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.

Base on the sequence of S genes, which encode spike proteins, we previously identified three different types (North American, S INDEL, and S large-DEL types) of porcine epidemic diarrhea virus (PEDV) that have re-emerged in Japan since 2013. Based on experimental infections with the North American and S large-DEL types, we also hypothesized that PEDV virulence may be linked to the S1 subunit of the S protein. To test this hypothesis, we have now assayed in gnotobiotic piglets various recombinant PEDVs generated by reverse genetics. Piglets inoculated with CV777 maintained in National Institute of Animal Health, along with piglets infected with a recombinant form of the same virus, developed subclinical to mild diarrhea. In contrast, severe watery diarrhea, dehydration, weight loss, astasia, and high mortality were observed in piglets inoculated with recombinant strains in which the S gene was partially or fully replaced with corresponding sequences from the highly virulent Japanese PEDV isolate OKN-1/JPN/2013. Indeed, symptoms resembled those in piglets inoculated with the OKN-1/JPN/2013, and were especially pronounced in younger piglets. Collectively, the data demonstrate that the S1 subunit of the S protein is an important determinant of PEDV virulence, and advance development of new vaccine candidate.

Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.

Bovine coronavirus (BCoV), a major causative pathogen of bovine enteric and respiratory diseases and a zoonotic pathogen transmissible between animals and humans, has led to severe economic losses in numerous countries. BCoV belongs to the genus Betacoronavirus, which is a model of a pathogen that is threatening human health and includes severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus. This study aimed to determine whether photocatalytic material effectively reduces CoVs in the environment. Using the film adhesion method of photocatalytic materials, we assessed its antiviral activity and the effect of visible light irradiation according to methods defined by the International Organization for Standardization. Consequently, photocatalytic material was found to have antiviral activity, reducing the viral loads by 2.7 log TCID₅₀ (tissue culture infective dose 50)/0.1 mL (500 lux), 2.8 log TCID₅₀/0.1 mL (1000 lux), and 2.4 log TCID₅₀/0.1 mL (3000 lux). Hence, this photocatalytic material might be applicable not only to reducing CoVs in the cattle breeding environment but also perhaps in other indoor spaces, such as offices and hospital rooms. To our knowledge, this study is the first to evaluate the antiviral activity of a photocatalytic material against CoV.