一株牛病毒性腹泻病毒BVDV-GSLY的分离鉴定

Isolation and Identification of Bovine viral diarrhea virus Strain GSLY

为了解甘肃地区牛病毒性腹泻病毒的流行情况及其分子特征,通过RT-PCR方法对甘肃地区某规模化养牛场的牛腹泻粪便样品进行了BVDV鉴定。将鉴定为BVDV阳性的牛腹泻粪便样品处理后接种到牛肾传代细胞(MDBK)进行病毒的分离,并通过RT-PCR、免疫荧光检测及病毒基因序列测定对分离的病毒进行鉴定及遗传进化分析。结果表明,成功分离到一株牛源BVDV,命名为BVDV-GSLY株;BVDV-GSLY毒株接种MDBK细胞培养可导致明显的致细胞病变效应,说明该毒株为致细胞病变型(CP)BVDV毒株;5′-UTR和Npro PCR扩增结果为阳性且扩增片段与预期大小相符;免疫荧光试验结果表明,接种BVDV-GSLY毒株的MDBK细胞可与FITC标记的BVDV抗体反应产生明亮的特异性绿色荧光,而正常MDBK对照细胞中没有荧光,表明该分离物是BVDV;BVDV-GSLY毒株在MDBK细胞上连续传12代,其病毒滴度可达10^(7.8)TCID_(50)/0.1 mL;基于5′-UTR和Npro核苷酸序列对BVDV-GSLY毒株进行序列比对和遗传进化分析,发现BVDV-GSLY毒株与美国的SD1株亲缘关系最近,5′-UTR核苷酸序列同源性为95.5%,Npro核苷酸序列同源性为91.5%,系统进化树分析也表明,BVDV-GSLY毒株与SD1株位于同一分支,说明BVDV-GSLY毒株属于BVDV-1a基因亚型。本研究成功分离到一株CP型BVDV-1a亚型毒株,不仅丰富了BVDV的分子流行病学数据,也为甘肃地区牛病毒性腹泻病的防控及相关疫苗的研发提供了一定的理论基础。

To understand the prevalence and molecular characteristic of Bovine viral diarrhea virus(BVDV)in Gansu Province,stool samples of cattle with diarrhea symptoms in a cattle farm in Gansu Province were collected and detected for BVDV by RT-PCR.The stool samples identified as BVDV positive were treated and then inoculated into MDBK cells for virus isolation and culture.Subsequently,the isolated virus was identified by RT-PCR,immunofluorescence detection and viral gene sequencing,and genetic evolution analysis was then carried out.The result revealed that a BVDV strain was successfully isolated and named BVDV-GSLY.BVDV-GSLY was identified as a cytopathogenic(CP)biotype since BVDV-GSLY could produce cytopathic effect(CPE)on MDBK cells.The 5′-UTR and the Npro of BVDV-GSLY were amplified by RT-PCR and the PCR products were the expected sizes,respectively.Immunofluorescence assay showed that BVDV-GSLY could react with BVDV antibody to generate specific fluorescence.The virus titer was 107.8TCID50/0.1 mL when BVDV-GSLY passaged 12 times in MDBK cells.The genetic evolution analysis of the 5′-UTR and Npro sequences indicated that BVDV-GSLY was closely related to SD1 strain,and the homology with SD1 strain was 95.5%in 5′-UTR and 91.5%in Npro,respectively.The phylogenetic tree also showed that BVDV-GSLY was in the same branch with SD1 strain,indicating that BVDV-GSLY belonged to BVDB-1a subgenotype.A BVDV subgenotype 1a strain was successfully isolated,and the research enriched the molecular epidemiology of BVDV and provided the theoretical foundation for prevention and control of BVDV in Gansu Province and the development of BVDV vaccine.