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.     

Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States

OBJECTIVE: To determine the incidence of bovine papillomavirus (BPV) type 1 or 2 in sarcoids and other samples of cutaneous tissues collected from horses in the western United States. ANIMALS: 55 horses with sarcoids and 12 horses without sarcoids. PROCEDURE: Tissue samples (tumor and normal skin from horses with sarcoids and normal skin, papillomas, and nonsarcoid cutaneous neoplasms from horses without sarcoids) were collected. Tissue samples were analyzed for BPV-1 or -2 DNA, using a polymerase chain reaction (PCR) and restriction fragment length polymorphism. The PCR products from 7 sarcoid-affected horses were sequenced to evaluate percentage homology with expected sequences for BPV-1 or-2. RESULTS: Most (94/96, 98%) sarcoids contained BPV DNA. Sixty-two percent of the tumors examined had restriction enzyme patterns consistent with BPV-2. Thirty-one of 49 (63%) samples of normal skin obtained from horses with sarcoids contained BPV DNA. All samples subsequently sequenced had 100% homology with the expected sequences for the specific viral type. All tissues from healthy horses, nonsarcoid neoplasms, and papillomas were negative for BPV DNA. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine papillomaviral DNA was detected in essentially all sarcoids examined. There appears to be regional variation in the prevalence of viral types in these tumors. The fact that we detected viral DNA in normal skin samples from horses with sarcoids suggests the possibility of a latent viral phase. Viral latency may be 1 explanation for the high rate of recurrence following surgical excision of sarcoids.     

Bovine papillomavirus infection in equine sarcoids and in bovine bladder cancers

Bovine papillomavirus (BPV) type 2 is involved in carcinogenesis of the urinary bladder in cattle, while BPV-1 is commonly associated with equine sarcoid tumours. In both cases the early viral proteins are expressed, but virion is not produced. Given the similarities in BPV biology between the tumours in cattle and horses, bovine bladder cancers and equine sarcoids were compared with respect to physical status, load of viral DNA and variability of the E5 open reading frame (ORF). Rolling circle amplification demonstrated that BPV-1 and BPV-2 genomes exist as double stranded, episomal, circular forms in the two tumours. Realtime quantitative PCR revealed that equine sarcoids contained higher viral DNA loads compared to bovine bladder cancers. The BPV-1 E5 ORF showed sequence variation but BPV-2 ORF did not. The presence of BPV-1 E5 variations or their absence in the BPV-2 E5 ORF does not appear to have an effect on viral DNA load in either tumour type.     

Lack of Correlation Between Papillomaviral DNA in Surgical Margins and Recurrence of Equine Sarcoids

Bovine papillomavirus (BPV) types 1 (BPV-1) and 2 (BPV-2) are causally associated with the development of equine sarcoid tumors. Recurrence rates after surgical excision of sarcoids are estimated to be 30%–40%. We hypothesized that the presence of BPV DNA in histologically tumor-free surgical margins of sarcoids is associated with risk of recurrence, and increased quantity of BPV DNA is associated with increased risk of recurrence. Formalin-fixed sarcoids classified as “completely excised” histologically were obtained from two institutions. A total of 25 tumors were included, eight of which recurred within 1 year of excision. Qualitative and quantitative polymerase chain reaction (PCR) tests for detection of BPV-1 and BPV-2 were performed on neoplastic tissue and tumor-free surgical margins in formalin-fixed paraffin-embedded biopsy specimens following DNA extraction. Bovine papillomavirus-1 was found in all tumor samples and in histologically “clean” margins of 21 samples, whereas BPV-2 was found in only two tumor samples. Although quantitative PCR was more sensitive than qualitative PCR in detecting BPV DNA in surgical margins, there was no significant difference in the presence of BPV-1 or BPV-2 DNA in margins of tumors that recurred versus those that did not recur for either test. Although this study is limited by sample size, our results suggest that PCR analysis of surgical margins for BPV DNA is not a reliable method to predict equine sarcoid recurrence after resection.     

Detection of bovine papillomavirus DNA in equine sarcoids using the polymerase chain reaction (PCR)]

Unfixed and formalin-fixed frozen sections and paraffin-sections of histopathologically confirmed sarcoids of 20 horses were studied in the PCR. The used set of primers was located in the E5 open reading frame fitting both to bovine papillomavirus 1 (BPV-1) and BPV-2. Independent of the quality of the used tissues BPV-DNA was detected in all 20 sarcoids. By cleaving with restriction endonuclease Bst XI it was shown that the DNA-sequences amplified by PCR were identical with that of BPV-1. The results support the general view that BPV play an important role in equine sarcoids.     

Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors

Bovine papillomavirus (BPV)-1 and -2 is linked to equine sarcoids, a commonly observed skin tumor in horses that is of considerable veterinary importance. Previous studies using in situ hybridization have detected BPV DNA only in fibroblasts and not in keratinocytes of sarcoids. In contrast, normal equine skin latently infected with BPV shows a dysplastic epithelium without dermal changes, similar to lesions induced by other papillomavirus types infecting the epithelium. The first goal of our study was to describe the epidermal and dermal characteristics of several stages in sarcoid development. Next, we explored whether BPV can infect epidermal cells in the horse using real-time PCR on laser-micro-dissected keratinocytes and fibroblasts. We found that latently infected normal skin samples and a subset of early stage sarcoids show dysplastic, koilocyte-like epithelial changes. BPV DNA was detected in keratinocytes in 40% of the samples with these particular epithelial properties, whereas advanced sarcoids only had BPV DNA in the fibroblasts. These data may indicate a novel and intriguing pathway of BPV infection in the horse composed of a first step of keratinocyte infection, followed by migration of viral material towards the dermis resulting in infection of sub-epidermal fibroblasts and their fully transformed phenotype. Additionally, an example of co-existence of a dermal BPV-1 and an epidermal BPV-2 infection in the same lesion is shown, indicating that horses can harbor infection with more than one BPV type at the same time.     

牛乳头瘤病毒基因1型广西GX01流行株全基因组克隆及序列分析

为了解牛乳头瘤病毒1型（bovine papillomavirus genotype 1,BPV-1）广西GX01株全基因组序列、结构特征及遗传变异情况,同时了解该毒株引起宿主产生的病理组织学变化情况,本研究选取广西贺州市患病牛皮肤肿瘤样物制作石蜡切片后镜检观察,提取病料DNA,以乳头瘤病毒L1基因的简并引物FAP59/FAP64进行PCR扩增以确定此病毒的基因型,根据GenBank中BPV参考株设计嵌套引物,对GX01株进行全基因组扩增、克隆测序及序列分析。病理组织学检查结果显示,可在病变部位发现表皮细胞增生、肿胀,角质过度及挖空细胞等乳头瘤病毒感染的特征性病变。序列分析结果表明,GX01株为BPV-1,其全基因组长为7 945 bp,包含E1、E2、E4、E5、E6、E7、L1、L2 8个开放阅读框,符合BPV-1型基因组的结构特征;GX01与BPV-1参考株全基因组核苷酸序列同源性为98.6%~99.6%,与BPV-2型参考株（M20219.1）、BPV-13型参考株（JQ798171.1）同源性分别为86.9%和87.2%。GX01株为广西地区首次经检测确认并测定全基因组序列的牛乳头瘤病毒。本研究为广西地区乃至全国的牛乳头状瘤的病原鉴定、流行规律、遗传变异、疫源追溯及科学防控提供了基础数据。     

Equine sarcoid of the glans penis with bovine papillomavirus type 1 in a miniature horse (Falabella)

A 23-year-old Falabella gelding kept in Tochigi, Japan, for more than 20 years presented with a recurrent mass of the glans penis that was first noticed about a year earlier. Partial phallectomy was performed with no adjunctive therapy for local regrowth of the mass. The horse was euthanized 3 months after surgery for urinary retention due to suspected regrowth. The resected mass affected the genital and urethral mucosa of the glans penis, and was diagnosed as equine sarcoid by histopathology and identification of bovine papillomavirus (BPV) DNA. Phylogenetic analysis of the BPV genome of the sarcoid showed high sequence homology to BPV type 1 (BPV-1) from Hokkaido, Japan, suggesting a geographical relationship for BPV-1 in Japan.     

