RESUMO
Infection with bovine papillomavirus (BPV) types 1 and 2 results in the most common skin tumor of horses, termed equine sarcoid. The persistent and recurrent nature of this tumor stands in contrast to the regressive nature of BPV-1/- 2 induced cutaneous papillomas in cattle. The circulation of horse-specific BPV-1/- 2 variants within equine populations has been suggested as a possible explanation for the difference in clinical presentation of BPV-1/- 2 infection between horses and cattle. In order to investigate this hypothesis, we identified 98 complete BPV-1/- 2 genomes using a Nanopore sequencing approach. Separate BPV-1/- 2 alignments were used to infer Bayesian phylogenetic trees. Phylogeny-trait association concerning host species was investigated using Bayesian Tip-association Significance software (BaTS) Overall, 179 unique BPV-1 and 128 BPV-2 substitutions were found. The E2 coding region in the viral genome exhibited an exceptionally high rate of non-synonymous mutations (81 %, n = 13/16). Interestingly, extensive deletions in the L1/L2 region (up to 1.5 kb) were found exclusively in horse-derived samples. Nevertheless, the most frequently detected single nucleotide polymorphisms were shared between equine and bovine hosts, which is in agreement with BaTS results indicating no phylogeny-host correlation. We found indications that horse-specific mutations might exist in subpopulations of equine derived BPV-1/- 2, but these did not result in horse-adapted genetic variants. Based on these observations, cross-species transmission from cattle to horses seems to be an ongoing process, rather than an ancient occurrence that has been followed by the circulation of horse-adapted BPV variants in the horse population..
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Quirópteros , Doenças dos Cavalos , Infecções por Papillomavirus , Neoplasias Cutâneas , Cavalos , Animais , Bovinos , Papillomavirus Bovino 1/genética , Filogenia , Teorema de Bayes , DNA Viral , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/veterinária , Genômica , Infecções por Papillomavirus/veterinária , Doenças dos Cavalos/patologiaRESUMO
Bovine papillomavirus (BPV) types 1 and 2 are causally associated with equine sarcoid, the most common mesenchymal neoplasm of horses, but the viral load (VL) differs between lesions. Sensitive and accurate BPV detection and quantification is essential for clinicians to confirm clinical suspicion, as well as in research settings for stratifying these skin lesions. Due to the limitations of histopathology in sarcoid diagnosis, PCR screening of superficial swabs constitutes the principal sampling method for BPV detection. This study aimed to investigate the ability of superficial swabs and fine-needle aspirates (FNA) to accurately detect the VL in equine sarcoids, considering the main clinical types: occult, nodular, verrucous and fibroblastic. Superficial swabs and FNAs from a series of sarcoid-affected horses were tested in parallel for BPV DNA quantification. Quantitative real-time PCR screening of postoperative tissue biopsies served as reference standard for the accuracy assessment of the viral titters. Our results indicate that VL is not a predictor of the clinical type. Student's t-test results gave evidence of a significant difference between both sample methods (P < 0.001) with FNA giving the best approximation of the actual VL (P < 0.01). In contrast to superficial swabs, the reference standard correlated moderately with FNA in general (P < 0.05; r = 0.39) and strongly with FNA results within the occult sarcoid group (P < 0.05; r = 0.59). In conclusion, the correlation of FNA with the reference standard was strong enough to suggest this is the preferred method for quantifying VL in sarcoids.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Cavalos , Neoplasias , Infecções por Papillomavirus , Sarcoidose , Dermatopatias , Neoplasias Cutâneas , Cavalos/genética , Animais , Carga Viral/veterinária , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/veterinária , DNA Viral/análise , Dermatopatias/veterinária , Neoplasias/veterinária , Sarcoidose/diagnóstico , Sarcoidose/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Doenças dos Cavalos/diagnóstico , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/veterinária , Papillomavirus Bovino 1/genéticaRESUMO
