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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing.

To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 zeta and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin beta, which are involved in cell motility and tissue invasion, as well as casein kinase 1 alpha, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin beta in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.

Kinetics of in vitro adsorption and entry of papillomavirus virions.

There has been much incongruence in reports addressing the rate at which papillomaviruses enter cultured cells. We used a recently developed QRT-PCR assay (J. Virol. Methods 111 (2003) 135) to analyze the expression, adsorption, and entry kinetics of human papillomavirus type 11 (HPV-11) in multiple cell lines. Parallel experiments with HPV-40 and cottontail rabbit papillomavirus (CRPV) were also performed with biologically relevant lines. Infection was determined by the expression of early transcripts containing the E1 E4 splice junction. Results support previous observations that papillomaviruses may enter cultured cells much more slowly than rates reported for similarly structured viruses (Virology 207 (1995) 136; Virology 307 (2003) 1; J. Virol. 75 (2001) 1565). Additionally, our data suggest that, following adsorption to the cell surface, capsomeric structure remains largely unchanged for many hours as HPV-11 virions remain equally susceptible to neutralization by a nonspecific microbicide and by L1-specific monoclonal antibodies (MAb) targeting both linear and conformationally sensitive epitopes.

The viral E4 protein is required for the completion of the cottontail rabbit papillomavirus productive cycle in vivo.

Expression of the papillomavirus E4 protein correlates with the onset of viral DNA amplification. Using a mutant cottontail rabbit papillomavirus (CRPV) genome incapable of expressing the viral E4 protein, we have shown that E4 is required for the productive stage of the CRPV life cycle in New Zealand White and cottontail rabbits. In these lesions, E4 was not required for papilloma development, but the onset of viral DNA amplification and L1 expression were abolished. Viral genome amplification was partially restored when mutant genomes able to express longer forms of E4 were used. These findings suggest that efficient amplification of the CRPV genome is dependent on the expression of a full-length CRPV E4 protein.

Amino acid residues in the carboxy-terminal region of cottontail rabbit papillomavirus E6 influence spontaneous regression of cutaneous papillomas.

Previous studies have identified two different strains of cottontail rabbit papillomavirus (CRPV) that differ by approximately 5% in base pair sequence and that perform quite differently when used to challenge New Zealand White (NZW) rabbit skin. One strain caused persistent lesions (progressor strain), and the other induced papillomas that spontaneously regressed (regressor strain) at high frequencies (J. Salmon, M. Nonnenmacher, S. Caze, P. Flamant, O. Croissant, G. Orth, and F. Breitburd, J. Virol. 74:10766-10777, 2000; J. Salmon, N. Ramoz, P. Cassonnet, G. Orth, and F. Breitburd, Virology 235:228-234, 1997). We generated a panel of CRPV genomes that contained chimeric and mutant progressor and regressor strain E6 genes and assessed the outcome upon infection of both outbred and EIII/JC inbred NZW rabbits. The carboxy-terminal 77-amino-acid region of the regressor CRPV strain E6, which contained 15 amino acid residues that are different from those of the equivalent region of the persistent CRPV strain E6, played a dominant role in the conversion of the persistent CRPV strain to one showing high rates of spontaneous regressions. In addition, a single amino acid change (G252E) in the E6 protein of the CRPV progressor strain led to high frequencies of spontaneous regressions in inbred rabbits. These observations imply that small changes in the amino acid sequences of papillomavirus proteins can dramatically impact the outcome of natural host immune responses to these viral infections. The data imply that intrastrain differences between separate isolates of a single papillomavirus type (such as human papillomavirus type 16) may contribute to a collective variability in host immune responses in outbred human populations.

A transactivator function of cottontail rabbit papillomavirus e2 is essential for tumor induction in rabbits.

Infection of domestic rabbits with cottontail rabbit papillomavirus (CRPV) causes local papillomas which progress to carcinomas in more than 80% of cases. This animal model system therefore allows the identification of molecular mechanisms required for the induction and progression of epithelial tumors. The viral E2 protein stimulates both viral DNA replication and transcription, and these functions can be genetically separated. We introduced the respective mutations into CRPV E2 and found, in line with published data for other papillomavirus E2 proteins, that mutation of the highly conserved amino acid 37 or 73 resulted in replication-competent but transactivation-deficient E2 proteins, whereas E2 proteins with mutations at residue 39 were replication deficient and transactivation competent. The R37A, I73L, and I73A E2 mutants, showing a loss of transactivation function, and the R37K E2 mutant, which is still transactivation competent, were introduced into the whole genome of CRPV, which was then injected into the skin of rabbits. Strikingly, the ability to induce tumors within 6 weeks was abolished by each of the E2 mutations, in contrast to the tumor induction rate (93%) obtained with wild-type CRPV DNA. Two small papillomas induced by mutant E2 I73A CRPV DNA appeared as late as 12 or 24 weeks postinjection, were significantly smaller, and showed no further extension of growth. These data suggest that functionally conserved amino acids in the transactivation domain of E2 are also required for the induction and growth of epithelial tumors in rabbits infected with CRPV.

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.