Isolation and complete genomic characterization of a Movar 33/63-like Japanese bovine herpesvirus 4 from a calf with respiratory disease.

Bovine herpesvirus 4 (BoHV-4) is an indigenous virus in cattle prevalent mainly in North and South American countries and European countries, but the genomic sequences and genetic characteristics of Japanese strains have not been reported. BoHV-4 is suspected, but not proven, to be associated with various diseases. In the present study, we isolated BoHV-4 from a 10-month-old Japanese Black calf with respiratory symptoms in Japan. To identify the genetic characteristics of the isolate named strain SG20, complete genome sequencing was performed using a combination of next-generation and Sanger sequencing technologies. The complete long unique coding region (LUR) of SG20 was found to comprise 108,819 nucleotides with 41.4% GC content and contain at least 78 open reading frames. It shares 83.4 to 99.3% overall nucleotide identity with six BoHV-4 strains available in the database. The deduced amino acid sequence alignment revealed that SG20 contains genotype 1-specific features of BoHV-4, such as amino acid substitutions and insertions within the glycoprotein B region. Phylogenetic analyzes based on the nucleotide sequences of ORF20 indicated that the virus belonged to genotype 1 (Movar 33/63-like group). The strain was also analyzed using the complete LUR and placed in the same clade as a strain recently isolated from China, but it was distinct from American and European BoHV-4 strains of genotype 1. Although further genomic and epidemiologic information is needed, our results help elucidate the molecular epidemiology of BoHV-4 and provide a foundation for future studies.

The possible role of haematophagous flies in the incidence of bovine teat papillomatosis.

The relationship between the incidence of bovine teat papillomatosis and the activity of haematophagous flies was investigated in Japan. A total of 15,737 flies consisting of 33 species were collected by dry ice-baited mosquito net (DMN) trap and a sweep net from udders of cattle. Simulium aokii (Takahasi) of Simuliidae (black flies) was the predominant species, followed by S. tobetsuense Ono and S. iwatense (Shiraki). Simulium aokii had the highest peak in October, followed by September. Numbers of blood spots from the bites per teat in nulliparous cattle were significantly correlated with numbers of S. aokii collected by DMN trap. Numbers of teats with warts and spots of blood from the bites per teat were significantly more abundant in anterior teats than posterior teats. The average incidence of teat papillomatosis in nulliparous cattle was significantly higher than that in parous cattle, and the highest incidence by month was in May, followed by April. Although bovine papillomavirus (BPV) DNA was not detected in flies examined, the presence of black flies and blood spots from their bites were associated with subsequent high incidence of growing warts. In particular, it would pay to give attention to species such as S. aokii that severely attack udders in the present locality. Further investigations for the detection of BPV DNA from flies parasitizing on teats are needed.

Upregulation of host genes during disease progression in bovine leukemia virus infection is independent of overexpression of viral transcriptional regulators in vitro.

Bovine leukemia virus (BLV) is a member of the genus Deltaretrovirus within the family Retroviridae that infects bovine B cells, causing persistent lymphocytosis and enzootic bovine leukosis (EBL) in a small fraction of infected cattle. As changes in the transcriptome of infected cells are important for BLV disease progression, comprehensive analysis of gene expression in different disease states is required. In this study, we performed an RNA-seq analysis using samples from non-EBL cattle with and without BLV infection. Subsequently, a transcriptome analysis was conducted in combination with previously obtained RNA-seq data from EBL cattle. We found several differentially expressed genes (DEGs) between the three groups. After screening and confirmation of target DEGs using real-time reverse transcription polymerase chain reaction, we found that 12 target genes were significantly upregulated in EBL cattle compared to BLV-infected cattle without lymphoma. In addition, the expression levels of B4GALT6, ZBTB32, EPB4L1, RUNX1T1, HLTF, MKI67, and TOP2A were significantly and positively correlated with the proviral load in BLV-infected cattle. Overexpression experiments revealed that these changes were independent of BLV tax or BLV AS1-S expression in vitro. Our study provides additional information on host gene expression during BLV infection and EBL development, which may be helpful for understanding the complexity of transcriptome profiles during disease progression.