Complete genome and phylogenetic analysis of bovine papillomavirus type 15 in Southern Xinjiang dairy cow

BackgroundBovine papilloma is a neoplastic disease caused by bovine papillomaviruses (BPVs), which were recently divided into 5 genera and at least 24 genotypes.ObjectivesThe complete genome sequence of BPV type 15 (BPV Aks-02), a novel putative BPV type from skin samples from infected cows in Southern Xinjiang China, was determined by collecting warty lesions, followed by DNA extraction and amplicon sequencing.MethodsDNA was analyzed initially by polymerase chain reaction (PCR) using the degenerate primers FAP59 and FAP64. The complete genome sequences of the BPV Aks-02 were amplified by PCR using the amplification primers and sequencing primers. Sequence analysis and phylogenetic analysis were performed using bio-informatic software.ResultsThe nucleotide sequence of the L1 open reading frame (ORF) of BPV Aks-02 was 75% identity to the L1 ORF of BPV-9 reference strain from GenBank. The complete genome consisted of 7,189 base pairs (G + C content of 42.50%) that encoded 5 early (E8, E7, E1, E2, and E4) and 2 late (L1 and L2) genes. The E7 protein contained a consensus CX₂CX₂₉CX₂C zinc-binding domain and a LxCxE motif. Among the different members of this group, the percentages of the complete genome and ORFs (including 5 early and 2 late ORFs) sequence identity of BPV Aks-02 were closer to the genus Xipapillomavirus 1 of the Xipapillomavirus genus. Phylogenetic analysis and sequence similarities based on the L1 ORF of BPV Aks-02 revealed the same cluster.ConclusionsThe results suggest that BPV type (BPV Aks-02) clustered with members of the Xipapillomavirus genus as BPV 15 and were closely related to Xipapillomavirus 1.     

BPV-1 infection is not confined to the dermis but also involves the epidermis of equine sarcoids

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive.     

Studies on vaccination against papillomaviruses: the immunity after infection and vaccination with bovine papillomaviruses of different types

Calves, free of antibodies to bovine papillomaviruses (BPV), were reared in isolation. One was infected with BPV-2, developed tumours and was resistant to homologous reinfection. Groups of calves were infected with BPV-2, BPV-5 or BPV-6; they all developed and subsequently rejected type-specific tumours. They were then infected with BPV-4; they were not immune and oral papillomas were induced. Groups of animals were vaccinated by intramuscular preparations of purified BPV-4 and BPV-6 and were challenged with homologous virus; all were immune to reinfection. An earlier experiment had shown this to be true for BPV-2. Two calves, immune to BPV-6, were not immune to BPV-1. These experiments, although they do not cover all the possibilities of reciprocal immunisation and challenge, indicate that prophylactic immunity to a range of papillomaviruses is type-specific. This is the first clear demonstration of this phenomenon in the papillomavirus group.     

Safety and immunogenicity of BPV-1 L1 virus-like particles in a dose-escalation vaccination trial in horses

Reasons for performing study: Infection with bovine papillomaviruses types 1 and 2 (BPV‐1, BPV‐2) can lead to the development of therapy‐resistant skin tumours termed sarcoids and possibly other skin diseases in equids. Although sarcoids seriously compromise the welfare of affected animals and cause considerable economic losses, no prophylactic vaccine is available to prevent this common disease. In several animal species and man, immunisation with papillomavirus‐like particles (VLP) has been shown to protect efficiently from papillomaviral infection. Hypothesis: BPV‐1 L1 VLPs may constitute a safe and highly immunogenic vaccine candidate for protection of horses against BPV‐1/‐2‐induced disease. Methods: Three groups of 4 horses each received 50, 100 or 150 µg of BPV‐1 L1 VLPs, respectively, on Days 0, 28 and 168. Three control horses received adjuvant only. Horses were monitored on a daily basis for one week after each immunisation and then in 2 week intervals. Sera were collected immediately before, 2 weeks after each vaccination and one and 2 years after the final boost and analysed by pseudovirion neutralisation assay. Results: None of the horses showed adverse reactions upon vaccination apart from mild and transient swelling in 2 individuals. Irrespective of the VLP dose, all VLP‐immunised horses had developed a BPV‐1‐neutralising antibody titre of ≥1600 plaque forming units (pfu)/ml 2 weeks after the third vaccination. Eight of 10 trial horses still available for follow‐up had neutralising antibody titres ≥1600 pfu/ml one year and ≥800 pfu/ml 2 years after the last immunisation. Conclusion: Intramuscular BPV‐1 L1 VLP vaccination in horses is safe and results in a long‐lasting antibody response against BPV‐1. Neutralisation titres were induced at levels that correlate with protection in experimental animals and man. Potential relevance: BPV‐1 L1 VLPs constitute a promising vaccine candidate for prevention of BPV‐1/‐2‐induced disease in equids.     

