Novel lambdapapillomavirus in northern sea otters Enhydra lutris kenyoni, associated with oral hyperplastic nodules.

A novel papillomavirus (PV) associated with hyperplastic nodules scattered over the muco-cutaneous border of the oral cavity of a dead, wild, subadult northern sea otter Enhydra lutris kenyoni (NSO) in 2004 in Homer, Alaska, USA, was genetically characterized. Primers for the amplification of 2 large overlapping DNA fragments that contained the complete genome of the NSO PV were designed. Sanger methodology generated sequences from which new specific primers were designed for the primer-walking approach. The NSO PV genome consists of 8085 nucleotides and contains an early region composed of E6, E7, E1, and E2 open reading frames (ORFs), an E4 ORF (contained within E2) lacking an in-frame proximal ATG start codon, an unusually long (907 nucleotide) stretch lacking any ORFs, a late region that contains the capsid genes L2 and L1, and a non-coding regulatory region (ncRR). This NSO PV has been tentatively named Enhydra lutris kenyoni PV2 (ElkPV2). Pairwise and multiple sequence alignments of the complete L1 ORF nucleotides and concatenated E1-E2-L1 amino acid sequences showed that the NSO PV is a novel PV, phylogenetically most closely related to southern sea otter PV1. The carboxy end of the E6 oncoprotein does not contain the PDZ-binding motif with a strong correlation with oncogenicity, suggesting a low-risk PV, which is in agreement with histopathological findings. However, the ElkPV2 E7 oncoprotein does contain the retinoblastoma (pRb) binding domain LXCXE (LQCYE in ElkPV2), associated with oncogenicity in some high-risk PVs. Further studies on the prevalence and clinical significance of ElkPV2 infections in NSO are needed.

Leopardus wiedii Papillomavirus type 1, a novel papillomavirus species in the tree ocelot, suggests Felidae Lambdapapillomavirus polyphyletic origin and host-independent evolution.

The limited knowledge on Papillomavirus diversity (particularly in wild animal species) influences the accuracy of PVs phylogeny and their evolutionary history, and hinders the comprehension of PVs pathogenicity, especially the mechanism of virus - related cancer progression. This study reports the identification of Leopardus wiedii Papillomavirus type 1 (LwiePV1), the first PV type within Lambdapapillomavirus in a Leopardus host. LwiePV1 full genome sequencing allowed the investigation of its taxonomic position and phylogeny. Based on results, LwiePV1 should be assigned to a novel PV species providing evidence for a polyphyletic origin of feline lambda PVs, and representing an exception to codivergence between feline lambda PVs and their hosts. Results improve our knowledge on PV diversity and pave the way to future studies investigating biological and evolutionary features of animal PVs.

Felis catus papillomavirus type-2 E6 binds to E6AP, promotes E6AP/p53 binding and enhances p53 proteasomal degradation.

E6 from high risk human papillomaviruses (HR HPVs) promotes ubiquitination and degradation of p53 tumour suppressor by mediating its binding to ubiquitin ligase E6AP in a ternary complex, contributing to cell transformation in cervical cancer. We have previously shown that Felis catus papillomavirus type -2 (FcaPV-2) E6 is expressed in feline squamous cell carcinoma (SCC) and displays the ability to bind p53 and decrease its protein levels in transfected CRFK cells. However, the mechanism of p53 downregulation has not yet been characterized. Here we show that FcaPV-2 E6 bound to E6AP, which in turn was bound by p53 exclusively in cells expressing the viral oncoprotein (CRFKE6). Furthermore, p53 was highly poly-ubiquitinated and underwent accumulation upon E6AP gene knockdown in CRFKE6. Half-life experiments and proteasome inhibition treatments indicated that down-regulation of p53 protein in CRFKE6 was due to accelerated proteasomal degradation. E6AP/p53 binding was also demonstrated in two feline SCC cell lines expressing FcaPV-2 E6, where p53 protein levels and poly-ubiquitination degree were proportional to E6 mRNA levels. The data obtained in both artificial and spontaneous in vitro models suggest that FcaPV-2 E6 degrades p53 through a molecular mechanism similar to HR HPVs, possibly contributing to the development of feline SCC.

Malignant transformation of canine oral papillomavirus (CPV1)-associated papillomas in dogs: An emerging concern?

