Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

Complete genome sequencing and genetic analysis of a Japanese porcine torovirus strain detected in swine feces.

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.

Phylogenetic analysis of novel posaviruses detected in feces of Japanese pigs with posaviruses and posa-like viruses of vertebrates and invertebrates.

Posaviruses and posa-like viruses are unclassified viruses with sequence similarity to viruses of the order Picornavirales. They have been reported in various vertebrates and invertebrates. We identified 11 posavirus-like sequences in porcine feces and performed phylogenic analysis. Previously reported Japanese posaviruses and those identified in this study clustered with posavirus 1, 4, and 7 and husavirus 1, while five viruses branched into three independent lineages, tentatively named posavirus 10, 11, and 12. Interestingly, posaviruses, except for posavirus 8 and 9, husaviruses, and rasaviruses, formed a cluster consisting of viruses only from pigs, humans, and rats, while posavirus 8 and 9, fisavirus, and basaviruses clustered with posa-like viruses from invertebrates.

Complete genomic analysis and molecular characterization of Japanese porcine sapeloviruses.

The Porcine Sapelovirus (PSV) is an enteric virus of pigs that can cause various disorders. However, there are few reports that describe the molecular characteristics of the PSV genome. In this study, almost the entire genomes of 23 PSVs detected in Japanese pigs were analyzed using bioinformatics. Analysis of the cis-active RNA elements showed that the predicted secondary structures of the internal ribosome entry site in the 5' untranslated region (UTR) and a cis-replication element in the 2C coding region were conserved among PSVs. In contrast, those at the 3' UTR were different for different PSVs; however, tertiary structures between domains were conserved across all PSVs. Phylogenetic analysis of nucleotide sequences of the complete VP1 region showed that PSVs exhibited sequence diversity; however, they could not be grouped into genotypes due to the low bootstrap support of clusters. The insertion and/or deletion patterns in the C-terminal VP1 region were not related to the topology of the VP1 tree. The 3CD phylogenetic tree was topologically different from the VP1 tree, and PSVs from the same country were clustered independently. Recombination analysis revealed that recombination events were found upstream of the P2 region and some recombination breakpoints involved insertions and/or deletions in the C-terminal VP1 region. These findings demonstrate that PSVs show genetic diversity and frequent recombination events, particularly in the region upstream of the P2 region; however, PSVs could currently not be classified into genotypes and conserved genetic structural features of the cis-active RNA elements are observed across all PSVs.

Complete genome analysis of porcine kobuviruses from the feces of pigs in Japan.

Porcine kobuviruses (PoKoVs) are ubiquitously distributed in pig populations worldwide and are thought to be enteric viruses in swine. Although PoKoVs have been detected in pigs in Japan, no complete genome data for Japanese PoKoVs are available. In the present study, 24 nearly complete or complete sequences of the PoKoV genome obtained from 10 diarrheic feces and 14 non-diarrheic feces of Japanese pigs were analyzed using a metagenomics approach. Japanese PoKoVs shared 85.2-100% identity with the complete coding nucleotide (nt) sequences and the closest relationship of 85.1-98.3% with PoKoVs from other countries. Twenty of 24 Japanese PoKoVs carried a deletion of 90 nt in the 2B coding region. Phylogenetic tree analyses revealed that PoKoVs were not grouped according to their geographical region of origin and the phylogenetic trees of the L, P1, P2, and P3 genetic regions showed topologies different from each other. Similarity plot analysis using strains from a single farm revealed partially different similarity patterns among strains from identical farm origins, suggesting that recombination events had occurred. These results indicate that various PoKoV strains are prevalent and not restricted geographically on pig farms worldwide and the coexistence of multiple strains leads to recombination events of PoKoVs and contributes to the genetic diversity and evolution of PoKoVs.

Identification of further diversity among posaviruses.

Recently, there have been reports of new members of posavirus-like viruses in the order Picornavirales. In this study, using a metagenomics approach, 11 posavirus-like sequences (>7,000 nucleotides) were detected in 155 porcine fecal samples. Phylogenetic analysis revealed that the newly identified virus sequences, together with other posavirus-like viruses, form distinct clusters within the order Picornavirales, composed of eight genogroups and unassigned sequences based on amino acid sequences of the helicase and RNA-dependent RNA polymerase regions, with <40 % and <50 % sequence identity, respectively. We propose further classifications of highly diverse posavirus populations based on newly identified sequences from Japanese pig feces.

Screening for Japanese Black cattle herds at risk of bovine leukemia virus transmission based on the presence of persistent lymphocytosis.