Bovine leukemia virus-associated B cell lymphoma with severe pleomorphism in a steer.

We examined a 26-month-old steer with neoplastic lesions in the spleen, lymph nodes, heart and kidneys, characterized by pleomorphic lymphoid cells that were immunohistochemically positive for CD20. The presence of bovine leukemia virus (BLV) at >200,000 copies per 100,000 cells by quantitative RT-PCR was considered to be due to random integration of the provirus into the neoplastic cells´ genomes. Inverse PCR identified the presence of one, two, two and three different malignant clones in the heart, spleen, mesenteric node and blood, respectively. Because BLV can rapidly induce lymphoma and a high proviral load facilitates B-cell carcinogenesis, multiclonal tumor development was suspected in the present case.

Whole-genome sequencing of live attenuated bovine adenovirus type 7 vaccine strain TS-GT suggests biomarkers for virulence attenuation.

Bovine adenovirus type 7 (BAdV-7) is one of the most important respiratory and enteric pathogens in the cattle industry. Although live attenuated vaccines are used to control the virus in Japan, limited information is available on the genomic regions that determine viral pathogenicity. We determined the complete genome sequence of the attenuated BAdV-7 strain TS-GT. The genome is 30,052 bp long and contains 45-bp inverted terminal repeats and 30 predicted genes. A genome sequence comparison showed that 99.9% of the TS-GT genome is identical to the prototypic and pathogenic BAdV-7 strain Fukuroi; however, the TS-GT genome contains a novel mutation and four indels. We describe here potential relationships between these genomic changes and the biological characteristics of BAdV-7.

Novel single nucleotide polymorphisms in the bovine leukemia virus genome are associated with proviral load and affect the expression profile of viral non-coding transcripts.

Bovine leukemia virus (BLV) infects bovine B-cells and causes malignant lymphoma, resulting in severe economic losses in the livestock industry. To control the spread of BLV, several studies have attempted to clarify the molecular mechanisms of BLV pathogenesis, but the details of the mechanism are still enigmatic. Currently, viral non-coding RNAs are attracting attention as a novel player for BLV pathogenesis because these transcripts can evade the host immune response and are persistently expressed in latent infection. One of the viral non-coding RNA, AS1, is encoded in the antisense strand of the BLV genome and consists of two isoforms, AS1-L and AS1-S. Although the function of the AS1 is still unknown, the AS1 RNA might also have some roles because it keeps expressing in tumor tissues. In the present study, we identified novel single nucleotide polymorphisms (SNPs) located in the AS1 coding region and indicated that individuals infected with BLV with minor SNPs showed low proviral load. To evaluate the effect of identified SNPs, we constructed infectious clones with these SNPs and found that their introduction affected the expression profile of AS1 RNA; the amount of AS1-L isoform increased compared with the wild type, although the total amount of AS1 RNA remained unchanged. Prediction analysis also suggested that the introduction of SNPs changed the secondary structure of AS1 RNA. These results explain part of the relationship between BLV expansion in vivo and the expression profile of AS1, although further analysis is required.

Complete Genome Sequence of Bovine Adenovirus Type 7 Strain Fukuroi, Isolated from a Cow with Respiratory Disease.

We determined the complete genome sequence of the bovine adenovirus type 7 prototype strain Fukuroi using next-generation sequencing technology. We found that the viral genome is 30,034 bp long and has the shortest inverted terminal repeats among known adenoviruses.

Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma of cattle caused by infection with bovine leukemia virus (BLV). It is defined by clonal and neoplastic expansion of BLV-infected B cells. Currently, multiple examinations are able to comprehensively diagnose this condition. Inverse polymerase chain reaction (PCR) is a useful method to determine retrovirus integration sites. Here, we established a simplified inverse PCR method, involving the evaluation of clonality and similarity of BLV integration sites, to clinically diagnose EBL, and we also assessed its reliability. We found that the novel BLV inverse PCR could detect clonal expansion of infected cells even if they constituted only 5% of the total number of cells, while not amplifying any fragments from BLV-uninfected cells, thus confirming its sufficient sensitivity and specificity for use in EBL diagnosis. Furthermore, 50 clinical cases of bovine leukemia were analyzed using BLV inverse PCR and other PCR-based methods, wherein our method most efficiently determined virus-dependent bovine leukemia, including unidentified clinical cases observed in a previous report. Following further clinical investigations to enhance its reliability, the proposed BLV inverse PCR method has the potential to be applied to EBL diagnosis.

Sequence and unique phylogeny of G genes of bovine respiratory syncytial viruses circulating in Japan.

Bovine respiratory syncytial virus (BRSV) is an etiologic agent of bovine respiratory disease. The rapid evolutionary rate of BRSV contributes to genetic and antigenic heterogeneity of field strains and causes occasional vaccine failure. We conducted molecular epidemiologic characterization of BRSV circulating in Japan to obtain genetic information for vaccine-based disease control. Phylogenetic analysis of G and F gene sequences revealed that all of the isolated Japanese BRSV strains clustered in the same genetic subgroup, which was distinct from the 9 known groups. We assigned the Japanese group to subgenotype X. The Japanese isolates formed 2 temporal clusters: isolates from 2003 to 2005 clustered in lineage A; isolates from 2017 to 2019 formed lineage B. The alignment of the deduced amino acid sequences of the G gene revealed that the central hydrophobic region responsible for viral antigenicity is conserved in all of the isolates; unique amino acid mutations were found mainly in mucin-like regions. Our results suggest that BRSV has evolved uniquely in Japan to form the new subgenotype X; the antigenic homogeneity of the viruses within this group is inferred.

Development of an in situ hybridization assay using an AS1 probe for detection of bovine leukemia virus in BLV-induced lymphoma tissues.

Enzootic bovine leukosis (EBL) is a malignant B cell lymphoma caused by infection with bovine leukemia virus (BLV). Histopathological examination is commonly used for diagnosis of the disease, but observation of lymphoma alone does not confirm EBL because cattle may be affected by sporadic forms of lymphoma that are not associated with BLV. Detection of BLV in tumor cells can be definitive evidence of EBL, but currently, there is no technique available for such a purpose. In this study, we focused on a viral non-coding RNA, AS1, and developed a novel in situ hybridization assay for the detection of BLV from formalin-fixed paraffin-embedded (FFPE) tissues. RNA-seq analysis revealed that all examined B lymphocytes derived from clinical EBL abundantly expressed AS1 RNA, indicating a possible target for detection. The in situ hybridization assay using an AS1 probe clearly detected AS1 RNA in fetal lamb kidney cells persistently infected with BLV. The utility of this assay in clinical samples was assessed using three EBL-derived lymph node specimens and one BLV-negative specimen, and AS1 RNA was detected specifically in the EBL-derived tissues. These results suggest that AS1 RNA is a useful target for the detection of BLV from FFPE specimens of tumor tissues. This technique is expected to become a powerful tool for EBL diagnosis.

Genomic characterization and distribution of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.

