Involvement of Felis catus papillomavirus type 2 in the tumorigenesis of feline Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine tumor. We recently demonstrated that cats with MCC often have other proliferative cutaneous lesions, such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Based on this finding, we hypothesize that Felis catus papillomavirus (FcaPV) is involved in the development of MCC in cats, similar to SCC and BCC. To investigate this hypothesis, the presence of FcaPV nucleic acid and immunoreactivity for tumor suppressor proteins were examined in 21 feline MCC cases. Polymerase chain reaction using FcaPV type-specific primers detected FcaPV2 DNA in 20/21 samples of MCC. The complete FcaPV2 sequence was characterized in one case. In situ hybridization for FcaPV2 E7 revealed punctate nuclear signals within tumor cells in 19/21 MCC. Increased immunoreactivity for p16CDKN2A protein and decreased immunoreactivity for retinoblastoma (pRb) and p53 proteins were observed in 20/21 MCC. These results suggest that feline MCC cases are infected with FcaPV2 and the subsequent inhibition of pRb and p53 induced by integrated viral oncogenes is associated with feline MCC tumorigenesis, similar to other PV-induced proliferative cutaneous lesions. On the other hand, the single case of FcaPV2-negative MCC showed strong p53 immunoreactivity, suggesting mutations in p53 caused by cancer inducers other than FcaPV2 infection in this case. The present study suggests FcaPV2 as a cause of feline MCC.

Vulvar squamous cell carcinoma associated with Equus caballus papillomavirus type 2 infection in a Japanese mare.

Equus caballus papillomavirus type 2 (EcPV2) infection has been associated with genital squamous cell carcinoma (SCC) development in horses. However, very few reports on EcPV2-associated disease in Asia exist. Our study characterizes pathological and virological features of an EcPV2-associated vulvar SCC from a Japanese mare. Conventional PCR, in situ hybridization, reverse-transcriptase PCR and immunohistochemistry confirmed the presence and distribution of EcPV2 within the lesion and suggested that p53 degradation may not be the mechanism by which this virus induces neoplastic transformation. The complete viral sequence in this Japanese case shows near perfect sequence homology with European reference strains of EcPV2, which may be useful when considering the target for future EcPV2 vaccine development. This report also serves to highlight the importance of EcPV2 in female (vulvar) neoplasia, which is less commonly recognized than EcPV2-induced male (penile or preputial) neoplasia. Finally, the SCC described in this mare was an unusual acantholytic variant that has not been reported previously in horses. It is the first report of EcPV2 identified from genital SCC in Asia and underscores the likely worldwide distribution of this virus and its consistent association with equine genital neoplasia.

Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle.

The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time.

Comparison of prevalence of Felis catus papillomavirus type 2 in squamous cell carcinomas in cats between Taiwan and Japan.

Felis catus papillomavirus (FcaPV), especially type 2 (FcaPV2) is considered as one of the causative agents in squamous cell carcinoma (SCC) in cats. However, our previous study detected FcaPV3 and FcaPV4, but not FcaPV2 in feline SCCs collected in Japan, suggesting that the prevalence of FcaPV2 in SCC may vary depending on geographic locations. To evaluate this hypothesis, two conventional PCR reactions targeting E1 and E7 genes were performed to detect FcaPV2 in feline SCC samples collected in Taiwan and Japan. While 46.9% (23/49) of feline SCC cases from Taiwan were PCR positive for FcaPV2, only 8.6% (3/35) cases from Japan were positive. Our result suggests that the prevalence of FcaPV2 in feline SCCs may depend on the region.

Full-genome characterization of a novel Felis catus papillomavirus 4 subtype identified in a cutaneous squamous cell carcinoma of a domestic cat.

The present study describes two full-genome sequences of Felis catus papillomavirus type 4 (FcaPV4) identified in squamous cell carcinoma (SCC) of two domestic cats. Two full-genome sequences of FcaPV4 were detected and characterized by PCR and sequencing. The L1 nucleotide sequence homology of one case showed 95.70% sequence identity to the reference FcaPV4, suggesting that this isolate should be classified as a subtype. Reverse-transcriptase PCR (RT-PCR) of two oncogenes, E6 and E7 was performed to confirm mRNA expression. Expression of E6 and E7 mRNA was detected in both cases, suggesting that FcaPV4 contributes to the development of SCC. This is the first report of FcaPV4 subtype. The present study will update the genomic features of FcaPV4 and contribute to deepening our knowledge about the etiological roles of FcaPV4 in feline cutaneous SCC.

