Ovine papillomaviruses: Diversity, pathogenicity, and evolution.

The family Papillomaviridae includes a plethora of viral species infecting virtually all vertebrates excluding amphibians, with astonishing impact on human and animal health. Although more than 250 species have been described in humans, the total number of papillomaviruses (PVs) discovered in animals does not reach up to this number. In animals, PV infections are mostly asymptomatic or can cause variable clinical conditions ranging from self-limiting papillomas and other cutaneous and mucosal benign lesions to cancer. Most of animal PV types have been discovered in cattle, dogs, horses, and cats with other farm host species remaining overlooked. In particular, the number of PV types so far identified in sheep is limited. This paper comprehensively reviews ovine PVs features, including viral taxonomy and evolution; genome organization; viral tropism and pathogenesis; macroscopical features and histopathological patterns, as well as available diagnostics tools. Data are critically presented and discussed in terms of impact on veterinary and public health. The development of future dedicated research is also discussed.

Evidence of a novel cross-species transmission by ovine papillomaviruses.

Ovine papillomavirus (OaPV) comprises four genotypes; OaPV1, OaPV2 and OaPV4 are fibropapillomaviruses within the genus Deltapapillomavirus, whereas OaPV3 is an epitheliotropic virus that belongs to the genus Dyokappapapillomavirus. To date, all of them have been known to infect sheep only. OaPV1, OaPV2 and OaPV4 have been associated with ovine cutaneous and mucosal fibropapillomas, whereas OaPV3 is a key factor in the squamous cell carcinoma pathway of the sheep skin. Whole blood samples obtained from 128 cattle at public slaughterhouses were investigated using droplet digital polymerase chain reaction (ddPCR). ddPCR is a new-generation PCR technique that enables an accurate and absolute quantification of target molecules with high sensitivity and specificity. All OaPVs were detected by identification and quantification of nucleic acids using specific fluorescent probes. Of 128 blood samples, 100 (∼78%) showed OaPV infections. Further, 42, 35 and 23 blood samples showed single, double and triple OaPV infections, respectively. OaPV1 was responsible for 22 single infections, OaPV2 caused 16 single infections and OaPV3 and OaPV4 caused two single infections each. OaPV1 and OaPV2 were the most frequent ovine viruses in dual and triple infections. In many blood samples, both ovine deltapapillomavirus and dyokappapapillomavirus were found to be transcriptionally active, as shown by the detection and quantification of E5 oncogene transcripts for OaPV1, L1 transcripts for OaPV2, E6 and E7 transcripts for OaPV3 and E6 for OaPV4. OaPVs were found in the blood samples from cattle that shared grasslands rich in bracken ferns known to contain immunosuppressant substances. Furthermore, OaPVs were also found in cattle from intensive livestock farming without any contact with sheep. Because OaPV DNA was detected in both grass hay and corn silage, it is conceivable that these feed may be the viral sources.

Molecular detection and genetic diversity of bovine papillomavirus in dairy cows in Xinjiang, China.

BACKGROUND: Bovine papillomatosis is a type of proliferative tumor disease of skin and mucosae caused by bovine papillomavirus (BPV). As a transboundary and emerging disease in cattle, it poses a potential threat to the dairy industry. OBJECTIVES: The aim of this study is to detect and clarify the genetic diversity of BPV circulating in dairy cows in Xinjiang, China. METHODS: 122 papilloma skin lesions from 8 intensive dairy farms located in different regions of Xinjiang, China were detected by polymerase chain reaction. The genetic evolution relationships of various types of BPVs were analyzed by examining this phylogenetic tree. RESULTS: Ten genotypes of BPV (BPV1, BPV2, BPV3, BPV6, BPV7, BPV8, BPV10, BPV11, BPV13, and BPV14) were detected and identified in dairy cows. These were the first reported detections of BPV13 and BPV14 in Xinjiang, Mixed infections were detected, and there were geographical differences in the distribution of the BPV genotypes. Notably, the BPV infection rate among young cattle (< 1-year-old) developed from the same supply of frozen sperm was higher than that of the other young cows naturally raised under the same environmental conditions. CONCLUSIONS: Genotyping based on the L1 gene of BPV showed that BPVs circulating in Xinjiang China displayed substantial genetic diversity. This study provided valuable data at the molecular epidemiology level, which is conducive to developing deep insights into the genetic diversity and pathogenic characteristics of BPVs in dairy cows.

