论文部分内容阅读
Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves and respiratory tract infections in calves and feedlot cattle.Although rarely fatal, BCoV infection is associated with dramatic reductions in milk production in dairy herds and loss of body condition in both calves and adults, resulting in severe economic losses tothe cattle industry.Since BCoV was first reported in newborn calves in 1973, the infection has been frequently reported in many countries.BCoV is an enveloped, positive-sense, single-stranded, RNA virus, which belongs to the genus Betacoronavirus, family Coronaviridae, order Nidovirales.The 31-kb genome of BCoV codes for the structural proteins hemagglutinin-esterase (HE), spike (S), nucleocapsid (N), transmembrane (M), and small envelope (sM).The S protein is widely used as targets for BCoV studies involving viral phylogenesis, diagnosis,vaccine development, and host-virus interactions.Although BCoV-induced diarrhea in calves has been frequently reported, especially in the past decades in China, there is still a great deal of information that remains unknown regarding the molecular phylogenesis of BCoV strains circulating in China.In our study, an outbreak of calf diarrhea with 91% mortality rate occurred in a large-scale farm in Heilongjiang Province, northeast China.The investigation and analysis of pathogens will be helpful for understanding and control of the outbreak.After the causative agent of diarrhea in calves, bovine coronavirus (BCoV) was determined by RT-PCR, the phylogenetic analysis, bioinformatic analysis, and virus isolation of the BCoV strain (HLJ-14/CHN) were conducted,respectively.HE, S, M, N, and sM genes of the BCoV strain HLJ-14/CHN exhibited 97.3%-98.8%, 97.5%-99.1%,97.7%-99.0%, 97.7%-99.0%, and 98.0%-100% nucleotide identities, respectively, when compared with reference sequences.The phylogenetic analysis of the HE, S, M, N, and sM genes revealed that the strain HLJ-14/CHN showed a close relationship with partial BCoV strains from the US.Furthermore, in the phylogenetic tree of S gene, the strain HLJ-14/CHN exhibited a close relationship with partial US strains and Cuba strains, and differed genetically from partial US strains and these strains of Korea, Japan, Canada, Germany, Sweden, Italy, Denmark,and Ireland;578threonine was specific for all S reference sequences belonged to GⅡ group, 578serine was specific for 91% S reference sequences (51/56) that belonged to GⅠ group.The S protein of the strain HLJ-14/CHN had a unique N-glycosylation site (499NGS502G), and its antigenic index decreased because of the 617Ile substitution in the neutralizing epitopes domain Ⅱ.The presence of viral RNA of the strain HLJ-14/CHN in MDBK cell lines could be confirmed by RT-PCR, but cytopathic effect (CPE) was not observed.The BCoV strain HLJ-14/CHN was genetically conservative, and has a close relationship with partial US strains, but was genetically different from Asia strains, and European strains.The S protein of the strain HLJ-14/CHN exhibited obviously differences in the N-glycosylation sites and antigenic indexes;the 578serine/threonine substitution of S protein may be a potential genetic marker for genotyping of BCoV.The BCoV strain HLJ-14/CHN could not cause the CPE in MDBK cell lines.