This study aimed to develop a screening method to identify Japanese Black (JB) cattle farms at high risk of bovine leukemia virus (BLV) transmission. We introduced a recently established lymphocyte count (LC) cut-off to detect cattle with persistent lymphocytosis (PL). To identify high-risk farms, we examined the relationship between the proportion of cattle with PL and the mean blood proviral loads (PVL) per farm. The results showed a strong correlation between the proportion of cattle with high LC, higher than the LC cut-off values for JB cattle, and the mean blood PVL per farm. This indicates that the mean blood PVL on a herd basis of JB cattle can be estimated from the proportion of cattle with high LC. Specifically, the mean blood PVL on farms with >30 % cattle having high LC was estimated to be greater than 100 copies/10 ng DNA. The higher the proportion of cattle with high LC per farm, the higher the proportion of cattle at risk as sources of BLV infection. The study demonstrated that the proportion of cattle with high LC, based on the new LC cut-off, serves as a practical index for screening high-risk farms with PL cattle that have high PVL. This finding is meaningful for prioritizing farms with a high proportion of cattle with high LC, necessitating BLV infection prevention measures, such as voluntary culling and segregation, in order to develop a regional, stepwise BLV eradication strategy.

Screening of persistently infected cattle with bovine viral diarrhea virus on dairy farms by using milk tanker and bulk tank milk samples for viral RNA and viral-specific antibody detection.

The objective of this study was to provide a screening scheme of persistently infected (PI) cattle on dairy herds by combining reverse-transcription polymerase chain reaction (RT-PCR) to detect bovine viral diarrhea virus (BVDV) in milk tanker samples and commercial enzyme-linked immunosorbent assay to detect BVDV antibodies in bulk tank milk. We conducted a pilot survey and regional survey targeting all dairy farms in Ibaraki Prefecture by using milk tanker and bulk tank milk samples to screen PI cattle. Farms with positive samples underwent a follow-up test to identify PI cattle. In the pilot study, all virus-positive samples in bulk tank milk were included in the positive milk tanker samples. The RT-PCR assay successfully detected BVDV at dilutions of 1:1,600 by using two PI cows' milk. In the regional survey, 5 of 79 milk tanker samples were virus-positive. The virus was detected in three PI lactating cows and one PI calf on three farms. Antibody screening using bulk tank milk samples revealed 15 of 363 samples were positive, and 12 of 348 farms were BVDV antibody-positive. Follow-up tests on one farm identified three PI calves. Thus, eight PI cattle on five farms were identified in this study. In conclusion, combining BVDV detection using milk tanker samples and antibody detection using bulk tank milk is a feasible and economical method to efficiently screen PI cattle and confirm the PI-free status among dairy herds.

Risk factors associated with the within-farm transmission of bovine viral diarrhea virus and the incidence of persistently infected cattle on dairy farms from Ibaraki prefecture of Japan.

For understanding the factors affecting bovine viral diarrhea virus (BVDV) transmission, this study investigated the distribution of BVDV and the epidemiological features of persistently infected (PI) cattle in Ibaraki Prefecture of Japan, and identified farm-level risk factors associated with BVDV infection, with a focus on within-farm transmission and PI animal detection. Among all 377 dairy farms, forty-four PI cattle were identified on 22 farms. Thirty-eight and six PI cattle were born on their current farms or purchased, respectively. Twenty-six PI cattle were born from pregnancies on their current farms, seven from pregnancies in summer pastures, and eight from pregnancies on other farms. The within-farm seroprevalence on farms with PI animals was significantly higher than that on farms without PI cattle. Of 333 farms holding homebred cattle without movement records, antibody-positivity in homebred cattle was observed on 194 farms; these cattle were likely infected by within-farm transmission. Herd size, summer pasturing, and BVDV infection status of the nearest dairy farm were risk factors associated with within-farm transmission. Likewise, herd size, summer pasturing, and the proportion of purchased cattle were related to PI animal occurrence. This study shows the risk of within-farm transmission and occurrence of PI animals after the introduction of BVDV via purchasing and summer pasturing, and illustrates the significant role of PI cattle in circulating BVDV. More effective measures for screening BVDV infection and PI animals, including intensive tests targeting moved cattle and newborn calves, and bulk milk surveillance, are required to control the spread of BVDV in Japan.

Spatial analysis of low pathogenic H5N2 avian influenza outbreaks in Japan in 2005.

We conducted a spatial analysis of low pathogenic H5N2 avian influenza (AI) outbreaks, that affected 41 chicken farms in Japan in 2005. A statistically significant (p=0.001) cluster of AI-positive farms was identified in the central part of Ibaraki Prefecture. Inside the AI cluster, the density was high for both chicken farms and chicken population, the proportion of layer finisher type farms was high and the farm size was large. We considered it important to take precautions for AI outbreaks in densely chicken-populated areas and to implement appropriate movement control around the affected farms to prevent transmission among farms located within small distances in the case of AI outbreaks. Spatial scan statistics are applicable in veterinary epidemiology to detection of high risk areas for animal diseases.

A hematologic key for bovine leukemia virus screening in Japanese black cattle.