Bovine papillomaviruses (BPVs) infect the basal layer of the epithelium of bovines, where they persist asymptomatically or produce benign fibroepithelial hyperplasia in the skin or mucosa. The aim of the present study was to describe the genotypes of bovine papillomas at the macroscopic and microscopic level. A descriptive study was carried out using non-probabilistic convenience sampling. Ninety-nine papillomas from 63 animals were collected on 32 farms, as well as information about age, gender, breed, and productive use of the bovines. The location, type, and degree of epithelial invasion of the papillomas were recorded. The samples were subjected to molecular and histopathological analysis. Papillomas were found most frequently on dairy farms (75.0%), in females (95.0%), in cattle of the Holstein breed (45.0%), and in animals over 24 months of age (50.0%). Most of the positive animals had from 1 to 15 papillomas (31.6%) and only one type of papilloma (79.4%). Cauliflower-like papillomas were found in 48.5% of the cases, while atypical papillomas were found in 11.1% of the cases. Cauliflower-like papillomas were found mainly on the udder (14.4%), head (10.0%), and neck (10.0%) and were associated with five BPV genotypes (BPV1, BPV2, BPV6, BPV7, and BPV10), while BPV2 and BPV6 were found to be associated with all types of papillomas (cauliflower, flat, pedunculated, and atypical). The presence of BPV11 in flat papillomas and BPV6 in atypical papillomas is reported here for the first time. Morphology and histopathological findings did not allow differentiation of the BPV genotypes.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Papiloma , Infecções por Papillomavirus , Feminino , Animais , Bovinos , Costa Rica , Doenças dos Bovinos/epidemiologia , Papillomavirus Bovino 1/genética , Papillomaviridae/genética , Genótipo , Infecções por Papillomavirus/veterináriaRESUMO
INTRODUCTION: Bladder tumors of cattle are very uncommon accounting from 0.1% to 0.01% of all bovine malignancies. Bladder tumors are common in cattle grazing on bracken fern-infested pasturelands. Bovine papillomaviruses have a crucial role in tumors of bovine urinary bladder. AIM OF THE STUDY: To investigate the potential association of ovine papillomavirus (OaPV) infection with bladder carcinogenesis of cattle. METHODS: Droplet digital PCR was used to detect and quantify the nucleic acids of OaPVs in bladder tumors of cattle that were collected at public and private slaughterhouses. RESULTS: OaPV DNA and RNA were detected and quantified in 10 bladder tumors of cattle that were tested negative for bovine papillomaviruses. The most prevalent genotypes were OaPV1 and OaPV2. OaPV4 was rarely observed. Furthermore, we detected a significant overexpression and hyperphosphorylation of pRb and a significant overexpression and activation of the calpain-1 as well as a significant overexpression of E2F3 and of phosphorylated (activated) PDGFßR in neoplastic bladders in comparison with healthy bladders, which suggests that E2F3 and PDGFßR may play an important role in OaPV-mediated molecular pathways that lead to bladder carcinogenesis. CONCLUSION: In all tumors, OaPV RNA could explain the causality of the disease of the urinary bladder. Therefore, persistent infections by OaPVs could be involved in bladder carcinogenesis. Our data showed that there is a possible etiologic association of OaPVs with bladder tumors of cattle.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Infecções por Papillomavirus , Neoplasias da Bexiga Urinária , Animais , Bovinos , Ovinos , Papillomavirus Bovino 1/genética , Neoplasias da Bexiga Urinária/veterinária , Neoplasias da Bexiga Urinária/etiologia , Neoplasias da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Reação em Cadeia da Polimerase , Carcinogênese , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/veterináriaRESUMO
Bovine papillomaviruses (BPVs) exhibit a high degree of genetic variability, and several viral types have been identified based on analysis of the L1 gene. The L1 is the main capsid protein and the main target for neutralizing antibodies. We performed a retrospective study on BPVs circulating in Rio Grande do Sul state, Southern Brazil, in 2016-2020. DNA from 43 bovine papilloma samples were amplified using two degenerate primer sets - FAP59/64 and MY09/11 - targeting the L1 region, and analyzed for phylogeny, mixed BPV infections (coinfections) and amino acid (aa) sequences. We also performed an in silico analysis with 114 BPV L1 sequences from the GenBank database to assess the agreement between the phylogeny obtained based on complete L1 sequences versus that based on the region amplified using the FAP59/64 and MY09/11 primer sets. Considering single and coinfections, we identified 31 BPV-1 (31/43; 72.1%), 27 BPV-2 (27/43; 62.8%) and 4 BPV-6 (4/43; 