New approach for genomic characterisation of equine sarcoid-derived BPV-1/-2 using nanopore-based sequencing

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.

Influenza virus vector iNS1 expressing bovine papillomavirus 1 (BPV1) antigens efficiently induces tumour regression in equine sarcoid patients

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.

Abortion storm of Yili Horses is Associated with Equus Caballus Papillomavirus 2 Variant Infection

Background: Nine different species of Equus caballus papillomavirus (EcPV) and three bovine papillomavirus (BPV) have been reported to infect horses, however, there are so far no describing such infections in China. In January 2021, an abortion storm occurred in Yili horses, as a result of which 50 out of 93 aborted fetus samples were found to be negative for equid herpesvirus (EHV) and equine arteritis virus (EAV).Results: In our pioneer study with Chinese horses, we first found EcPV-2 in the nasal swabs (4/230, 1.7%) of Yili horses, and semen (3/18, 16.7%) of the Thoroughbred horses. This indicated that EcPV can be indeed hosted by horses in China, and that EcPV-2 might be transmitted though breeding. Further detection of EcPVs in the lung tissues of aborted fetus in Yili horses, which were originally negative for equid herpesviruses, established that EcPV-2 was positive in 19 of 50 samples, thereby indicating that EcPV-2 might be a new pathogen causing of abortions. Thereafter, the sequence analyses for L1 genes sequences of 26 China’s EcPV-2 were performed which indicated that EcPV-2, that primarily infected the horses in China, shared 98.3%-99.9% nt identity with the already published sequences for EcPV-2. These observations indicated that EcPV-2 identified in the current study were highly similar variants of the previously identified strains of EcPV-2. Phylogenetic analysis based on L1 genes in GenBank showed that EcPV-2, found in the Chinese horses, was closely related to and clustered together with an already known EcPV-2a lineage. Conclusion: Our study provides the first evidence related to EcPV-2 infection in the Chinese horses, which can serve as a causative agent for Yili horse abortions, and thus can possibly lay the foundation for a systematic and detailed epidemiological study of this infection in the Chinese horses.

Pteridium spp. and Bovine Papillomavirus: Partners in Cancer

Bovine papillomavirus (BPV) are a cause for global concern due to their wide distribution and the wide range of benign and malignant diseases they are able to induce. Those lesions include cutaneous and upper digestive papillomas, multiple histological types of urinary bladder cancers—most often associated with BPV1 and BPV2—and squamous cell carcinomas of the upper digestive system, associated with BPV4. Clinical, epidemiological and experimental evidence shows that exposure to bracken fern (Pteridium spp.) and other related ferns plays an important role in allowing viral persistence and promoting the malignant transformation of early viral lesions. This carcinogenic potential has been attributed to bracken illudane glycoside compounds with immune suppressive and mutagenic properties, such as ptaquiloside. This review addresses the role of BPV in tumorigenesis and its interactions with bracken illudane glycosides. Current data indicates that inactivation of cytotoxic T lymphocytes and natural killer cells by bracken fern illudanes plays a significant role in allowing viral persistence and lesion progression, while BPV drives unchecked cell proliferation and allows the accumulation of genetic damage caused by chemical mutagens. Despite limited progress in controlling bracken infestation in pasturelands, bracken toxins remain a threat to animal health. The number of recognized BPV types has steadily increased over the years and now reaches 24 genotypes with different pathogenic properties. It remains essential to widen the available knowledge concerning BPV and its synergistic interactions with bracken chemical carcinogens, in order to achieve satisfactory control of the livestock losses they induce worldwide.

Evidence from a Series of 104 Equine Sarcoids Suggests That Most Sarcoids in New Zealand Are Caused by Bovine Papillomavirus Type 2, although Both BPV1 and BPV2 DNA Are Detectable in around 10% of Sarcoids

Equine sarcoids are common mesenchymal neoplasms of horses that are caused by cross-species infection by deltapapillomaviruses. While bovine papillomavirus (BPV) 1 and 2 are the most common causes, there are differences between countries regarding which of these BPV types cause the majority of sarcoids. Additionally, no causative PV can be detected in a subset of sarcoids, suggesting that other PV types could be rarer causes of these neoplasms. In the present study, consensus PCR primers and PCR primers specific for the five deltapapillomavirus types currently known to cause mesenchymal neoplasia (BPV1, BPV2, BPV13, BPV14, and Ovis aries PV2 DNA) were used to investigate the presence of PV DNA in 104 sarcoids from three defined regions in New Zealand and from two distinct time periods separated by 15 years. PV DNA was detected in 94 (90.4%) sarcoids. Of the sarcoids containing PV DNA, 83 (88.3%) contained only BPV2 DNA, 9 (9.6%) BPV1 and BPV2 DNA, and 2 (2.1%) only BPV1 DNA. No other PV types were detected. The predominance of BPV2 is consistent with studies of sarcoids from North America but dissimilar to studies of sarcoids from Europe and Australia. Detection rates of BPV1 and BPV2 were similar in sarcoids from different regions of New Zealand and in sarcoids from different time periods. These results suggest that most equine sarcoids in New Zealand are caused by BPV2 and thus if vaccines are developed to prevent sarcoids, vaccines that provide good protection against BPV2 will be required in this country.