Genomic characterisation of bovine papillomavirus types 1 and 2 identified in equine sarcoids in Japan.

BACKGROUND: Bovine papillomavirus types 1 and 2 (BPV1/2) infection in horses has been associated with the development of equine sarcoids. Previous findings revealed the presence of sarcoid-associated BPV sequence variants that have been proposed as a key factor of cross-species infection in horses. To verify this hypothesis, sarcoid-associated BPV variants should be identified regardless of geographic location. OBJECTIVES: Sequence analyses of BPV1/2 derived from both horses and cattle were conducted to clarify the sarcoid-associated sequence variants. The aim of this study was to clarify the correlation between BPV phylogeny and the geographic origin/host species. STUDY DESIGN: Cross-sectional study. METHODS: Conventional PCR to detect BPV1/2 was performed with genomic DNA extracted from equine sarcoid (n = 10) and bovine papilloma (n = 10) samples collected in Japan. Direct sequencing results were compared between equine and bovine (equine/bovine)-derived BPV to identify sarcoid-associated variants of two early regions (E2, E5), one late region (L1) and the long control region (LCR). Phylogenetic and phylogeny-trait correlation were analysed using Bayesian Markov chain Monte Carlo (MCMC) method and Bayesian tip-association significance testing (BaTS). RESULTS: Seven BPV1 and three BPV2 were identified from equine sarcoids using PCR and direct sequencing. Sequence analysis of equine/bovine-derived samples showed no sarcoid-associated variants in four regions (E2, E5, L1 and LCR) of either BPV1 or BPV2. The phylogenetic tree of BPV1 E2, L1 and LCR tended to cluster within its geographic origins. BaTS analysis demonstrated that BPV1 sequence variability may be due to the geographic origin rather than host species difference. MAIN LIMITATIONS: There was a limitation in sample numbers. CONCLUSIONS: This study supports the geographic-specific hypothesis of sequence variability, suggesting that BPV1 is shared between local equids and bovids. However, more extensively collected sequences worldwide and functional evaluations are needed to verify the geographic-specific sequence variability of BPV1/2 between equine- and bovine-derived sequence.

Anogenital-Associated Papillomaviruses in Animals: Focusing on Bos taurus Papillomaviruses.

In contrast to the diverse studies on human papillomaviruses (HPVs), information on animal PVs associated with anogenital lesions is limited. In the animal kingdom, papillomas occur more commonly in cattle than in any other animals, and diverse types of Bos taurus papillomaviruses (BPVs) exist, including the very recently discovered BPV type 29 (BPV29). From this perspective, we will review previous studies describing PV types associated with anogenitals in animals, with a focus on BPVs. To date, two classical BPV types, classified into Deltapapillomavirus (BPV1 and BPV2) and Dyokappapapillomavirus (BPV22), and two novel Xipapillomaviruses (BPV28 and BPV29) have been identified from anogenital lesions and tissues of the domestic cow. Due to the limited reports describing anogenital-associated PVs in animals, the relationships between their phylogenetic and pathogenetic properties are still undiscovered. Animal studies are valuable not only for the veterinary field but also for human medicine, as animal diseases have been shown to mimic human diseases. Studies of anogenital-associated PVs in animals have a positive impact on various research fields.

Characterization of Bovine papillomavirus 28 (BPV28) and a novel genotype BPV29 associated with vulval papillomas in cattle.

Papillomavirus (PV) infections are associated with the development of cutaneous and mucosal tumors in humans and various animal species. In humans, infection of high-risk human PVs (HPVs) causes anogenital cancers, while in animals, anogenital-associated PVs are not well understood. Among animal PVs, Bos taurus PVs (BPVs) have the most diverse genotypes, up to 28 of them. The present study will report two unique BPVs identified in vulval papilloma lesions from two Holstein Friesian cattle by conventional PCR and sequencing. In the first case, BPV28 harboring two L1 open reading frames (ORFs) due to a five-nucleotide deletion was identified. In the second case, histologically diagnosed as papilloma, an unclassified BPV genotype was detected. However, in both cases, the immunohistochemistry against PV antigen was negative. The full genome of the unclassified BPV was amplified by inverse PCR and analyzed by genome-walking sequencing. The L1 nucleotide sequence was most identical to BPV genotype 6 (BPV6), showing 78 % identity, indicating that this novel BPV should be classified as species Xipapillomavirus 1, genotype BPV29. The mRNA expression of three early genes (E1, E2, E10), but not L1, was confirmed in both BPV28- and BPV29-detected papilloma lesions. The present study suggests the involvement of novel types of BPV in vulval papilloma. The alteration of BPV28 pathogenicity due to the frameshift mutation of L1 needs to be elucidated in the future.