9.3%). Coinfections with BPV-1 and BPV-2 were observed in 61.3% of the samples. Our results are supported by in silico analyses that demonstrate that the classification using FAP59/64 or MY09/11 matches the complete L1 results, except for BPV-17 and -18, which may be mistakenly classified depending on the primers used. Furthermore, we found unique or rare amino acids in at least one L1 sequence of each BPV type identified in our study, some of which have been identified previously in papillomavirus epitopes, suggesting immune-mediated selection. Finally, our study provides an overview of BPVs circulating in Southern Brazil over the last five years and point to the combined use of primers FAP59/64 and MY09/11 for analysis of BPV coinfections and putative epitopes.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Coinfecção , Infecções por Papillomavirus , Animais , Bovinos , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/veterinária , Filogenia , Brasil/epidemiologia , Aminoácidos/genética , Estudos Retrospectivos , DNA Viral/genética , Papillomaviridae/genética , Doenças dos Bovinos/epidemiologiaRESUMO
Bovine papillomaviruses are related to cause fibroepithelial proliferations in the skin and mucosae and are associated with economic loss mainly related to poor body condition and reduced milk production. This study aimed to investigate the presence and types of bovine papillomaviruses (BPVs) in cattle sampled in different areas of Costa Rica using molecular techniques. A descriptive study with a non-probability convenience sampling was carried out. A total of 99 papillomatous lesions were collected from 63 animals in 32 farms, and analyzed by polymerase chain reaction, rolling circle amplification (RCA), sequencing, and restriction enzymes digestion. Seven bovine papillomavirus types (BPV1, BPV2, BPV4, BPV6, BPV7, BPV10, BPV11) and two putative novel viral variants (BPV-CR1 and BPV-CR2) were identified for the first time in Costa Rica. BPV6 was the most frequently detected virus in lesions (31.2%), followed by BPV2 (25%) and BPV1 (25%). BPV1 and BPV2 were the most widely distributed in the Country. Coinfections were recorded in two animals (BPV1 / BPV2 and BPV4 / BPV6). Restriction analyses allowed differentiating BPV1 from BPV2, BPV4, and BPV7, but failed to identify BPV6, BPV10, and BPV11. Results suggest that a great PVs diversity is harbored by bovines in Costa Rica and indicate the need for further investigations aimed to uncover PV diversity at the full genomic level.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Animais , Bovinos , Papillomavirus Bovino 1/classificação , Papillomavirus Bovino 1/genética , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Costa Rica/epidemiologia , Tipagem Molecular/veterinária , Infecções por Papillomavirus/veterinária , Infecções por Papillomavirus/virologia , Pele/patologiaRESUMO
Sarcoids are the most common cutaneous neoplasm of the horse, arising as a result of a neoplastic proliferation of fibroblasts associated with infection with bovine papillomavirus, most notably types 1 and 2. Although they do not metastasise, they are locally invasive and aggressive, and can lead to important welfare concerns, interfere with tack and therefore impede athleticism, and undoubtedly lead to a reduction in the value of affected horses. This review discusses the evidence behind the most commonly used treatments for equine sarcoids. The most commonly used treatments are discussed. No one treatment is universally successful, and there are many treatments with varying level of scientific evaluation and reported success rates.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Cavalos , Infecções por Papillomavirus , Sarcoidose , Neoplasias Cutâneas , Cavalos , Animais , Infecções por Papillomavirus/terapia , Infecções por Papillomavirus/veterinária , Doenças dos Cavalos/terapia , Neoplasias Cutâneas/terapia , Neoplasias Cutâneas/veterinária , Sarcoidose/veterináriaRESUMO
Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 106-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four passages. Infected cells displayed typical features of equine sarcoid cells, including hyperproliferation, and loss of contact inhibition. Neither multiple passaging nor storage negatively affected cell hyperproliferation, viral DNA replication, and gene transcription, suggestive for infection-mediated cell immortalization. Intriguingly, extracellular vesicles released by BPV1-infected fibroblasts contained viral DNA that was most abundant in the fractions enriched for apoptotic bodies and exosomes. This viral DNA is likely taken up by non-infected fibroblasts. We conclude that equine primary fibroblasts stably infected with BPV1 and BPV2 virions constitute a valuable near-natural model for the study of yet unexplored mechanisms underlying the pathobiology of BPV1/2-induced sarcoids.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Cavalos , Infecções por Papillomavirus , Cavalos , Animais , Papillomavirus Bovino 1/genética , DNA Viral/genética , Replicação do DNA , Replicação Viral , Vírion , Fibroblastos/patologiaRESUMO
