mm that were ROPV positive by both in situ hybridization and Southern analyses and were also viral capsid antigen positive by immunohistological staining. Infection of rabbit genital tissues with ROPV may be a useful animal model for the study of genital tissue-targeting papillomaviruses.