The diagnostic value of the droplet digital PCR for the detection of bovine deltapapillomavirus in goats by liquid biopsy.

In the present study, the highly pathogenic bovine deltapapillomavirus (δPV) was investigated by liquid biopsy in blood samples of 168 clinically normal goats using both droplet digital PCR (ddPCR) and quantitative real-time PCR (qPCR). Overall, ddPCR discovered BPV E5 DNA in ~ 61.3% of the blood samples examined, while real-time qPCR revealed the virus in ~ 10.7% of the same samples. Moreover, ddPCR showed BPV E5 DNA in ~ 78.8% of blood samples from goats that were in close contact with cattle and in 20% of blood samples from goats living in closed pens without any contact with cattle. In addition, ddPCR unmasked a single BPV genotype in ~ 59.2% and multiple genotypes in ~ 40.8% of goats harbouring BPV DNA, while real-time qPCR detected single genotypes in ~ 17% and multiple genotypes in ~ 1%. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes and six (~14%) four genotypes. In contrast, real-time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three or four genotypes. BPV2 and BPV13 were the most prevalent viruses responsible for single and multiple co-infections, respectively. The present study showed that ddPCR is promising method for circulating bovine papillomavirus DNA detection and quantification and suggested that animal husbandry practices contribute to cross-species transmission of BPVs.

A Comprehensive Study of Cutaneous Fibropapillomatosis in Free-Ranging Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus): from Clinical Manifestations to Whole-Genome Sequencing of Papillomaviruses.

Papillomaviruses (PVs) are an extremely large group of viruses that cause skin and mucosa infections in humans and various animals. In roe deer and red deer, most PVs belong to the Deltapapillomavirus genus and cause neoplastic changes that are generally described as fibropapillomas. Despite the wide distribution of roe and red deer throughout Europe and beyond, the data in the scientific literature regarding the widespread distribution of PVs and the genetic variability of PV genomes in these species are rather scarce. This study describes cutaneous fibropapillomatosis cases in roe and red deer with clinical manifestations that are typical of infections with PVs. In all cases, the presence of PV DNA was confirmed using PCR, followed by Sanger sequencing of the partial L1 gene. The complete PV genomes were determined in all the investigated samples using next-generation sequencing technology, revealing infections of roe deer with the CcaPV1-type and red deer with the CePV1v-type variant. A comparison of the complete CcaPV1-type and CePV1v-type variant genome sequences reported here with already available complete genome sequences in GenBank revealed their great genetic stability across time and space.

Transforming properties of ovine papillomaviruses E6 and E7 oncogenes.

An increasing number of studies suggest that cutaneous papillomaviruses (PVs) might be involved in skin carcinogenesis. However, only a few animal PVs have been investigated regard to their transformation properties. Here, we investigate and compare the oncogenic potential of 2 ovine Delta and Dyokappa PVs, isolated from ovine skin lesions, in vitro and ex vivo. We demonstrate that both OaPV4 (Delta) and OaPV3 (Dyokappa) E6 and E7 immortalize primary sheep keratinocytes and efficiently deregulate pRb pathway, although they seem unable to alter p53 activity. Moreover, OaPV3 and OaPV4-E6E7 expressing cells show different shape, doubling time, and clonogenic activities, providing evidence for a stronger transforming potential of OaPV3 respect to OaPV4. Also, similarly to high-risk mucosal and cutaneous PVs, the OaPV3-E7 protein, constantly expressed in sheep squamous cell carcinomas, binds pRb with higher affinity compared to the E7 encoded by OaPV4, a virus associated to fibropapilloma. Finally, we found that OaPV3 and OaPV4-E6E7 determine upregulation of the pro-proliferative proteins cyclin A and cdk1 in both human and ovine primary keratinocytes. Collectively, results provide evidence for implication of ovine PVs in cutaneous proliferative lesions and skin cancer progression, and indicate sheep as a possible animal model for the study of cutaneous lesions and malignancies.

Detection and Characterization of Okapi (Okapia johnstoni)-specific Papillomavirus type 1 (OjPV1).