Association of bovine papillomavirus type 2 (BPV-2) and urinary bladder tumours in cattle from the Azores archipelago

Urinary bladder tumours in cattle are caused by chronic ingestion of bracken fern and BPV-1/2 infection. The objective of the present study was to assess if BPV-2 was present in urinary bladder lesions from cattle with chronic enzootic haematuria (CEH) from the Azores archipelago (Portugal), in order to gain further information regarding the epidemiologic distribution of this virus. Samples were analysed using PCR specific primers for BPV-2 DNA and an immunohistochemistry for BPV E5 oncoprotein detection. We found a 28% incidence rate of BPV-2 DNA in different types of tumours and cystitis cases (13 out of 46 samples). Tested positive samples for PCR were also positive for the viral E5 oncoprotein; protein immunolabeling was mainly detected within the cytoplasm of urothelial cells, displaying a juxtanuclear distribution. This is the first report of BPV-2 detection in urinary bladder tumours associated with CEH in cattle from the Azores archipelago.     

Consistent detection of bovine papillomavirus in lesions, intact skin and peripheral blood mononuclear cells of horses affected by hoof canker

Reasons for performing the study: Equine hoof canker is a chronic proliferative pododermatitis of as yet unknown aetiology. Like equine sarcoid disease, canker is a therapy‐resistant disorder characterised by hyperkeratosis, acanthosis and a marked tendency to recur. Hypothesis: There is an association of sarcoid‐inducing bovine papillomaviruses of types 1 and 2 (BPV‐1, BPV‐2) with hoof canker disease. Methods: Using PCR‐based techniques, we assessed canker tissue, intact skin and/or peripheral blood mononuclear cells (PBMCs) of 25 canker‐affected horses for the presence of sarcoid‐associated BPV‐1 and ‐2. Results: Conventional PCR revealed BPV‐1/‐2 DNA in 24/24 canker, 12/13 skin and 10/11 PBMC DNA isolates. Using inverse PCR, full‐length BPV episomes were detected in 1/5 canker specimens. Sequencing of viral early and late genes amplified from canker, intact skin and PBMC DNA of 2 cases revealed an overall identity of 98% to BPV‐1. Viral DNA loads amounted to ≤16 copies per cell in canker tissue and intact skin, and to ≤0.35 copies per PBMC, as determined by quantitative PCR. Using RT‐PCR, the viral major oncogene E5 was shown to be transcribed in 2/4 canker tissue specimens and 5/7 PBMC isolates. Immunocapture PCR from 7 canker and 6 skin extract supernatants revealed capsomere‐associated viral DNA in one canker and one skin sample. Hoof tissue, skin and PBMCs collected from 13 individuals with no signs of canker or BPV‐related malignancies scored negative throughout the experiments. Conclusion: These findings suggest that the observed presence of BPV‐1/‐2 in canker‐affected horses is not coincidental but indicative of an active contribution to hoof canker disease. Potential relevance: The use of antivirals and/or immune modulators may help improving canker therapy.     

Detection and characterisation of papillomavirus in skin lesions of giraffe and sable antelope in South Africa

Papillomavirus was detected electron microscopically in cutaneous fibropapillomas of a giraffe (Giraffa camelopardalis) and a sable antelope (Hippotragus niger). The virus particles measured 45 nm in diameter. Histopathologically, the lesions showed histopathological features similar to those of equine sarcoid as well as positive immunoperoxidase-staining of tissue sections for papillomavirus antigen. Polymerase chain reaction (PCR) detected bovine papillomavirus (BPV) DNA. Bovine papillomavirus-1 was characterised by real-time PCR in the sable and giraffe, and cloning and sequencing of the PCR product revealed a similarity to BPV-1. As in the 1st giraffe, the lesions from a 2nd giraffe revealed locally malignant pleomorphism, possibly indicating the lesional end-point of papilloma infection. Neither virus particles nor positively staining papillomavirus antigen could be demonstrated in the 2nd giraffe but papillomavirus DNA was detected by real-time PCR which corresponded with BPV-1 and BPV-2.     