Canine oral papillomavirus (CPV1, also known as COPV), the most common cause of non-neoplastic papillomas, has not been shown to cause squamous cell carcinomas (SCC). Furthermore, malignant transformation of benign papillomas to SCC has only been reported in a single group of dogs with severe combined immunodeficiency infected with CPV2. Here, we report a series of 7 dogs with benign CPV1-associated papillomas with histologic evidence of CPV1 causing malignant transformation to carcinoma in situ and ultimately SCC. Expression of p53 and p16 proteins in CPV1-infected cells within the benign papillomas and lesions that progressed into SCC also supported an association between papillomavirus and malignant transformation. Moreover, our retrospective analysis indicated that while there have been increased numbers of viral papillomas with malignant transformation, the number of annually diagnosed canine viral papillomas has remained constant over the past decade in our laboratory. We speculate that either an altered host immunity from increased usage of immunosuppressive drugs or changing environmental factors, e.g. increase exposure to UV radiation, may cause an increased oncogenic potential of this "low-risk" virus. This study aims to raise awareness of the malignant potential of CPV1 and to encourage further investigations into the cause of this suspected change in its oncogenic potential.

Malignant Transformation of a Canine Papillomavirus Type 1-Induced Persistent Oral Papilloma in a 3-Year-Old Dog.

This case report describes a rare case of a persistent canine papillomavirus type 1 (CPV-1)-induced oral papilloma that underwent malignant transformation into an oral squamous cell carcinoma (OSCC) in a 3-year-old Labrador retriever cross. Initially, the patient had multiple and multifocal verrucous lesions populating the oral cavity exclusively. The papillomas persisted despite multiple surgical ablations, azithromycin, interferon α-2b, alternative medicines, and off-label drug use of an immunostimulant. After 1 year and 6 months, an aggressive lesion developed at the level of the left mandibular first molar (309) and progressed to a well-differentiated invasive OSCC. The presence of CPV-1 DNA in the OSCC, and the known oncogenic abilities of CPV-1, suggests that this virus might have played a significant role in the emergence of the OSCC that ultimately led to the patient's euthanasia due to poor quality of life. This is the first well-documented case where OSCC has developed from an oral papilloma caused by CPV-1 in which the presence of coinfection by another papillomavirus was excluded by multiple polymerase chain reaction tests using various primers.

Canine oral papillomavirus outbreak at a dog daycare facility.

This report describes an outbreak at a dog daycare facility where 13 of 52 dogs developed suspected canine papillomavirus (CPV) infection. Based on contact tracing, subclinical CPV shedding was speculated. Active surveillance, exclusion of animals with active or recent infection and cohort formation may have been effective in stopping the outbreak.

Éclosion du papillomavirus oral canin dans une garderie canine. Ce rapport décrit l'éclosion dans une garderie canine où 13 des 52 chiens ont développé une infection suspectée par le virus du papillome canin (VPC). En se basant sur le retraçage des contacts, on a émis la supposition d'une excrétion subclinique du VPC. Une surveillance active, l'exclusion des animaux avec une infection active ou récente et la formation d'une cohorte ont pu être efficaces pour freiner l'éclosion.(Traduit par Isabelle Vallières).

Lambdapapillomavirus 2 in a Gray Wolf ( Canis lupus ) from Minnesota, USA with Oral Papillomatosis and Sarcoptic Mange.

Oral papillomatosis was diagnosed in a gray wolf ( Canis lupus ) with sarcoptic mange from Minnesota, US found dead in February 2015. Intranuclear inclusion bodies were evident histologically, and papillomaviral antigens were confirmed using immunohistochemistry. Sequencing of the L1 papillomavirus gene showed closest similarity to Lambdapapillomavirus 2.

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.

Genomic characterisation of canine papillomavirus type 17, a possible rare cause of canine oral squamous cell carcinoma.

Squamous cell carcinomas (SCCs) are the second most common cancer of the canine oral cavity resulting in significant morbidity and mortality. Recently a dog with multiple oral SCCs that contained a novel papillomavirus (PV) was reported. The aim of the present study was to determine the genome of this novel PV. To do this a short section of PV DNA was amplified from an oral SCC and 'back-to-back' primers were designed. Due to the circular nature of PV DNA, these primers were then used to amplify the remainder of the genome by inverse PCR. The PCR product was sequenced using next generation sequencing and the full genome of the PV, consisting of 8007 bp, was assembled and analysed. As this is the seventeenth PV identified from the domestic dog, the novel PV was designated Canis familiaris papillomavirus (CPV) type 17. Similar to other CPV types, the putative coding regions of CPV-17 were predicted to produce 5 early and 2 late proteins. Phylogenetic analysis of ORF L1 revealed greater than 70% similarity to CPV-2 and CPV-7 and we propose that CPV-17 also be classified as a Taupapillomavirus 1. While it appears CPV-17 is only rarely present in canine oral SCCs, evidence suggests that this PV could influence the development of oral SCCs in this species.

