A new equine coronavirus was isolated from the feces of adult horses with pyrogenic and enteric disease. The disease outbreak was mainly observed among 2- to 4-year-old horses living in stables of a draft-horse racetrack in Japan. On comparing the isolated virus (isolate Tokachi09) with the equine coronavirus NC99 strain, no significant differences were observed in several biological properties such as hemagglutinating activity, antigenicity (in indirect immunofluorescence and neutralization tests), and one-step growth (in cell culture). The sequences of the nucleocapsid and spike genes of isolate Tokachi09 showed identical size (1341 and 4092 nucleotides, 446 and 1363 amino acids, respectively) and high similarity (98.0% and 99.0% at the nucleotides, 97.3% and 99.0% at the amino acids, respectively) to those of strain NC99. However, the isolate had a 185-nucleotide deletion from four bases after the 3'-terminal end of the spike gene, resulting in the absence of the open reading frame predicted to encode a 4.7-kDa nonstructural protein in strain NC99. These results suggest that the 4.7-kDa nonstructural protein is not essential for viral replication, at least in cell culture, and that the Japanese strain probably originated from a different lineage to the North American strain. This is the first equine coronavirus to be isolated from adult horses with pyrogenic and enteric disease.

ABSTRACT. Equine coronavirus (ECoV) outbreaks have occurred three times at Obihiro Racecourse in Hokkaido, Japan. The third ECoV outbreak occurred between late February and early April 2012. The main clinical signs of affected horses were anorexia, pyrexia and leucopenia; gastrointestinal disease was observed in about 10% of affected horses. Two ECoV strains were isolated from diarrheal samples. All paired sera (9/9) collected from febrile horses showed seroconversion by neutralization test. Sequence and phylogenetic analysis of the ECoV isolated showed that putative amino acid sequences in S and N genes were highly conserved among ECoV strains. In contrast, sequences of the region coding 4.7kDa non-structural protein (p4.7) differed among the strains. Because of the diversity of the p4.7 region, this region should be useful for epidemiological investigation of ECoV.

Recently, outbreaks associated with equine coronavirus (ECoV) have occurred in Japan and the United States. While ECoV is likely to be pathogenic to horses, it has not been shown that experimental inoculation of horses with ECoV produces clinical signs of disease. In this study, we inoculated three Japanese draft horses with an ECoV-positive diarrheic fecal sample to confirm infection after inoculation and to investigate the clinical course and virus shedding patterns of ECoV. Virus neutralization tests showed that all three horses became infected with ECoV. Two of the three horses developed clinical signs similar to those observed during ECoV outbreaks, including fever, anorexia, and gastrointestinal dysfunction. All horses excreted a large amount of virus into their feces for more than 9 days after inoculation regardless of the presence or absence of clinical signs, which suggests that feces are an important source of ECoV infection. ECoV was also detected in nasal swabs from all horses, suggesting that respiratory transmission of ECoV may occur. Both symptomatic horses developed viremia, while the asymptomatic horse did not. White blood cell counts and serum amyloid A concentrations changed relative to the clinical condition of the inoculated horses; these may be useful markers for monitoring the clinical status of horses infected with ECoV. This is the first report of induction of clinical signs of ECoV infection in horses by experimental inoculation. These clinical and virological findings should aid further investigation of the pathogenesis of ECoV.

Here, we used a sheep bioassay to determine the effect of freezing colostrum to prevent the transmission of bovine leukemia virus (BLV) among neonatal calves. Leukocytes were isolated from the colostrum of a BLV-infected Holstein cow and were then either left untreated (control) or freeze-thawed. A sheep inoculated intraperitoneally with the untreated leukocytes was infected with BLV at 3 weeks after inoculation, whereas the sheep inoculated with treated leukocytes did not become infected. The uninfected sheep was inoculated again with leukocytes isolated from the colostrum of another BLV-infected Holstein cow after freezing treatment, and again it did not become infected with BLV. Finally, this sheep was inoculated with the leukocytes isolated from the colostrum of another virus-infected cow without freezing treatment, and it became infected with BLV at 4 weeks after inoculation. The results indicate that colostrum should be frozen as a useful means of inactivating the infectivity of BLV-infected lymphocytes.

Equine coronavirus has been responsible for several outbreaks of disease in the United States and Japan. Only one complete genome sequence (NC99 isolated in the US) had been reported for this pathogenic RNA virus. Here, we report the complete genome sequences of three equine coronaviruses isolated in 2009 and 2012 in Japan. The genome sequences of Tokachi09, Obihiro12-1 and Obihiro12-2 were 30,782, 30,916 and 30,916 nucleotides in length, respectively, excluding the 3'-poly (A) tails. All three isolates were genetically similar to NC99 (98.2-98.7%), but deletions and insertions were observed in the genes nsp3 of ORF1a, NS2 and p4.7.