Phylogenetic and antigenic analysis of bovine parainfluenza virus type 3 isolated in Japan between 2002 and 2019.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory pathogens of cattle. In addition to the classical BPIV3 genotype A (BPIV3a), new genetic groups, genotype B (BPIV3b) and C (BPIV3c), have been identified and isolated in certain parts of the world. The present study aimed to investigate the genetic and antigenic characteristics of BPIV3 circulating in Japan. Seventy-three BPIV3 field strains were isolated from nasal samples of cattle between 2002 and 2019. Phylogenetic analysis of the phosphoprotein and hemagglutinin-neuraminidase genes showed that the isolates clustered into two genotypes, BPIV3a (49 %) and BPIV3c (51 %). The BPIV3a strains had more wide genetic variation than the rest of the genotypes. Additionally, new variants were obtained and designated them tentatively as subgroup 4 of the BPIV3a. The first Japanese BPIV3c was isolated in 2012, but here the BPIV3c NM2 strain was isolated from a sample collected four years earlier than the previous report. The antigenicity of ten BPIV3 strains including all three genotypes was assessed with a viral cross-neutralization test. Anti-sera against BPIV3a and BPIV3b cross-reacted well with both homologous and heterologous viruses. On the other hand, anti-sera against BPIV3c had reduced cross-reactivity to the heterologous viruses. Overall, our findings showed that genetically and antigenically divergent BPIV3 is prevalent in cattle in Japan. These results could provide a reference for molecular epidemiological characterization of BPIV3 and vaccine development.

Production of immunogenic recombinant L1 protein of bovine papillomavirus type 9 causing teat papillomatosis.

Bovine papillomavirus type 9 (BPV9) is a causative agent of severe teat papillomatosis. Considering the lack of efficient BPV culture methods, recombinant proteins such as virus-like particles developed through genetic engineering may serve as a useful tool for developing effective vaccines against BPV infection. In this study, we successfully produced immunogenic particles composed of recombinant L1 protein of BPV9 (rBPV9-L1), using a baculovirus expression system. rBPV9-L1-immunized mice produced BPV9-specific IgG, which did not cross-react with BPV type 6, which is another causative agent of teat papillomatosis. Hence, immunogenic rBPV9-L1 is potentially applicable as a vaccine candidate for teat papillomatosis.

The chemokines CCL2 and CXCL10 produced by bovine endometrial epithelial cells induce migration of bovine B lymphocytes, contributing to transuterine transmission of BLV infection.

Bovine leukemia virus (BLV) causes a lymphoproliferative disease in cattle and is transmitted horizontally and vertically via infected lymphocytes. Although transplacental infection is considered the predominant route of vertical transmission of BLV, the molecular mechanisms of this process remain to be elucidated. Notably, how BLV passes through the blood-placental barrier remains unclear, given that BLV is transmitted primarily by cell-to-cell contact. One hypothesis is that B cell migration to the placenta may be induced by certain endometrium-expressed chemokines. To test this hypothesis, we performed an in vitro cell migration assay using bovine B cell lines and endometrial epithelial cells. Cell migration assays showed that two bovine B cell lines, BL2M3 and BL3.1 cells, were attracted to the supernatant of bovine endometrial epithelial cells (BEnEpCs). Quantitative real-time RT-PCR showed that expression levels of mRNAs encoding the chemokines CCL2 and CXCL10 were higher in BEnEpCs than in MDBK cells. Additionally, an inhibition assay using immune serum against CCL2 and CXCL10 showed suppression of migration of bovine B cell lines. A syncytium assay showed that cells expressing BLV envelope (Env) protein fused with BEnEpCs. Here we found that bovine B cells are attracted by chemokines produced in the endometrium and that cells expressing BLV Env protein fused with endometrium epithelial cells. These results explain part of the molecular mechanism of transplacental transmission during BLV infection, although further analysis will be required. Advances in these areas are expected to contribute to controlling the spread of BLV.

Laryngeal squamous cell carcinoma and papilloma associated with Equus caballus papillomavirus 2 in a horse.