Molecular and immunohistochemical studies do not support a role for papillomaviruses in canine oral squamous cell carcinoma development.

Oral squamous cell carcinomas (OSCCs) are common neoplasms of dogs and are of unknown cause. Whereas papillomaviruses (PVs) are an established cause of human OSCCs, few studies have investigated canine OSCCs for a PV aetiology. In humans, a PV aetiology can be determined by detecting PV DNA and PV-induced increased p16(CDKN2A) protein (p16) within the OSCC. In this study, PCR, using four different primer sets and p16 immunohistochemistry, was used to evaluate 28 canine OSCCs for a possible PV aetiology. None of the primers amplified PV DNA from any of the OSCCs although four neoplasms contained intense p16 immunostaining. Intense p16 immunostaining would indicate a PV aetiology in a human OSCC but the absence of PV DNA suggests that the increase in p16 was not due to PV infection. Overall the results indicated that PVs are not a significant cause of canine OSCCs.

Antibody titres against canine papillomavirus 1 peak around clinical regression in naturally occurring oral papillomatosis.

BACKGROUND: Most forms of canine papillomatosis are believed to be associated with papillomavirus infections. Canine papillomavirus type 1 (CPV1) is considered to be responsible for most oral cases and several forms of cutaneous papillomatosis. HYPOTHESIS/OBJECTIVES: The aim of this study was to evaluate cases of naturally occurring oral papillomatosis with regard to the type of virus involved, antibody induction and remission time. METHODS: Forty dogs showing different degrees of classical oral papillomatosis were included as a single study group. Tissue and serum samples were acquired upon initial presentation; serum samples were collected again upon remission (n = 13) and after 3 months of convalescence (n = 4). None of the dogs underwent antiviral therapy. Tissue samples were tested by PCR to detect CPV DNA, while serum samples were tested using a specific enzyme-linked immunosorbent assay for antibodies against the L1 capsid protein of CPV1. RESULTS: All tissue samples were positive for CPV1 DNA, and 87.5% of all serum samples contained measurable levels of antibody against the virus (cut-off value 0.3). The average optical density measured in the enzyme-linked immunosorbent assay was 0.51 at initial presentation, 1.65 upon remission and 0.83 at 3 months postrecovery. Time to clinical regression varied between 1 month and 1 year. CONCLUSIONS AND CLINICAL IMPORTANCE: These data support existing evidence for a high prevalence of CPV1 in canine oral papillomatosis. The healing process seems to correlate with a strong antibody response, and antibody titres peaked around the time of clinical recovery. In contrast to previous data from laboratory settings, the variation in remission time was very high.

Canine papillomatosis: a retrospective study of 24 cases (2001-2011) and immunohistochemical characterization / Papilomatose em cães: estudo retrospectivo de 24 casos (2001-2011) e caracterização imuno-histoquímica

A retrospective study of 24 cases of papillomas in dogs was performed from January 2001 to March 2011. Additionally, immunohistochemistry (IHC) was used to characterize and evaluate the samples. We found that disease was observed more in mixed breed dogs, ages ranging from 6 months to 10 years (mean 3.1 years), and there was no gender predilection. The main lesion sites were the skin (75%), lips (16.7%), and eyelids (8.3%). Upon histological evaluation, we observed papillary exophytic proliferation of squamous epithelium and papillary endophytic proliferation (inverted) in 87.5% and 12.5% of cases, respectively. The tumors were characterized by spinous layer hyperplasia (87.5%) with koilocytes (70.8%) and intranuclear pale basophilic inclusions bodies (8.3%), prominent granular layer with large amounts of keratohyalin granules (95.8%), and hyperkeratosis in the stratum corneum (100%). Positive immunostaining for Papillomavirus was found in 83.3% of cases, which were distributed between the granular layer and the stratum corneum. These findings indicate the following: that papillomas in dogs are caused by Papillomavirus, the viral cytopathic effect induces epithelial lesions, viral particles are found inside the cell nuclei, and inclusions bodies are rare.

