Genomic Characterization and Phylogenetic Classification of Bovine Coronaviruses Through Whole Genome Sequence Analysis.

Bovine coronavirus (BCoV) is zoonotically transmissible among species, since BCoV-like viruses have been detected in wild ruminants and humans. BCoV causing enteric and respiratory disease is widespread in cattle farms worldwide; however, limited information is available regarding the molecular characterization of BCoV because of its large genome size, despite its significant economic impact. This study aimed to better understand the genomic characterization and evolutionary dynamics of BCoV via comparative sequence and phylogenetic analyses through whole genome sequence analysis using 67 BCoV isolates collected throughout Japan from 2006 to 2017. On comparing the genomic sequences of the 67 BCoVs, genetic variations were detected in 5 of 10 open reading frames (ORFs) in the BCoV genome. Phylogenetic analysis using whole genomes from the 67 Japanese BCoV isolates in addition to those from 16 reference BCoV strains, revealed the existence of two major genotypes (classical and US wild ruminant genotypes). All Japanese BCoV isolates originated from the US wild ruminant genotype, and they tended to form the same clusters based on the year and farm of collection, not the disease type. Phylogenetic trees on hemagglutinin-esterase protein (HE), spike glycoprotein (S), nucleocapsid protein (N) genes and ORF1 revealed clusters similar to that on whole genome, suggesting that the evolution of BCoVs may be closely associated with variations in these genes. Furthermore, phylogenetic analysis of BCoV S genes including those of European and Asian BCoVs and human enteric coronavirus along with the Japanese BCoVs revealed that BCoVs differentiated into two major types (European and American types). Moreover, the European and American types were divided into eleven and three genotypes, respectively. Our analysis also demonstrated that BCoVs with different genotypes periodically emerged and predominantly circulated within the country. These findings provide useful information to elucidate the detailed molecular characterization of BCoVs, which have spread worldwide. Further genomic analyses of BCoV are essential to deepen the understanding of the evolution of this virus.

Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs.

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.

A provisional complete genome-based genotyping system for rotavirus species C from terrestrial mammals.

Rotaviruses C (RVCs) have been detected in pigs, humans, cows, ferrets and dogs. Despite their zoonotic impact and pathogenicity, the genetic characterization of RVCs is incomplete, unlike rotaviruses A (RVAs), whose genetics are well studied. Several studies reported partial and complete genomic sequences for multiple porcine and canine RVCs. We aimed to establish a complete genome-based genotyping system for RVCs, by analysing complete genome data from 22 porcine RVCs identified in Japan from 2002 to 2010, along with those from multiple human, bovine, porcine, canine and ferret RVCs reported in several previous studies. Comparative sequence analyses among RVCs from various host species demonstrated that porcine RVCs had a high level of genetic diversity. In addition, phylogenetic analyses of all 11 RNA segments indicated that porcine RVCs could be classified into multiple genotypes. Notably, the VP4 dendrogram divided bovine RVCs into multiple genotypes. Consequently, the provisional genotype classification for RVCs from terrestrial mammals revealed the existence of genotypes 18G, 21P, 13I, 4R, 6C, 6M, 9A, 8N, 6T, 5E and 4H for the genes VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively, based on the cut-off values as defined by the Rotavirus Classification Working Group. The system established in this study deepens our understanding of RVC evolution and facilitates the discovery of genetic events (gene reassortment and interspecies transmission) among RVCs.

Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex.

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

Analysis of genetic divergence among strains of porcine rotavirus C, with focus on VP4 and VP7 genotypes in Japan.

Porcine rotavirus C (RVC) has been often detected in sporadic cases or outbreaks of diarrhoea in suckling and weaned pigs. Surveillance studies of RVCs have demonstrated high prevalence in the United States, and Japan, and some other countries. To date, the zoonotic impact and pathogenicity of RVCs are not well understood, and only a few complete sequences of RVCs are available. The aim of this study was to perform sequence and phylogenetic analyses for the VP4 and VP7 genes of the 22 porcine RVCs identified in Japan from 2002 to 2010. The genetic classification of the VP4 genes of the 22 porcine RVCs revealed the presence of six clusters including one cluster each from human and bovine RVCs with a cut-off value of 80%. In addition, VP7 genes of the 22 porcine RVCs were grouped into four of the seven known clusters on the basis of cut-off values of 85% at the nucleotide level reported previously. The data presented here demonstrate that multiple porcine RVC strains with distinctive genotypes based on a combination of the VP4 and VP7 genes are widely distributed and circulated among farms throughout Japan. According to establishment of dual genetic classification for VP4 and VP7 genotypes of porcine RVCs, furthermore, we discovered a possible event of gene reassortment between different rotavirus strains from the same farm. Our findings should advance the understanding of the evolution and pathogenicity of RVCs.

Phylogenetic characterization of VP6 gene (inner capsid) of porcine rotavirus C collected in Japan.

Porcine rotavirus C (RVC) has been often detected in sporadic cases or outbreaks of diarrhea in suckling and weaned pigs. Previous surveillance studies using both enzyme-linked immunosorbent assays and reverse-transcription polymerase chain reaction in some countries including Japan and the United States have demonstrated a high prevalence of porcine RVCs. In order to understand the phylogenetic relatedness of RVCs, we performed genetic analysis of VP6 gene encoding inner capsid protein by using 22 porcine RVC strains collected in Japan from 2002 to 2010. Comparative analyses of the VP6 nucleotide and amino acid sequences from these porcine RVCs exhibited lower sequence identities than those from human and bovine RVCs. The phylogenetic analysis of VP6 gene of RVC indicated the presence of seven clusters (tentatively assigned I1-I7) according to host species with cut-off values of 87% at the nucleotide level, and VP6 genes of porcine RVCs were divided into five genotypes. These findings indicate that multiple porcine RVC strains with distinctive genotypes are broadly spreading and circulating among farms in Japan. Our data may provide important insights in understanding evolutionary dynamics of RVCs.