牦牛感染牛病毒性腹泻病毒的诊断与病毒分离鉴定

Diagnosis of bovine viral diarrhea virus infection in yak by isolation and identification of the virus

为了探讨牦牛养殖场发病犊牛的致病原及其生物学特征,采用临床诊断和病毒分离方法对犊牛进行诊断,并对分离毒株E0基因测序分析后进行同源性比较和遗传进化分析。发病犊牛临床主要表现出体温升高、食欲减退、口腔黏膜溃疡、水样腹泻、粪便带有血液和肠黏膜等典型牛病毒性腹泻病毒（BVDV）感染症状。病牦牛的鼻黏液、粪便样品接种牛肾传代细胞（MDBK）出现明显的细胞病变。该分离毒株的效价达到TCID（50）为10^（-5.19）/0.1 mL,能被BVDV阳性血清所中和,BVDV间接免疫荧光试剂盒检测为阳性。其E0基因核苷酸序列与GenBank上登录的BVDV-1b亚型同源性高达99.5%。结果表明：该牦牛养殖场疫情为BVDV感染,分离获得的毒株为我国BVDV毒株资源与分子流行病学提供了资料。

To explore the agent of morbid yak calves on yak farms and elucidate its biological traits,clinical diagnosis and virus isolation were performed in this study. The E0 gene of virus was amplified and sequenced,which was then used to analyze homology and construct a phylogenetic tree. The clinical signs of the yak calves were fever,anorexia,oral ulcer,diarrhea,stool with blood and intestinal mucosa and other typical symptoms of BVDV infection. Then,MDBK cells were used to incubate virus in and isolate it from nasal mucus and fecal samples which resulted in obvious cytopathic effect. The titer of the isolated virus was as high as TCID（50） at 10^（-5. 19）/0. 1 mL,which was neutralized by BVDV positive serum and tested to be positive by indirect immunofluorescence detection kit. The E0 nucleotide sequence of the virus showed 99. 5% homology with the subtype BVDV-1b deposited in Gen Bank. Our results proved the epidemic on the yak farms was BVDV infection. The virus strain isolated here provided reference for effective prevention and control BVDV infection of yak calves,added to the resources of BVDV strains and molecular epidemiology in China.