An important component of tissues is the extracellular matrix (ECM), which not only forms a tissue scaffold, but also provides the environment for numerous biochemical reactions. Its composition is strictly regulated, and any irregularities can result in the development of many diseases, including cancer. Sarcoid is the most common skin cancer in equids. Its formation results from the presence of the genetic material of the bovine papillomavirus (BPV). In addition, it is assumed that sarcoid-dependent oncogenic transformation arises from a disturbed wound healing process, which may be due to the incorrect functioning of the ECM. Moreover, sarcoid is characterized by a failure to metastasize. Therefore, in this study we decided to investigate the differences in the expression profiles of genes related not only to ECM remodeling, but also to the cell adhesion pathway, in order to estimate the influence of disturbances within the ECM on the sarcoid formation process. Furthermore, we conducted comparative research not only between equine sarcoid tissue bioptates and healthy skin-derived explants, but also between dermal fibroblast cell lines transfected and non-transfected with a construct encoding the E4 protein of the BP virus, in order to determine its effect on ECM disorders. The obtained results strongly support the hypothesis that ECM-related genes are correlated with sarcoid formation. The deregulated expression of selected genes was shown in both equine sarcoid tissue bioptates and adult cutaneous fibroblast cell (ACFC) lines neoplastically transformed by nucleofection with gene constructs encoding BPV1-E1^E4 protein. The identified genes (CD99, ITGB1, JAM3 and CADM1) were up- or down-regulated, which pinpointed the phenotypic differences from the backgrounds noticed for adequate expression profiles in other cancerous or noncancerous tumors as reported in the available literature data. Unravelling the molecular pathways of ECM remodeling and cell adhesion in the in vivo and ex vivo models of epidermal/dermal sarcoid-related cancerogenesis might provide powerful tools for further investigations of genetic and epigenetic biomarkers for both silencing and re-initiating the processes of sarcoid-dependent neoplasia. Recognizing those biomarkers might insightfully explain the relatively high capacity of sarcoid-descended cancerous cell derivatives to epigenomically reprogram their nonmalignant neoplastic status in domestic horse cloned embryos produced by somatic cell nuclear transfer (SCNT).
Assuntos
Papillomavirus Bovino 1 , Doenças dos Cavalos , Infecções por Papillomavirus , Sarcoidose , Dermatopatias , Neoplasias Cutâneas , Animais , Papillomavirus Bovino 1/genética , Transformação Celular Neoplásica , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Doenças dos Cavalos/metabolismo , Cavalos/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/veterináriaRESUMO
Papilloma and fibropapilloma cases are quite common in cattle breeding, which cause economic losses due to decrease in the production of milk, meat, and also impair the quality of hide. In this study, we aimed to determine viral etiology and investigate p53 expression levels with immunohistochemical methods from a total of 30 cases. The study material was collected between 2013 and 2021 in Kars, Turkey. Paraffin embedded tissues were used for earlier cases in which the freshly specimens could not be provided. Cases were investigated for papillomavirus etiology with polymerase chain reaction (PCR) using FAP59/FAP64 and MY09/MY11 primer pairs. In 20 of the 30 cases papillomaviruses were identified, and 10 cases were identified as Bovine papillomavirus-1 (BPV-1), 1 case as BPV-2, 1 case as BPV-12, and 1 case as equus caballus papillomavirus 2 (EcPV-2) after sequence analysis. p53 immunostaining was also performed, and the cases were graded according to the positively stained cells. In conclusion BPV-12 was detected for the first time in our country, EcPV-2 was detected first time in cattle indicating cross species infection and p53 was staining most evident in BPV-1 and BPV-2 cases and BPV-12 and EcPV-2 was not stained.