Low prevalence of equine coronavirus in foals in the largest thoroughbred horse breeding region of Japan, 2012-2014.

BACKGROUND: Equine coronavirus (ECoV) is considered to be a diarrheic pathogen in foals. In central Kentucky in the United States, it has been shown that approximately 30 % of thoroughbred foals are infected with ECoV and thus it is considered widely prevalent. In contrast, the epidemiology of ECoV and its relationship to diarrhea in foals are poorly understood in Japan. We investigated ECoV in rectal swabs collected from thoroughbred foals in Japan. RESULTS: We collected 337 rectal swabs from 307 diarrheic foals in the Hidaka district of Hokkaido, the largest thoroughbred horse breeding region in Japan, between 2012 and 2014. In addition, 120 rectal swabs were collected from 120 healthy foals in 2012. These samples were tested by reverse transcription loop-mediated isothermal amplification and a real-time reverse transcription-polymerase chain reaction. All samples collected from diarrheic foals were negative, and only three samples (2.5 %) collected from healthy foals were positive for ECoV. Compared with central Kentucky, ECoV is not prevalent among thoroughbred foals in the Hidaka district of Hokkaido. CONCLUSION: ECoV is not prevalent and was not related to diarrhea in thoroughbred foals in the Hidaka district of Hokkaido between 2012 and 2014.

Complete genome analysis of equine coronavirus isolated in Japan.

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.

Experimental inoculation of equine coronavirus into Japanese draft horses.

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.

Effect of freezing treatment on colostrum to prevent the transmission of bovine leukemia virus.

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.

Epidemic of equine coronavirus at Obihiro Racecourse, Hokkaido, Japan in 2012.

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 4.7 kDa non-structural protein (p 4.7) differed among the strains. Because of the diversity of the p4.7 region, this region should be useful for epidemiological investigation of ECoV.

Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) as an antiinflammatory target: discovery and in vivo activity of selective pyrazolo[1,5-a]pyrimidine inhibitors using a focused library and structure-based optimization approach.

A novel class of mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) inhibitors was discovered through screening a kinase-focused library. A homology model of MAPKAP-K2 was generated and used to guide the initial SAR studies and to rationalize the observed selectivity over CDK2. An X-ray crystal structure of a compound from the active series bound to crystalline MAPKAP-K2 confirmed the predicted binding mode. This has enabled the discovery of a series of pyrazolo[1,5-a]pyrimidine derivatives showing good in vitro cellular potency as anti-TNF-α agents and in vivo efficacy in a mouse model of endotoxin shock.

Identification of a quantitative trait locus for spontaneous osteoarthritis in STR/ort mice.

Osteoarthritis (OA) is the most common joint disorder in humans. Most of the animal models of OA were developed by surgical destabilization of joints or through transgenic approaches, and information from naturally occurring models of OA is very limited. The mouse strain STR/ort is recognized as a spontaneous model of OA. This mouse is unique in that it develops late onset cartilage degeneration of the tibio-femoral joint, similar to human OA. The purpose of this study was to identify quantitative trait loci (QTL) for the OA phenotype in STR/ort. Whereas the trait had been reported to be recessive, a significant population of the F1 generation exhibited OA phenotype. Thus, backcrossed (BC) mice generated by crossing F1 male to C57BL/6N female mice were used for genetic analysis. Degeneration of articular cartilage in BC mice was evaluated by scanning electron microscopy. Linkage analysis was carried out using microsatellite markers covering the entire genome. Cartilage degeneration in STR/ort mice was a polygenic trait. A QTL for the OA phenotype was mapped to a region 20 centimorgans proximal to the centromere of chromosome 4 (LOD = 3.37, p = 0.0065). A QTL associated with the onset of cartilage degeneration in C57BL/6N mice was also identified on chromosome 5 (LOD = 3.04, p = 0.0147). These results suggest that multiple loci are involved in the OA phenotype in mice.

[Animal models for bone and joint disease. Mouse models develop spontaneous osteoarthritis].

Osteoarthritis (OA) is a major joint disorder that significantly decreases the quality of life of elderly. Animal models, especially mice, have contributed in studies on a number of diseases. Among mice develop OA spontaneously, genetically-modified and naturally mutated mice have been reported. STR/ort is a well recognized model, which pronounces natural onset OA. Here we introduce the several models possessed spontaneous OA with focusing the feature and utilization of STR/ort.

Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain.

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.