Genomic characterization and distribution of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.

Whole Genomic Analysis and Comparison of Two Canine Papillomavirus Type 9 Strains in Malignant and Benign Skin Lesions.

Papillomaviruses (PVs) usually cause benign proliferative lesions in the stratified epithelium of various animal species. However, some high-risk types of PVs have been proven to lead to malignant transformations. In dogs, several canine papillomaviruses (CPVs) have been identified in malignant lesions and are suggested as one of the risk factors for the development of squamous cell carcinomas (SCCs). In the present study, the full genomes of two CPV9 strains from recurrent SCCs of Dog 1 and skin viral papilloma (viral plaque) of Dog 2 were sequenced. Alignment of the two CPV9 sequences with the genome of the reference CPV9 strain (accession no. JF800656.1) derived from a solitary pigmented plaque was performed. Compared with the reference strain, a 27 bp in-frame insertion in the E1 gene was identified in both CPV9 strains in this study. In comparison with the CPV9 strains derived from benign lesions, the CPV9 from the SCCs of Dog 1 exhibited a 328 bp deletion at the 3' end of the E2 and spacer sequence, which encoded a truncated deduced E2 protein and a chimeric E8^E2 protein. However, there was no difference in the mRNA expression levels of viral oncoproteins of E6 and E7 between the two CPV9 cases, suggesting that the oncogenesis of CPV9 for malignant transformation might be different from that of human papillomaviruses. The roles of E2 and E8^E2 deleted CPV9 in the oncogenesis of benign and malignant lesions should be further investigated.

Genomic characterization of a novel bovine papillomavirus type 28.

Infection of bovine papillomavirus (BPV) has been associated with mucosal and/or cutaneous tumor development in bovids. To date, up to 27 genotypes of BPVs have been identified and classified based on the nucleotide sequence identity of L1 open reading frame. In the present study, the complete sequence of a novel BPV concurrently identified with BPV1 and BPV2 in the facial cutaneous papilloma lesion of a domestic cattle was characterized. The whole genome of the unclassified BPV was 7263 base pairs in full length with GC ratio of 42.9%. In comparison with published BPV sequences, L1 nucleotide sequence of the novel BPV shared 75% identity with BPV15, and was suggested to be classified in the genus, Xipapillomavirus. According to the criteria established by the International Committee on the Taxonomy of Viruses, the novel BPV was designated as BPV type 28.

Detection and genotyping of bovine leukemia virus (BLV) in Vietnamese cattle.

Bovine leukemia virus (BLV) belongs to the genus, Deltaretrovirus of the family, Retroviridae and it is the causative agent of enzootic bovine leukosis. The prevalence of BLV in three provinces in the Red River Delta Region in the North of Vietnam, Hanoi, Vinhphuc and Bacninh was studied from April 2017 to June 2018. A total of 275 blood samples collected from cattle were used for serum isolation and DNA extraction. Of these samples, 266 sera were subjected to ELISA test for detecting antibody against BLV gp51 protein and 152 DNA samples were used to detect the 444 bp fragment corresponding to a part of the gp51 region of the env by nested PCR. The results showed that 16.5% (n=44) and 21.1% (n=32) of samples were positive for BLV gp51 antibody and BLV proviral DNA, respectively. Phylogenetic analysis of the partial (423 bp) and complete (913 bp) BLV env-gp51 gene indicated that Vietnamese strains were clustered into genotypes 1, 6 and 10 (G1, G6 and G10). Of those genotypes, G1 genotype was dominant; G6 strains were designated as G6e and G6f subgenotypes; the existence of genotype 10 was confirmed for the first time in Vietnam. The present study provides important information regarding the prevalence of BLV infection and genetic characteristics of BLV strains identified in Vietnam, contributing to promote the establishment of disease control and eradication strategies in Vietnam.

Inactivation of human norovirus and its surrogate by the disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals.