Papillomavirus-specific DNA was detected in skin lesions collected from an okapi (Okapia johnstoni) in the Zoo Basel. According to the nucleotide sequence analysis, the virus belongs to the genus Deltapapillomavirus. Based on bioinformatics analysis, we propose to designate the newly identified virus as Okapia johnstoni Papillomavirus type 1 (OjPV1). OjPV1 is genetically most closely related to a recently described giraffe (Giraffa camelopardalis) -specific papillomavirus (GcPV1). Of note, the putative oncogenic E5 proteins from OjPV1 and GcPV1 are more conserved than the L1 proteins. This indicates, that the selection pressure on E5 may be more pronounced than that on the otherwise most conserved major capsid protein L1.

[Feline sarcoid in a 1-year-old domestic short-haired cat caused by bovine papillomavirus type 14 in Switzerland]. / Felines Sarcoid bei einer 1-jährigen europäischen Hauskatze ausgelöst durch bovines Papillomavirus Typ 14 in der Schweiz.

INTRODUCTION: A 1-year- old domestic short haired cat, living on a farm in Switzerland, was presented to the veterinarian with a 5 cm in diameter mass, bulging from her left nostril. The mass was only incompletely removed because of its unfavourable location. Histologically, the lesion consisted of an infiltrative growing spindeloid proliferation in close approximation to the epidermis and was diagnosed as a feline sarcoid tumour. The presence of Bovine Papillomavirus type 14 (BPV-14) specific DNA could be identified in the tissue by using two PCR assays. The amplified sequences of 194 and 549 base pairs (bp) were 99% and 100% identical with a virus isolated after autopsy, from a cat with feline sarcoid in the USA. The cat recovered completely after an even incomplete surgical excision and no recurrence could be observed 10 months later.

Host cell tropism, genome characterization, and evolutionary features of OaPV4, a novel Deltapapillomavirus identified in sheep fibropapilloma.

Investigating papillomavirus (PV) diversity is crucial to fully comprehend pathogenicity, genetic features, and evolution of taxa hosted by domestic and wild animal species. This study reports the identification of OaPV4, a novel ovine PV type within Deltapapillomaviruses 3. The study of OaPV4 genomic features combined to in situ hybridization and immunohistochemistry investigations allowed extrapolating several general biological features of ovine PVs, such as their cellular tropism, pathogenicity, and evolutionary history. Based on results, ovine PVs can be grouped into a polyphyletic ancient group of viruses, which splits in two main subgroups having peculiar cellular tropism and pathogenicity. Results add up to animal PV diversity and are crucial to future studies aimed to investigate the correlation between animal PV and cutaneous benign and malign proliferations.

Identification of a novel species of papillomavirus in giraffe lesions using nanopore sequencing.

Papillomaviridae form a large family of viruses that are known to infect a variety of vertebrates, including mammals, reptiles, birds and fish. Infections usually give rise to minor skin lesions but can in some cases lead to the development of malignant neoplasia. In this study, we identified a novel species of papillomavirus (PV), isolated from warts of four giraffes (Giraffa camelopardalis). The sequence of the L1 gene was determined and found to be identical for all isolates. Using nanopore sequencing, the full sequence of the PV genome could be determined. The coding region of the genome was found to contain seven open reading frames (ORF), encoding the early proteins E1, E2 and E5-E7 as well as the late proteins L1 and L2. In addition to these ORFs, a region located within the E2 gene is thought, based on sequence similarities to other papillomaviruses, to encode an E4 protein, although no start codon could be identified. Based on the sequence of the L1 gene, this novel PV was found to be most similar to Capreolus capreolus papillomavirus 1 (CcaPV1), with 67.96% nucleotide identity. We therefore suggest that the virus identified here is given the name Giraffa camelopardalis papillomavirus 1 (GcPV1) and is classified as a novel species within the genus Deltapapillomavirus, in line with the current guidelines for the nomenclature and classification of PVs.

Detection of bovine papillomavirus type 14 DNA sequences in urinary bladder tumors in cattle.