The European Community's leukosis key (EC key) is a well-known hematologic method for detecting bovine leukemia virus (BLV) infection in dairy cattle. The key identifies infected cattle with persistent lymphocytosis via a combination of lymphocyte count (LC) and age. Using the EC key to identify BLV-infected Japanese black (JB) cattle is problematic, however, given the inherently lower LCs of JB cattle compared to dairy cattle. We analyzed the LC in BLV-positive and -negative JB cattle and estimated LC cutoff values by age using receiver operating characteristic curve analysis. Among the 716 JB blood samples collected, 452 (63%) JB cattle were confirmed as BLV-positive by an antibody ELISA for ≥1-y-old cattle and by real-time PCR for <1-y-old cattle. The cutoff values for the LC in each age group were calculated as 6.3 × 109/L for <1 y, 5.9 × 109/L for 1 to <2 y, 5.5 × 109/L for 2 to <3 y, 4.5 × 109/L for 3 to <6 y, 4.3 × 109/L for 6 to ≤10 y, and 3.7 × 109/L for >10 y. The sensitivity and specificity of the estimated cutoff values were 0.49 (95% confidence interval: 0.44-0.53) and 0.81 (0.75-0.85), whereas those of the EC key were 0.20 (0.16-0.24) and 0.99 (0.97-1.00). Our LC cutoff values for screening JB cattle for BLV infection appear to be preferable to those of the EC key.

First identification of Sapoviruses in wild boar.

Sapoviruses (SaVs) are enteric viruses that have been detected in human and animals previously; however, SaVs have not been identified in wild boar yet. Using a metagenomics approach, we identified SaVs in fecal samples of free-living wild boars in Japan for the first time. Six of the 48 specimens identified belonged to one genogroup (G)III, one GV and four GVI SaV sequence reads. We successfully determined complete genome of GV and GVI SaV strains using the long reverse transcription PCR strategy and the 5' rapid amplification of cDNA end method. Phylogenetic tree analysis and pairwise distance calculation revealed that GV SaV detected from wild boar was related to recently assigned GV.5 strains from pig, while GVI SaV was assigned to a new genotype within GVI. Moreover, wild boar may act as a reservoir for transmission of SaVs to the pig population (and vice versa) because GIII, GV, and GVI SaVs were all detected in pigs previously.

Genetic diversity and recombination of enterovirus G strains in Japanese pigs: High prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus G population.

To study the genetic diversity of enterovirus G (EV-G) among Japanese pigs, metagenomics sequencing was performed on fecal samples from pigs with or without diarrhea, collected between 2014 and 2016. Fifty-nine EV-G sequences, which were >5,000 nucleotides long, were obtained. By complete VP1 sequence analysis, Japanese EV-G isolates were classified into G1 (17 strains), G2 (four strains), G3 (22 strains), G4 (two strains), G6 (two strains), G9 (six strains), G10 (five strains), and a new genotype (one strain). Remarkably, 16 G1 and one G2 strain identified in diarrheic (23.5%; four strains) or normal (76.5%; 13 strains) fecal samples possessed a papain-like cysteine protease (PL-CP) sequence, which was recently found in the USA and Belgium in the EV-G genome, at the 2C-3A junction site. This paper presents the first report of the high prevalence of viruses carrying PL-CP in the EV-G population. Furthermore, possible inter- and intragenotype recombination events were found among EV-G strains, including G1-PL-CP strains. Our findings may advance the understanding of the molecular epidemiology and genetic evolution of EV-Gs.

Whole genome analysis of porcine astroviruses detected in Japanese pigs reveals genetic diversity and possible intra-genotypic recombination.

Porcine astroviruses (PoAstVs) are ubiquitous enteric virus of pigs that are distributed in several countries throughout the world. Since PoAstVs are detected in apparent healthy pigs, the clinical significance of infection is unknown. However, AstVs have recently been associated with a severe neurological disorder in animals, including humans, and zoonotic potential has been suggested. To date, little is known about the epidemiology of PoAstVs among the pig population in Japan. In this report, we present an analysis of nearly complete genomes of 36 PoAstVs detected by a metagenomics approach in the feces of Japanese pigs. Based on a phylogenetic analysis and pairwise sequence comparison, 10, 5, 15, and 6 sequences were classified as PoAstV2, PoAstV3, PoAstV4, and PoAstV5, respectively. Co-infection with two or three strains was found in individual fecal samples from eight pigs. The phylogenetic trees of ORF1a, ORF1b, and ORF2 of PoAstV2 and PoAstV4 showed differences in their topologies. The PoAstV3 and PoAstV5 strains shared high sequence identities within each genotype in all ORFs; however, one PoAstV3 strain and one PoAstV5 strain showed considerable sequence divergence from the other PoAstV3 and PoAstV5 strains, respectively, in ORF2. Recombination analysis using whole genomes revealed evidence of multiple possible intra-genotype recombination events in PoAstV2 and PoAstV4, suggesting that recombination might have contributed to the genetic diversity and played an important role in the evolution of Japanese PoAstVs.