Human norovirus is one of the major causes of foodborne gastroenteritis, and it can be easily transmitted from infected person, virus-contaminated foods and environmental surfaces. Effective disinfection method is needed to stop the transmission of human norovirus. CAC-717 is a new disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals. We aimed to evaluate the efficacy of CAC-717 against human norovirus. This study used human norovirus derived from fecal specimens and cultured murine norovirus, which is one of the surrogate viruses for human norovirus. The disinfection effect against murine norovirus was estimated by infectivity assay and transmission electron microscopy. The inactivation effect against human norovirus was assessed by reverse transcription polymerase chain reaction. Disinfection effect of CAC-717 against the infectivity of murine norovirus was shown within 100 s after the CAC-717 treatment, presenting the destruction of viral capsids. The treatment of CAC-717 significantly reduced human norovirus genomic RNA (3.25-log reduction) by the presence of the mesoscopic structure of calcium hydrogen carbonate. CAC-717 stably inactivated human norovirus in stool suspensions. The inactivation effect of CAC-717 against human norovirus was less susceptible to organic substances than sodium hypochlorite. CAC-717 would be a useful alternative for disinfecting human norovirus in contaminated environmental surfaces.

Identification of bovine papillomavirus type 1 and 2 from bovine anogenital fibropapillomas.

Papillomavirus (PV) is a well-known pathogen associated with epithelial and mucosal neoplastic diseases. In contrast to human PVs, characterization of animal PVs from the aspect of anogenital neoplasm is still on a learning curve. In the present study, two vulval and one anal warts, histologically diagnosed as fibropapillomas, excised from dairy cattle were analyzed. PCR and sequencing revealed that bovine papillomavirus type 1 (BPV-1) and BPV-2 were detected from anal and vulval fibropapillomas, respectively. Immunohistochemistry detected PV antigen in a few differentiated keratinocytes of one vulval case. Reverse-transcriptase PCR detected the early region, but not the late region of BPV mRNA in all three cases. The present study will provide new insight into the relationship between BPV and anogenital papilloma in cattle.

Pathologic characterization of Felis catus papillomavirus type 5 (FcaPV-5)-associated viral plaques and Bowenoid in situ carcinoma in a Domestic Shorthair cat.

The present paper describes Felis catus papillomavirus (FcaPV) type 5-associated cutaneous mass in a Domestic Shorthair cat. Histological examination revealed multicentric epidermal acanthosis with papillomavirus-associated cytopathic changes, which progressed to a tumor lobule with intact basement membrane. An association between FcaPV-5 and the cutaneous lesions was confirmed by detection of virus antigen and genes using immunohistochemistry, polymerase chain reaction (PCR), sequencing analysis, and in situ hybridization. Based on these findings, the lesions were diagnosed as FcaPV-5-associated viral plaques and Bowenoid in situ carcinoma (BISC). To date, this is the first reported case of FcaPV-5 infection in a cat in Japan, and the second case reported worldwide. For the first time this papillomavirus type is associated with BISC development.

Full genome analysis of bovine papillomavirus type 1 derived from a calf with severe cutaneous multiple papillomatosis.

Severe papillomatosis occasionally causes astasia leading to euthanizing cattle. There are currently a limited number of reports on virologic approach in severe bovine papillomatosis. Here we report a full genome characterization of bovine papillomavirus type 1 (BPV-1) from the case of severe papillomatosis. A calf developed numerous papillomas on the skin and some nodules in the upper gastrointestinal tract at seven months old. The skin lesion was diagnosed as the epithelial papilloma with BPV antigen expression, while the gastrointestinal lesions were diagnosed as the fibropapilloma without BPV antigen. Full genome analysis revealed that BPV-1s detected in all the lesions were exactly the same. Compared with the reference BPV-1 sequence, there was a single nucleotide insertion in the upstream regulatory region.

Detection of felis catus papillomavirus type 3 and 4 DNA from squamous cell carcinoma cases of cats in Japan.

Association of felis catus papillomaviruses (FcaPVs) with feline squamous cell carcinoma (SCC) has been reported worldwide, while there is limited information about FcaPVs in Asia. In this study, 21 feline SCC biopsy samples from cats in Japan were analyzed by PCR with PV consensus primers and type-specific primers for FcaPV type 2 (FcaPV-2), FcaPV-3 and FcaPV-4 designed in this study. Sequence analysis revealed that one sample was FcaPV-3, and two were FcaPV-4. In both FcaPV-4 positive samples, 334th tryptophan in L1 ORF was deleted compared with the reference sequence. Moreover, immunohistochemistry showed that p16 protein was positive in both FcaPV-4 detected samples. This study would contribute to the molecular epidemiological and pathological understanding of FcaPV in Japan.