Foi realizado um estudo retrospectivo de 24 casos de papilomas em cães diagnosticados no período de janeiro 2001 a março de 2011, bem como a sua caracterização imuno-histoquímica (IHQ). Cães sem raça definida foram os mais afetados, a idade média foi de 3,1 anos, com variação de 6 meses a 10 anos e não houve predileção sexual. Quanto à localização das lesões, 75,0% estavam na pele, 16,7% no lábio e 8,3% em pálpebra. Na avaliação histológica havia proliferação papilar exofítica do epitélio escamoso em 87,5% e papilar endofítica (invertido) em 12,5%. O tumor era caracterizado por hiperplasia do estrato espinhoso (87,5%) com coilócitos (70,8%) e inclusões intranucleares basofílicas pálidas (8,3%); o estrato granular estava proeminente com grande quantidade de grânulos de querato-hialina (95,8%); e havia hiperqueratose do estrato córneo (100%). Na avaliação IHQ para Papillomavirus houve marcação nos estratos granuloso e córneo em 83,3%. Estes achados indicam que os papilomas em cães são causados por Papillomavirus, as lesões epiteliais são decorrentes do efeito citopático viral, as partículas virais estão no núcleo das células e corpúsculos de inclusão são raros.

Characterization of canine oral papillomavirus by histopathological and genetic analysis in Korea.

In August 2008, forty dogs out of 400 developed oral warts in a breeding farm in Korea. Canine oral papilloma infection is a common disease in dogs. However, there has been no report of an outbreak of canine oral papillomavirus (COPV) in a group of dogs or in dog breeding farms in Korea, and the genetic analysis of COPV in Korea has yet to be performed. This study diagnosed canine oral papilloma from the oral samples of these dogs based on histopathological examination and immunohistochemistry. Polymerase chain reaction was applied to amplify the corresponding products using preexisting primer sets for COPV and a universal human papillomavirus targeting L1 gene. Further genetic analysis of the major viral capsid gene L1 confirms the sequences of Korean COPV, which shows a close relationship to previously reported COPV. This study describes the histopathological and immunohistochemical characteristics of canine oral papilloma in a group of breeding dogs in Korea and discloses the complete L1 gene sequences of Korean COPV.

Canine inverted papillomas associated with DNA of four different papillomaviruses.

Inverted papillomas are uncommon papillomavirus (PV)-induced canine skin lesions. They consist of cup- to dome-shaped dermal nodules with a central pore filled with keratin. Histologically they are characterized by endophytic projections of the epidermis extending into dermis. Cytopathic effects of PVs infection include the presence of clumped keratohyalin granules, koilocytes and intranuclear inclusion bodies. Different DNA hybridization studies carried out with a canine oral papillomavirus (COPV) probe suggested that a different PV than COPV might cause these lesions. Canine papillomavirus 2 (CPV2) was discovered a few years ago in inverted papillomas of immunosuppressed beagles. Two other cases, presenting with distinct clinical and histological features have also been described. This study was carried out on four dogs with clinical and histological signs of inverted papillomas. Molecular biological analyses confirmed that PV DNA was present in all four lesions but demonstrated that the sequences in each case were different. One corresponded to COPV, the second to CPV2, and the third and fourth to unknown PVs. These findings suggest that inverted papillomas are not caused by one single PV type. Similar observations have also been made in human medicine.

Detection of canine oral papillomavirus DNA in conjunctival epithelial hyperplastic lesions of three dogs.

Papillomavirus infections are responsible for plaques and papillomas in various locations on the skin and in mucous membranes. The aim of this report was to describe morphologic features of a viral pigmented conjunctival plaque and 2 conjunctival squamous papillomas in 3 dogs, and to investigate these lesions for the presence of papillomavirus DNA by polymerase chain reaction (PCR), DNA sequence analysis, and in situ hydridization (ISH). Histopathology revealed in all neoplasms various degrees of epithelial hyperplasia, acanthosis, and hyperkeratosis with koilocytosis. In all lesions E6, E7, and L1 gene fragments of canine oral papillomavirus (COPV) DNA were detected by PCR and sequencing analysis. ISH revealed COPV DNA in a highly specific pattern within nuclei of the hyperplastic epithelium. The presence of canine papillomavirus in ocular conjunctival plaques and papillomas suggests these benign lesions may have the potential for malignant transformation. This is the first time that the lambdapapillomavirus COPV has been detected in ocular epithelial hyperplastic lesions.