A case of laryngeal squamous cell carcinoma (SCC) and squamous papilloma in a 19-year-old Thoroughbred stallion is described. The animal exhibited severe wheezing caused by laryngopharyngeal stenosis. Histological examination identified laryngeal, laryngotracheal, and guttural pouch tumor masses consisting of areas of SCC. In the epiglottic lesion, the overlying epithelium was replaced by papilloma cells, and superficial cells frequently had nuclear inclusion bodies that expressed oncoprotein E6, which is characteristic of high risk human papillomaviruses. The papillomatous epithelium was continuous with epithelium composed of SCC cells. Equus caballus papillomavirus 2 (EcPV2) DNA was detected in the guttural pouch tumor. These findings suggest that laryngeal SCC and papilloma are a continuum of EcPV2-induced neoplastic lesions in horses.

Full genome analysis of bovine papillomavirus type 1 derived from a calf with severe cutaneous multiple papillomatosis.

Severe papillomatosis occasionally causes astasia leading to euthanizing cattle. There are currently a limited number of reports on virologic approach in severe bovine papillomatosis. Here we report a full genome characterization of bovine papillomavirus type 1 (BPV-1) from the case of severe papillomatosis. A calf developed numerous papillomas on the skin and some nodules in the upper gastrointestinal tract at seven months old. The skin lesion was diagnosed as the epithelial papilloma with BPV antigen expression, while the gastrointestinal lesions were diagnosed as the fibropapilloma without BPV antigen. Full genome analysis revealed that BPV-1s detected in all the lesions were exactly the same. Compared with the reference BPV-1 sequence, there was a single nucleotide insertion in the upstream regulatory region.

Detection of bovine papillomavirus type 2 DNA in calf conjunctival myofibroblastoma.

An 8-month-old male Japanese Black calf was referred for the evaluation of a slow-growing conjunctival mass in the right eye. A superficial keratectomy was performed followed by recurrence on two occasions. No metastases were found in surrounding tissues. Histological, immunohistochemical, and ultrastructural investigation revealed that both the primary and the recurrent lesions were benign, conjunctival, myofibroblastomas. Interestingly, bovine papillomavirus type 2 (BPV-2) DNA was detected in both myofibroblastoma lesions. Archival bovine myofibroblastomas from the vulva and neck were also analyzed for papillomaviral genomes. BPV-2 DNA was also amplified from these lesions. To the best of our knowledge, this is the first report describing a potential causal relationship between BPV-2 infection and conjunctival myofibroblastoma.

A long-term animal experiment indicating persistent infection of bovine coronavirus in cattle.

A long-term animal experiment involving inoculation with bovine coronavirus (BCoV) was conducted to verify its persistent infection in cattle. Three colostrum-deprived Holstein calves were housed separately in individual rooms of a high-containment facility and inoculated with the BCoV strain Kumamoto/1/07. Until the end of the experiment (1,085, 700 and 280 days, respectively), viral RNAs were detected sporadically by RT-PCR and nested PCR from plasma, nasal discharge, and feces. Seroconversion and titer changes were validated by hemagglutination inhibition tests and neutralization tests. Among the samples, nasal discharge showed a higher viral positivity than feces, which seemed to be associated with positive detection in the plasma. These data demonstrate the existence of persistent infection of BCoV in the respiratory tissues of cattle.

Production of highly immunogenic virus-like particles of bovine papillomavirus type 6 in silkworm pupae.

Bovine papillomaviruses (BPVs) are the causative agent of bovine teat papillomatosis, which can lead to severe economic losses in dairy cattle. Among the 14 identified BPV genotypes, BPV type 6 (BPV6) is the most frequently detected in teat papilloma lesions, and is therefore thought to play a major role in teat papillomatosis. To develop an effective vaccine against BPV6 infection, we produced virus-like particles of BPV6 (BPV6-VLP) in silkworm (Bombyx mori) pupae and purified these by heparin affinity chromatography using a single column. About 0.7mg purified BPV6-VLP was obtained from one pupa. BPV6-VLP-immunized mice produced a specific IgG to BPV6 that recognized BPV6 antigen with high sensitivity in an immunohistochemical analysis. Thus, silkworm pupae are a useful bioreactor for the production of BPV6-VLP, which can potentially be used as a vaccine for bovine teat papillomatosis.

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

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