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.

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.

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.

Rusa alfredi papillomavirus 1 - a novel deltapapillomavirus inducing endemic papillomatosis in the endangered Visayan spotted deer.

We describe a novel papillomavirus - Rusa alfredi papillomavirus 1 (RalPV1) - which causes endemic fibropapillomatosis in the European conservation breeding population of the highly endangered Visayan spotted deer (Rusa alfredi). Degenerated papillomavirus-specific primers were used to amplify and sequence parts of the viral DNA. Subsequently, the complete genomic DNA was cloned and the sequence was determined. The RalPV1 genome has a length of 8029 bp, encodes the early proteins E6, E7, E1, E2 and E5, the two late proteins L1 and L2 and contains an upstream regulatory region. Highest sequence identities were observed with two deltapapillomaviruses, the Capreolus capreolus PV1 and Cervus elaphus PV1. Pairwise comparisons and phylogenetic analysis based on the ORF L1 suggested that RalPV1 is a putative new type of the papillomavirus species Deltapapillomavirus 5.

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.

Spontaneous cutaneous papillomatosis in yaks and detection and quantification of bovine papillomavirus-1 and -2.

Seven clinical cases of cutaneous papillomatosis in yaks were studied in Arunachal Pradesh, India. Sporadic, single or a chain of multiple varying size warts appeared around the eyes or on the body. Predominant site of warts was around eyes. Histopathologically, these cases were diagnosed as fibropapilloma. It was confirmed by the detection of BPV-1 and BPV-2 or their mixed infection by PCR and sequencing. Quantitative SYBR Green real-time PCR detected comparatively lower viral DNA copy number in cutaneous warts (CWs). Cases of CWs and its causative agent as bovine papillomavirus (BPVs) are reported for the first time in yaks.

Prevalence of BPV genotypes in a German cowshed determined by a novel multiplex BPV genotyping assay.

Bovine papillomaviruses (BPV) induce benign tumours of the cutaneous or mucosal epithelia in cattle, but are also involved in the development of cancer of the urinary bladder and of the upper gastrointestinal tract. Current BPV genotyping assays employ techniques developed originally for the detection of human papillomaviruses. These methods rely on consensus PCR amplification and subsequent sequencing and are cumbersome and limited in their analytic sensitivity to detect BPV, especially in multiple infections. In this study, a novel multiplex BPV genotyping assay is described to detect sensitively and specifically BPV-1 to -10 as well as BaPV-11. The assay is based on a multiplex PCR using novel broad-spectrum bovine papillomavirus (BSBP) primers followed by multiplex bovine genotyping (MBG) by Luminex xMAP technology. The detection limit of the assay was shown to be between 10 and 100 BPV genomes. In a first application, BPV was detected in 100% of wart preparations with BPV-8 being most prevalent, followed by types 6, 1 and 10. The majority of warts were positive for at least four BPV types. In conclusion, BSBP-PCR/MBG is a powerful high-throughput method suitable for the study of the natural history of BPV and could be useful to veterinarians for the monitoring of the efficacy of future BPV vaccines.

Molecular identification of bovine papillomaviruses associated with cutaneous warts in southern Brazil.

Bovine papillomaviruses (BPVs) are widespread pathogens mainly associated with benign, self-limiting, cutaneous lesions (warts). At least 8 viral types, defined by serology or nucleotide sequences of the L1 gene, have been identified to date. Different serotypes are associated with the specific type and morphology of the lesion and with particular geographical regions. This article describes the molecular identification of papillomaviruses from Brazilian cattle (n = 48) and horses (n = 1) through partial amplification and sequencing of the L1 gene. Bovine papillomavirus-1 (BPV-1) was identified in warts from 29 cattle (59%), BPV-6 from 9 cattle (18%), and BPV-2 in 8 lesions (16%). Warts of 2 cattle harbored L1 sequences of a new BPV type (BAA5), otherwise identified almost exclusively in healthy skin. The newly proposed BPV type "BR-UEL-4" was identified in a sarcoid tumor of a horse. Thus, the present report provides information on the main types of BPV involved in bovine papillomatosis in Brazil and reveals a new viral type associated with equine sarcoid, which to date has been attributed exclusively to BPV-1 and BPV-2.

Identification of unreported putative new bovine papillomavirus types in Brazilian cattle herds.

The amplification by degenerate primers FAP59/FAP64 and sequencing allowed the detection of 15 putative new BPV types in cutaneous warts as well as in healthy skin. Four of these isolates were recently recognized as new BPV types (BPV-7, -8, -9, and -10) after determination of their complete genome sequences. In Brazil, investigations involving the definition of BPV types present in skin warts are still rare. The aim of the current study was to identify the BPV types associated with cutaneous papillomatosis observed in Brazilian cattle herds. Twenty-two cutaneous papilloma specimens were submitted to PCR assay employing the FAP primer pair. All PCR products with approximately 480 bp were submitted to direct sequencing. Cloning was performed for the amplicons which prior analysis revealed as putative new BPV types. From 16 cutaneous lesions, BPV-1, -2, and -6 were identified in two, six, and eight papilloma specimens, respectively. In addition, four putative new BPV types were identified in other six skin warts, and then designated as BPV/BR-UEL2 to -5. The detection of the BPV-1, -2, and -6 types in skin wart specimens supports the existence of these BPV types throughout the Brazilian cattle herd. In addition, the identification of four putative new BPV types is the first report of the presence of different BPV types in the American continent.

Characterisation of the first complete genome sequence of the roe deer (Capreolus capreolus) papillomavirus.

The complete genomic DNA of a novel roe deer (Capreolus capreolus) papillomavirus (CcPV1) was amplified and sequenced from fibropapillomatous skin lesions of a Hungarian roe deer. Viral DNA was detected by a pair of degenerate primers and the remaining genomic sequence was amplified by a long-template high-fidelity PCR and sequenced. The CcPV1 genome was 8032 bp long and contained open reading frames (ORFs) typical for Delta-papillomaviruses (E6, E7, E1, E2, E4, E5, E9, L2, and L1) and a 799 bp long untranslated regulatory region (URR). Phylogenetic analysis based on the 3861 bp long concatenated sequence of the E1-E2-L2-L1 ORFs and on separate alignments of all major ORFs using both neighbour-joining and maximum parsimony methods placed CcPV1 on a distinct branch between Ovine papillomavirus 1 and the other deer papillomaviruses within the Delta-papillomavirus genus, although pairwise nucleotide alignments of L1 ORF sequences determined highest identities with European Elk Papillomavirus (71.2%) and Reindeer Papillomavirus (70.3%).