Ancient papillomavirus-host co-speciation in Felidae.

BACKGROUND: Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1. RESULTS: The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 x 10(-8) (95% confidence interval 1.32 x 10(-8) to 2.47 x 10(-8)) nucleotide substitutions per site per year for the viral coding genome. CONCLUSION: Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.

Amplicon-plus targeting technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants.

High-level expression of transgenes is essential for cost-effective production of valuable pharmaceutical proteins in plants. However, transgenic proteins often accumulate in plants at low levels. Low levels of protein accumulation can be caused by many factors including post-transcriptional gene silencing (PTGS) and/or rapid turnover of the transgenic proteins. We have developed an Amplicon-plus Targeting Technology (APTT), by using novel combination of known techniques that appears to overcome both of these factors. By using this technology, we have successfully expressed the highly-labile L1 protein of canine oral papillomavirus (COPV L1) by infecting transgenic tobacco plants expressing a suppressor of post-transcriptional gene silencing (PTGS) with a PVX amplicon carrying a gene encoding L1, and targeting the vaccine protein into the chloroplasts. Further, a scalable "wound-and-agrospray" inoculation method has been developed that will permit high-throughput Agrobacterium inoculation of Nicotiana tabacum, and a spray-only method (named "agrospray") for use with N. benthamiana to allow large-scale application of this technology. The good yield and short interval from inoculation to harvest characteristic of APTT, combined with the potential for high-throughput achieved by use of the agrospray inoculation protocol, make this system a very promising technology for producing high value recombinant proteins, especially those known to be highly labile, in plants for a wide range of applications including producing vaccines against rapidly evolving pathogens and for the rapid response needed to meet bio-defense emergencies.

An epidermotropic canine papillomavirus with malignant potential contains an E5 gene and establishes a unique genus.

A novel canine papillomavirus, CfPV-2, was cloned from a footpad lesion of a golden retriever. Unlike the known canine oral papillomavirus (COPV), which has a double-stranded DNA genome size of 8607 bps, the genome of CfPV-2 is 8101 bps. Some of this size difference is due to an abbreviated early-late region (ELR), which is 1200 bps shorter than that of COPV. However, CfPV-2 has other differences from COPV, including the presence of an E5 ORF between the E2 gene and the ELR and an enlarged E4 ORF (one of the largest PV E4 open reading frames). The genome of CfPV-2 shares low homology with all the other papillomaviruses and, even in the most highly conserved ORF of L1, the nucleotide sequence shares only 57% homology with COPV. Due to this highly divergent DNA sequence, CfPV-2 establishes a new PV genus, with its closest phylogenetic relatives being amongst the Xi and Gamma genuses. CfPV-2 also has unique biological features; it induces papillomas on footpads and interdigital regions which, if infection is persistent, can progress to highly metastatic squamous cell carcinoma. CfPV-2 does not induce oral papillomas in immunocompetent animals and antibodies generated against COPV and CfPV-2 are type-specific. The availability of a new canine papillomavirus with differing genetic and biological properties now makes it possible to study type-specific host immune responses, tissue tropism and the comparative analysis of viral gene functions in the dog.

Cell-mediated immune responses to COPV early proteins.

Cell-mediated immunity plays a key role in the regression of papillomavirus-induced warts and intra-epithelial lesions but the target antigens that induce this response are not clear. Canine oral papillomavirus (COPV) infection of the oral cavity in dogs is a well-characterized model of mucosal papillomavirus infection that permits analysis of the immune events during the infectious cycle. In this study we show that during the COPV infectious cycle, systemic T cell responses to peptides of several early proteins particularly the E2 protein, as assayed by delayed type hypersensitivity, lymphoproliferation and IFN-gamma ELISPOT, can be detected. The maximal response occurs in a narrow time window that coincides with maximal viral DNA replication and wart regression: thereafter, systemic T cell responses to early proteins decline quite rapidly. Vaccination using particle-mediated immunotherapeutic delivery (PMID) of codon-modified COPV E2 and E1 genes induces strong antigen-specific cell-mediated immune responses in the vaccinated animals. These data show that therapeutic immunization by PMID with codon-modified E2 is completely effective, that to E1 is partially protective, that this correlates with the intensity of antigen-specific cell-mediated immune responses and, further, they emphasize the importance of these responses and the route of immunization in the generation of protective immunity.