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.

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.

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.

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.

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.

Antigenic variation among recent Japanese isolates of bovine coronaviruses belonging to phylogenetically distinct genetic groups.

Bovine coronaviruses (BCoVs) isolated in Japan consist of four genetic groups, as determined by phylogenetic analysis using the polymorphic region (aa 456-592) of the S glycoprotein gene. Japanese field isolates of BCoV, reference Kakegawa strain, and vaccine strain 66/H were analyzed for their antigenic properties by indirect immunofluorescence and neutralization testing. There were no significant differences observed among these BCoVs in direct immunofluorescence tests. However, antigenic differences were observed between BCoVs in the neutralization tests, although there was no clear indication of a distinct serotype. A monoclonal antibody, 4H4, against the Kakegawa strain belonging to group 1 lacked significant neutralizing activity for viruses of groups 2, 3, and 4. Therefore, we speculate that the genetic differences between these groups may have altered their antigenicity. Analysis of mutant viruses resistant to neutralization by 4H4 revealed that the antigenic site of the Kakegawa strain maps to amino acid position 284 of the S glycoprotein. This site is not homologous to a known antigenic site (aa 528) of the Quebec strain belonging to group 1, and it is not located in the conformational domain comprising domain I (aa 351-403) and domain II (aa 517-621). This amino acid constitutes a neutralization epitope of BCoV, which is distinct from aa 528 of the Quebec strain. These results indicate antigenic evolution of BCoV between the genetic groups circulating in Japan.

Characterization of novel bovine papillomavirus type 12 (BPV-12) causing epithelial papilloma.

Bovine papillomavirus type 12 (BPV-12, putative type BAA1) was detected in epithelial papilloma located on the tongue of an infected cow. Then, the whole genome was sequenced, and phylogenetic analysis illustrated that it should be classified as a member of the genus Xipapillomavirus. The viral genome is 7197 base pairs in length and contains five early ORFs (E1, E2, E4, E7 and E8), three late ORFs (L1, L2 and L3), and a long control region that possesses replication regulatory elements. Meanwhile, mRNA of each gene was detected in the papilloma sample. The papilloma was identified as epithelial papilloma by histological and immunohistochemical examination. Based on the genome information and pathological properties, BAA1 was designated as BPV-12 in this study.

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.

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.

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone.

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.

Detection of a novel bovine papillomavirus type 11 (BPV-11) using xipapillomavirus consensus polymerase chain reaction primers.

Polymerase chain reaction-based bovine papillomavirus (BPV) detection methods using a combination of two primer sets, subAup/subAdw and subBup/subBdw, have enabled the broad-spectrum detection of most characterized BPV types. These methods were used to detect the partial L1 nucleotide sequence of BPV types from 167 cutaneous warts in cattle. Three potentially new viruses were detected using subBup/subBdw primer sets. The partial nucleotide sequences of these viruses were most similar to BPV-4, -6 and -9. Whole genome sequencing of one sample defines a new BPV type in the genus Xipapillomavirus, designated BPV-11.

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.

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.

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.

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.

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.

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.

Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD.

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB - encoded by a prophage in S. Typhimurium DT104 - are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [(32)P]NAD and ArtA, and the label was released by HgCl(2), which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA(6Arg-Ala) and ArtA(115Glu-Ala), in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

Bovine papillomavirus type 9 induces epithelial papillomas on the teat skin of heifers.

Experiments were carried out to investigate whether papillomas could be induced on the teat skin of heifers by intradermal injection with bovine papillomavirus type 9 (BPV-9). Three heifers (#1 and 2, two 0.5-year-old Holsteins; #3, a 1.5-year-old Japanese Black) were injected with BPV-9 and one heifer (#4, a 0.5-year-old Holstein) was mock-infected. Viral DNA load in the inocula was quantified by real-time polymerase chain reaction assay and adjusted to 1.56x10(12) copies per injection. Papillomas appeared at the injection sites in the BPV-9-injected heifers #1, 2 and 3 and grew over the 8 (#1 and 2) and 4 (#3)mo observation period, respectively. However, no papillomas were found in the mock-infected heifer #4. The experimentally induced papillomas were excised and examined. Histologically, the lesions were characterized by hyperplasia of the epidermis with hyperkeratosis and marked acanthosis and were morphologically similar to naturally occurring lesions. BPV-9 DNA and bovine papillomavirus capsid antigen were abundant in the lesions. Therefore, we conclude that BPV-9 is an etiological agent causing epithelial papillomas on the teat skin of heifers.