High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoid-affected and healthy horses

Bovine papillomavirus (BPV), the causative agent of papillomas in cattle, has been shown to play a major role in the pathogenesis of equine sarcoids in horses. BPV has also been detected occasionally in normal equine skin. In this study, presence and activity of BPV in normal skin and peripheral blood of 4 groups of horses were evaluated: sarcoid-affected horses, horses living in contact with sarcoid-affected horses, horses living in contact with papilloma-affected cattle and control horses. From each horse, 3 samples on 4 locations were collected: a swab of the intact skin surface and both a swab and a biopsy after decontamination. BPV DNA was found in the normal skin of 24 of 42 horses (57%). Mainly sarcoid-affected horses and horses living in contact with cattle were carriers (73%), but BPV DNA was also detected in 50% of the horses living in contact with sarcoid-affected horses and in 30% of the control population. BPV mRNA was detected in 58% of the samples positive for BPV DNA, although in a much lower quantity compared to sarcoids. In most of the BPV DNA positive samples mild acanthosis, slight basophilic cytoplasmic swelling of the epidermal layers and/or thickening of the basal membrane were noticed, but these observations were also present in several BPV DNA negative normal skin samples. BPV DNA could not be detected in peripheral blood. These findings suggest latent infection and a wide-spread occurrence of BPV in the horse population.     

Polymerase chain reaction analysis of the surgical margins of equine sarcoids for bovine papilloma virus DNA

OBJECTIVE: To examine apparently normal skin around equine sarcoids for evidence of bovine papilloma virus (BPV) DNA, and to relate this finding to the observed recurrence after surgery. STUDY DESIGN: Prospective study. ANIMALS OR SAMPLE POPULATION: Forty-one equine sarcoids from 19 horses. MATERIALS AND METHODS: The tumors were surgically excised at a measured distance of 8, 12, or 16 mm. Samples from the tumor and of the entire surrounding skin were taken at 4, 8, 12, and 16 mm from the tumor border and analyzed for the presence of BPV DNA using polymerase chain reaction (PCR) amplification. The samples were grouped per examined sarcoid, and a tumor was considered positive at a certain distance as soon as at least one of the samples at that distance was positive. The clinical outcome was recorded for each sarcoid after a minimal follow-up of 6 months. RESULTS: All sarcoids were positive for BPV(1) or BPV(2). The tumor margin was positive at 4, 8, 12, and 16 mm in, respectively, 95%, 73%, 39%, and 33% of the examined sarcoids. Local recurrence was observed in 3 sarcoids on 3 different horses. From survival analysis, there was a greater likelihood for local recurrence when sarcoids had a surgical margin that was positive for BPV DNA. CONCLUSIONS AND CLINICAL RELEVANCE: BPV DNA is often detected in visibly normal skin around sarcoids, and there is a significantly greater probability for local recurrence when the surgical margins are positive for the presence of BPV DNA.     

Bovine papillomavirus E5 oncoprotein upregulates parkin-dependent mitophagy in urothelial cells of cattle with spontaneous papillomavirus infection: A mechanistic study

This study aimed to provide mechanistic insights into mitophagy pathway associated with papillomavirus infection in urothelial cells of cattle. The elimination of mitochondria via autophagy, termed mitophagy, is an evolutionarily conserved mechanism for mitochondrial quality control and homeostasis. PINK1/parkin-mediated mitophagy, a ubiquitin-dependent selective autophagy of dysfunctional mitochondria, has been described here, for the first time, in urothelial cells from 25 bladder cancers in cattle infected by bovine papillomavirus (BPV). The expression of BPV-2 and BPV-13 E5 oncoprotein was detected by RT-PCR. Abnormal mitochondria delimited by expanding phagophores, were peculiar ultrastructural features of neoplastic urothelial cells. High levels of mitochondrial phosphorylated PINK1/parkin were observed in neoplastic urothelial cells infected by BPVs. Phosphoparkin interacted with mitofusin 2 (Mfn2) and ubiquitin (Ub), which confirmed that Mfn2 is a parkin receptor at the mitochondrial level, where parkin interacted also with Ub. Furthermore, parkin established a complex that was comprised of optineurin, p62, LC3, laforin, and embryonic stem cell-expressed Ras (ERAS), that interacted with BPV E5 oncoprotein, and Bag3, which, in turn, regulated the formation of a complex composed of Hpc70/Hsp70, CHIP, an HSC70-interacting E3 ubiquitin ligase. It is conceivable that ERAS is involved in mitophagosome maturation via phosphatidylinositol 3-kinase (PI3K) pathway. Bag3, in association with Hsc70/Hsp70, may contribute to the transport and degradation of CHIP-ubiquitinated cargo as this complex recognises ubiquitinated cargos and transports them to aggresomes to be degraded. Furthermore, Bag3 may be involved in mitophagosome formation as it interacted with synaptopodin 2, which is known to play a role in mitophagosome biogenesis.