Bovine papillomavirus type 14 (BPV-14) is a novel Deltapapillomavirus (δPV) which is most closely related to BPV-1, -2, and -13, well-known members of the δPV genus. So far BPV-14 has been detected in cutaneous neoplastic lesions in cattle and in feline sarcoids. As BPV-14 may share biological and pathological properties with BPV-1, -2 and -13, it has been hypothesized that, like other δPVs, BPV-14 could be associated with bovine bladder neoplasia. In this study, 50 tumors of the urinary bladder of cattle were diagnosed. DNA was extracted from all tumor samples as well as from 25 normal bladder samples and submitted to BPV-14 L1 PCR and subsequent amplicon sequencing analysis. BPV-14 L1 DNA sequences of specific 195bp amplicons were obtained from 17 of 50 (34%) tumor DNA isolates; no BPV-14 DNA was detected from 25 normal samples. Amplicons revealed a 99% homology with the corresponding BPV-14 L1 DNA region (GenBank accession number KP276343.1). Co-infections by two or three δPV types were also seen. This study reveals the presence of BPV-14 DNA alone or in combination with other δPV DNA in bovine bladder tumors alone and suggests that BPV-14 could also be involved in bladder neoplasia as its E5 oncoprotein has the potential to induce cell proliferation. Furthermore, this is the first study to show the presence of BPV-14 in Europe, suggesting that BPV-14, like other δPVs, has a worldwide distribution.

Genomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bos taurus papillomavirus type 14.

Feline sarcoids are rare mesenchymal neoplasms of domestic and exotic cats. Previous studies have consistently detected short DNA sequences from a papillomavirus (PV), designated feline sarcoid-associated papillomavirus (FeSarPV), in these neoplasms. The FeSarPV sequence has never been detected in any non-sarcoid sample from cats but has been amplified from the skin of cattle suggesting that feline sarcoids are caused by cross-species infection by a bovine papillomavirus (BPV). The aim of the present study was to determine the genome of the PV that contains the FeSarPV sequence. Using the circular nature of PV DNA, four specifically designed 'outward facing' primers were used to amplify two approximately 4,000 bp DNA segments from a feline sarcoid. The two PCR products were sequenced using next generation sequencing and the full genome of the PV, consisting 7,966 bp, was assembled and analysed. Phylogenetic analysis revealed the PV was closely related to the species 4 delta BPVs-1, -2, and -13, but distantly related to any carnivoran PV genus. These results are consistent with feline sarcoids being caused by a BPV type and we propose a classification of BPV-14 for this novel PV. Initial analysis suggests that, like other delta BPVs, the BPV-14 E5 protein could cause mesenchymal proliferation by binding to the platelet derived growth factor beta receptor. Interestingly BPV-14 has not been detected in any equine sarcoid suggesting that BPV-14 has a host range that is limited to bovids and felids.

Development of a novel PCR-RFLP assay for improved detection and typing of bovine papillomaviruses.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and type bovine papillomaviruses (BPVs) from tumors in cattle. Two degenerate primer sets targeting the BPV L1 gene, subAup/subAdw and subBup/subBdw, and one restriction enzyme RsaI were used in this assay. In silico analyses of the restriction enzyme sites in the PCR fragments of 13 BPV sequences (BPV-1 to -13) revealed that all known BPVs are differentiated by the PCR-RFLP assay. Analyses of 63 previously typed clinical samples, that included teat papillomas and both esophageal and urinary bladder cancer biopsies, show that the assay clearly differentiates between eight clinically important BPV types (BPV-1 to -6, -9, -10), and discriminates between single and multiple infections. To further assess the reliability of the PCR-RFLP method amplified fragments were sequenced. A high correlation (95%) was observed when the results of the PCR-RFLP method were compared with PCR-sequencing. Differences in typing occurred for 3 of 63 specimens; PCR-RFLP identified additional BPV types in these specimens, while the PCR-sequencing identified only one. These results indicate that the PCR-RFLP method reported here is simpler and more reliable in the detection and typing of BPVs from bovine tumor samples than PCR-sequencing.

Phylogenetic classification and clinical aspects of a new putative Deltapapillomavirus associated with skin lesions in cattle.