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Papiloma , Animais , Papillomavirus Bovino 1/genética , Bovinos , Doenças dos Bovinos/diagnóstico , DNA Viral/química , DNA Viral/genética , Expressão Gênica , Papiloma/veterinária , Papillomaviridae/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. METHODS: In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. RESULTS: We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. CONCLUSIONS: By generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Cavalos , Sequenciamento por Nanoporos , Nanoporos , Infecções por Papillomavirus , Neoplasias Cutâneas , Animais , Teorema de Bayes , Papillomavirus Bovino 1/genética , DNA Viral/genética , Genômica , Cavalos , Filogenia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/veterináriaRESUMO
Bovine papillomaviruses types 1 and 2 (BPV1, BPV2) commonly induce skin tumours termed sarcoids in horses and other equids. Sarcoids seriously compromise the health and welfare of affected individuals due to their propensity to resist treatment and reoccur in a more severe form. We have developed influenza (Flu) A and B virus vectors that harbour a truncated NS1 gene (iNS) assuring interferon induction and co-express shuffled BPV1 E6 and E7 antigens for sarcoid immunotherapy. In a safety trial involving 12 healthy horses, intradermal administration of iNSA/E6E7equ and iNSB/E6E7equ was well tolerated, with the only transient side effect being mild fever in four horses. Repeated screening of secretions and faeces by RT-PCR and plaque assay revealed no virus shedding, thus also confirming biological safety. In a patient trial involving 29 horses bearing BPV1-induced single or multiple sarcoids, at least one lesion per horse was intratumourally injected and then boosted with iNSA/E6E7equ and/or iNSB/E6E7equ. The treatment induced a systemic antitumour response as reflected by the synchronous regression of injected and non-injected lesions. Irrespective of vaccination schemes, complete tumour regression was achieved in 10/29 horses. In 10/29 horses, regression is still ongoing (May 2021). Intriguingly, scrapings collected from former tumour sites in two patients tested negative by BPV1 PCR. Nine severely affected individuals with a history of unsuccessful therapeutic attempts did not (6/29) or only transiently (3/29) respond to the treatment. INSA/E6E7equ and iNSB/E6E7equ proved safe and effective in significantly reducing the tumour burden even in severe cases.
Assuntos
Papillomavirus Bovino 1/imunologia , Neoplasias/imunologia , Neoplasias/virologia , Sarcoidose/patologia , Sarcoidose/virologia , Animais , Chlorocebus aethiops , Vetores de Doenças , Feminino , Cavalos , Humanos , Influenza Humana/imunologia , Masculino , Orthomyxoviridae/imunologia , Vacinação/métodos , Células Vero , Eliminação de Partículas Virais/imunologiaRESUMO
We have previously reported that bovine papillomavirus type 1 (BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae (budding yeast) cultures (Zhao and Frazer 2002, Journal of Virology, 76:3359-64 and 76:12265-73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication (day 3-16), middle weak replication (day 23-34/45) and late stable replication (day 45-82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs (cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.
Assuntos
Papillomavirus Bovino 1 , Papillomavirus Bovino 1/genética , Replicação do DNA , DNA Viral/genética , Saccharomyces cerevisiae/genética , Vírion/genética , Replicação ViralRESUMO
Sarcoids are the most common cutaneous tumor of equids and are caused by bovine papillomavirus (BPV). Different clinical subtypes of sarcoids are well characterized clinically but not histologically, and it is not known whether viral activity influences the clinical or histological appearance of the tumors. The aim of this study was to verify whether the development of different clinical types of sarcoids or the presence of certain histological features were associated with BPV distribution within the tumor. The presence of BPV was assessed by polymerase chain reaction (PCR) and visualized in histological sections by chromogenic in situ hybridization (CISH) in 74 equine sarcoids. Furthermore, to better characterize the molecular features of neoplastic cells, immunohistochemistry for S100, smooth muscle actin-α (αSMA), and fibroblast-associated protein-α (FAPα) was performed. The presence of BPV was confirmed in all tissues examined by either or both PCR and CISH (72/74, 97% each). Of 70/74 