The Development of Novel Primer Sets to Specifically Amplify Each of the Five Different Deltapapillomaviruses That Cause Neoplasia after Cross-Species Infection

Bovine papillomavirus (BPV) types 1 and 2 are recognized as the main cause of equine sarcoids. However, some studies report that up to a quarter of these tumors do not contain detectible BPV1 or BPV2 DNA. The absence of detectible BPV1 or BPV2 in these sarcoids suggests the possible involvement of other papillomavirus types. Currently, five deltapapillomaviruses are recognized to cause mesenchymal neoplasia after cross-species infection. In addition to BPV1 and BPV2, BPV13 has been associated with equine sarcoids in Brazil, BPV14 has been associated with feline sarcoids, and Ovis aries papillomavirus 2 caused a sarcoid-like lesion in a pig. To investigate the cause of equine sarcoids, PCR primers were developed to specifically amplify each of the five different deltapapillomaviruses that have been associated with mesenchymal neoplasia. The specificity of these primers was confirmed using samples of formalin-fixed tissue known to contain each PV type. These primers allow rapid and sensitive detection of deltapapillomavirus DNA in equine sarcoids. As studies have revealed marked regional variability in the cause of equine sarcoids, these primers will be useful to determine the predominant PV type causing sarcoids in a region. Additionally, there is a single report describing mixed infections by BPV1 and BPV2 in equine sarcoids. The specific primer sets are expected to enable more sensitive detection of mixed infections in equine sarcoids. Determining the cause of equine sarcoids is important as vaccines are developed to prevent these common malignant neoplasms.

Molecular characterization and pathological identification of a novel strain of delta papillomavirus-4 (bovine papillomavirus-2) not viruses in Egypt

Background and Aim: Bovine papillomaviruses (BPV) are a heterogeneous group of oncoviruses, distributed globally, which produce major economic losses. In the current study, we compared the results of different diagnostic approaches and compared the strains identified in this study with previously characterized strains at local and international levels. Materials and Methods: Samples of skin warts were collected from five bovines with generalized papillomatosis from two Egyptian provinces, Menya and Ismailia, in 2020. Electron microscopy, molecular characterization, histopathological, and immunohistochemical examination were performed. Results: BPV was detected using electron microscopy in the collected samples. Using molecular characterization, BPV-2 was successfully identified for 1st time in Egypt. The strain has 99.6% identity with the BPV-2 reference strains obtained from GenBank. These results were supported by histopathology and immunohistochemistry examination. Partial nucleotide sequences of the L1 gene were submitted to GenBank with accession numbers MW289843 and MW289844. Conclusion: BPV-2 was reported for 1st time in the current study. The strain was identified grossly, microscopically, and pathologically and confirmed using molecular approaches. All results were consistent. The sequence analysis revealed that this strain has high sequence similarity to the reference Deltapapillomavirus-4, BPV-2 strains from Brazil and China.

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture

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.

Detection of different types of papillomavirus and co-infection in cattle in the State of Goiás - Brazil

Bovine papilomavírus (BPVs) is the etiological agent of bovine papillomatosis, a disease that triggers warts throughout the skin, udder, roofs, genitalia and in more severe cases can develop extensive papillomas, cause neoplasia in the digestive tract and bladder, cause losses in productivity and losses to livestock. In Brazil, the occurrence of BPV infection is relatively common, but the identification of viral types is still sporadic. The present study is a research report that aimed to describe the occurrence of BPV infections in dairy cattle affected by papillomatosis, based on the nucleotide sequences of the ORF L1, the most conserved sequence. Twenty-five samples of cutaneous wart from nine cattle clinically diagnosed as cutaneous papillomatosis were analyzed in the state of Goiás, central-western Brazil. Amplification was obtained in 11 samples (papilloma) from different cattle. PCR reactions followed by sequencing revealed the presence of BPV-1 in 60%, BPV-5 in 40%, and BPV-14 in 20% of the samples analyzed. The presence of coinfection was verified in 60% of the amplified samples. These data suggest that several types of BPV can infect a lesion simultaneously and demonstrate the possibility that BPV infection in epithelial tissue can occur without restriction to one or two viral types, demonstrating the region's genetic diversity. As far as we know, this is the first registry of typification of BPVs of the central-western region of Brazil. This analysis provides important information for bovine papillomavirus (BPV) research in Brazil.