Bovine papillomaviruses (BPVs) are recognized as causal agents of benign and malignant tumors in cattle. Thirteen types of BPVs have already been described and classified into 3 distinct genera. Divergences in the nucleotide sequence of the L1 gene are used to identify new viral types through the employment of PCR assays with degenerated primers. In the present study, a method for identifying BPVs based on PCR-RFLP and DNA sequencing allowed the identification of a new putative Deltapapillomavirus, designated JN/3SP (JQ280500.1). The analysis of the L1 gene showed that this strain was most closely related to the BPVs -1, -2, -13 , and OaPV1 (71-73% genetic similarity). In this study, we describe the detection of this new putative Deltapapillomavirus type and verify its phylogenetic position within the genus.

Bovine papillomavirus type 10 with a deletion associated with a lingual papilloma in a cow.

Different types of papillomavirus usually cause papillomas in specific tissues. Previously, bovine papillomavirus (BPV) type 10 has been associated specifically with cutaneous papillomas in cattle. In this study, BPV-10 was detected in a papilloma on the tongue of a cow. Whole genome analysis demonstrated that the sequence of this BPV-10 strain had a 129 base pair deletion in the E1 open reading frame, which was confirmed by Southern blot analysis, PCR and reverse transcriptase-PCR.

Bovine papillomavirus in Brazil: detection of coinfection of unusual types by a PCR-RFLP method.

Bovine papillomavirus (BPV) is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil.

The E5 proteins.

The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 protein is the PDGF ß receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis.

Genetic characterization of a novel bovine papillomavirus member of the Deltapapillomavirus genus.

This report describes the complete genomic sequence and taxonomic position of BPV type 13. The BPV13 genome was amplified using the multiply primed rolling-circle amplification technique and long-template PCR employing two specific primers. The two long PCR fragments obtained were cloned and sequenced via primer walking. The complete genomic sequence of the BPV13 contains 7961 bp encoding eight proteins, E1, E2, E4, E5, E6, E7, L1, and L2. Similarly to the E5 gene in BPVs 1 and 2, the putative BPV13 E5 ORF encodes a small transforming protein that contains a hydrophobic transmembrane domain. Meanwhile, the retinoblastoma tumor suppressor-binding domain is absent in the putative BPV13 E7 protein. The presence of these two specific molecular features has been recognized as a distinct marker for the development of fibropapilloma in artiodactyl PV-induced lesions. The phylogenetic analysis demonstrated that BPV13 is a new member of the Deltapapillomavirus genus, to be classified as the third representative of the Delta 4 species. The characterization of the genomic sequence of this novel PV will aid in the interpretation of the pathologies described to be related to this virus and provide support for the development of diagnostic tools for epidemiological surveillance of BPV13 in its potential natural hosts.

Initial detection of a circular genome deletion in a naturally bovine papillomavirus-infected sample.

Bovine papillomavirus type 12 (BPV-12) was recently identified in epithelial papillomas on the cattle tongue in our previous study. Along with the full-length genome, one deleted circular genome, named BPV-12-del, was detected in the same papilloma lesion. BPV-12-del is 3363 base pairs in length, and a total of 3,384 base pairs, including E1, E2 and E4 genes and partial E7 and L2 ORFs, were deleted compared with the complete genome. Real-time PCR results showed that the percentage of BPV-12-del was 42% of the total genomes in the sample. Southern Blot analysis also confirmed the presence of the deleted genome. This is the first report describing a circular genome deletion detected in a naturally BPV-infected sample.

Bos grunniens papillomavirus type 1: a novel deltapapillomavirus associated with fibropapilloma in yak.

Papillomaviruses (PVs) have been widely identified among vertebrates, but have not yet been reported to infect yaks. We report, for the first time, a novel deltapapillomavirus that was associated with fibropapilloma in yak herds on the Qinghai-Tibetan Plateau. Six skin papilloma samples were collected and examined using histopathology, immunohistochemistry and PCR assays. The samples were identified as fibropapilloma and were found to contain PV antigens. Sequencing of diagnostic PCR products and the full-length genome revealed that all samples were infected with the same PV type. The whole virus genome was 7946 bp in length and possessed the common PV genomic organization. The virus was identified as a novel PV type and designated Bos grunniens papillomavirus type 1 (BgPV-1) based on the nucleotide sequence alignment of the L1 ORF. It is classified in the Delta-4 species of the genus Deltapapillomavirus based on phylogenetic analysis of the L1 ORF. Identification of this novel PV type provides further information about the pathology, development of diagnostic methods and evolutionary studies of the family Papillomaviridae.