CISH-positive cases, signal distribution appeared as either diffuse (61/70, 87%) or subepithelial (9/70, 13%); the latter was more frequently observed in the verrucous subtype. However, no statistically significant association was found between clinical subtypes and specific histological features or hybridization pattern. Moreover, CISH signal for BPV was not detected in the epidermis overlying sarcoids nor in the tissue surrounding the neoplasms. By immunohistochemistry, αSMA confirmed the myofibroblastic differentiation of neoplastic cells in 28/74 (38%) sarcoids. Using tissue microarrays, FAPα labelling was observed in neoplastic fibroblasts of all sarcoids, suggesting this marker as a potential candidate for the immunohistochemical diagnosis of sarcoids.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Cavalos , Ácidos Nucleicos , Infecções por Papillomavirus , Neoplasias Cutâneas , Animais , Papillomavirus Bovino 1/genética , DNA Viral , Fibroblastos , Cavalos , Infecções por Papillomavirus/veterinária , Neoplasias Cutâneas/veterináriaRESUMO
We have previously reported that bovine papillomavirus type 1 (BPV1) can replicate its genome and produces infectious virus-like particles in short-term BPV1 virion-infected Sacharomyces cerevisiae (Zhao and Frazer, 2002). Here, we report viral RNA transcription and L1 capsid protein expression in long-term BPV1 virion-infected S. cerevisiae culture. Northern blot hybridization showed that viral RNA was detected in long-term BPV1-infected S. cerevisiae cultures (82-108 days). The levels of the viral RNA transcription varied significantly over the long time period, which showed active transcription at an early stage (Day 3 to Day 16), weak transcription at a middle stage (Day 23 to Day 45) and stable transcription at the late stage of culture (Day 55 to Day 82/85/95). Three major BPV1 transcripts of 4.3, 2.6 and 1.8 Kb were identified, with 4.3 Kb a minor transcript and the 1.8 Kb the most prominent transcript compared with the 2.6 Kb species. Immunoblotting showed that L1 capsid protein was expressed, with its variable amounts corresponding to the levels of RNA transcription over the time period. 35S-methionine/cysteine labeling and immunoprecipitation proved that the detected L1 protein was newly synthesized in BPV1-infected S. cerevisiae cultures. 33.3-54.2% of the cell colonies expressed L1 protein. Thus, the S. cerevisiae system, as a promising model, may be used not only for the study of virus like particle formation of BPV1 in vitro, but also for further functional analysis of individual viral genes in BPV1 life cycle. Keywords: BPV1; viral RNA transcription; expression of L1 capsid protein; virion-infected Saccharomyces cerevisiae.
Assuntos
Papillomavirus Bovino 1 , Papillomavirus Bovino 1/genética , Capsídeo , Proteínas do Capsídeo/genética , Saccharomyces cerevisiae/genética , VírionRESUMO
Highly pathogenic bovine papillomaviruses (BPVs) were detected and quantified for the first time using digital droplet polymerase chain reaction (ddPCR) by liquid biopsy in 103 clinically healthy sheep. Overall, ddPCR detected BPVs in 68 blood samples (66%). BPV infection by a single genotype was revealed in 61.8% of the blood samples, and BPV coinfection by double, triple or quadruple genotypes was observed in 38.2% of liquid biopsies. The BPV-2 genotype was most frequently seen in sheep, whereas BPV-1 was the least common. Furthermore, ddPCR was very useful for detection and quantification; the BPV-14 genotype was observed for the first time in ovine species, displaying the highest prevalence in some geographical areas (Apulia). In 42 of the positive samples (61.8%), a single BPV infection was observed, 26 of which were caused by BPV-2 (61.9%) and 7 by BPV-13 (16.7%). BPV-14 was responsible for 7 single infections (16.7%) and BPV-1 for 2 single infections (4.7%). Multiple BPV coinfections were observed in the remaining 26 positive samples (38.2%), with dual BPV-2/BPV-13 infection being the most prevalent (84.6%). BPV infection by triple and quadruple genotypes was also observed in 11.5% and 3.8% of cases, respectively. The present study showed that ddPCR, a biotechnological refinement of conventional PCR, is by far the most sensitive and accurate assay for BPV detection compared to conventional qPCR. Therefore, ddPCR displayed an essential diagnostic and epidemiological value very useful for the identification of otherwise undetectable BPV genotypes as well as their geographical distributions and suggesting that animal husbandry practices contribute to cross-species transmission of BPVs.
Assuntos
Papillomavirus Bovino 1/genética , DNA Viral/sangue , Reação em Cadeia da Polimerase/veterinária , Ovinos/virologia , Animais , Bovinos , Genes Virais , Genótipo , Biópsia Líquida , Reação em Cadeia da Polimerase/métodosRESUMO
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.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Cavalos , Infecções por Papillomavirus , Neoplasias Cutâneas , Animais , Papillomavirus Bovino 1/genética , DNA Viral , Cavalos , Japão , Masculino , Infecções por Papillomavirus/veterinária , Pênis/cirurgia , Filogenia , Neoplasias Cutâneas/veterináriaRESUMO
Equine sarcoids (ES) were diagnosed in 12 Somali wild asses (SWA) (Equus africanus somaliensis) from 10 different institutions of the SWA European Endangered Species Programme from 1976 to 2019. Samples of surgically excised masses, biopsies, or necropsy samples were submitted for histologic and virologic analysis. In addition, tissue samples from one onager (Equus hemionus onager), one kulan (Equus hemionus kulan), and two Hartmann's mountain zebras (HMZ) (Equus zebra hartmannae) were examined. Histology confirmed the diagnosis of ES exhibiting the typical microscopic features. Polymerase chain reaction detected bovine papillomavirus type 1 (BPV1) DNA in eight SWA samples and bovine papillomavirus type 2 (BPV2) DNA in one SWA sample. The onager, kulan, and one HMZ sample tested positive for BPV1. The other HMZ tested positive for BPV1 and BPV2. This is the first report of ES in an onager. Surgical excision was the treatment elected by most veterinarians. A follow-up survey of the cases over several years after clinical diagnosis and therapy revealed variable individual outcome with ES recurrence in four cases. Three SWA and the kulan were euthanized due to the severity of the lesions. Nine affected SWA were males with seven having a sarcoid located at the prepuce. Because a genetic disposition is a risk factor for the development of ES in horses, this may also be true for endangered wild equids with few founder animals in their studbook history. Innovative approaches regarding therapy and prevention of ES in wild equids are therefore highly encouraged.
Assuntos
Equidae , Neoplasias Cutâneas/veterinária , Animais , Animais de Zoológico , Papillomavirus Bovino 1/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Espécies em Perigo de Extinção , Feminino , Predisposição Genética para Doença , Masculino , Neoplasias/veterinária , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
Equine sarcoid is the most common skin tumor of horses. Clinically, it occurs as a locally invasive, fibroblastic, wart-like lesion of equine skin, which has 6 clinical classes: occult, verrucose, nodular, fibroblastic, mixed, and malignant. Sarcoids may be single but multiple lesions are more frequent. The typical histological feature is increased density of dermal fibroblasts which form interlacing bundles and whorls within the dermis. Lesions are mostly persistent, resist therapy, and tend to recur following treatment. In general, sarcoids are not fatal but their location, size, and progression to the more aggressive form may lead to the withdrawal of a horse from use and serious infringement of their welfare leading to the loss of valuable animals. Bovine papillomavirus (BPV) type 1 and less commonly type 2 contribute to the development of equine sarcoid. The viral genome and proteins are detected in a high percentage of cases. Furthermore, viral oncoprotein activity leads to changes in the fibroblastic tissue similar to changes seen in other types of tumors. Equine sarcoids are characterized by a loss of tumor suppressor activity and changes allowing abnormal formation of the affected tissue, as well as y immune defense abnormalities that weaken the host's immune response. This impaired immune response to BPV infection appears to be crucial for the development of lesions that do not spontaneously regress, as occurs in BPV-infected cows.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Doenças dos Cavalos , Infecções por Papillomavirus , Animais , Papillomavirus Bovino 1/genética , Bovinos , DNA Viral , Cavalos , Recidiva Local de Neoplasia/veterinária , Infecções por Papillomavirus/veterináriaRESUMO
Sarcoids are the most commonly diagnosed skin tumours in equines. Bovine papillomaviruses (BPVs) are the primary causative agent of sarcoids. There has been intensive research to discover the molecular mechanisms that may contribute to the aetiopathogenesis of this disease and tumour suppressors and proto-oncogenes known to play a role in human neoplastic conditions have been investigated in equine sarcoids. Current approaches include the identification of gene expression profiles, characterising sarcoid and normal skin tissues, and an assessment of epigenetic alterations such as microRNA differential expression and DNA methylation status. This review focuses on selected groups of genes that contribute to the molecular mechanisms of sarcoid formation. These genes have the potential to complement current clinical examinations of equine sarcoid disease in diagnosis, prognosis, therapeutic response and screening.