延边地区牛病毒性腹泻病毒YBHC株的分离鉴定与遗传进化分析

Isolation and genetic evolution analysis of bovine viral diarrhea virus YBHC strain

延边朝鲜族自治州地理位置特殊,边境畜产品贸易往来日见频繁,为了解延边地区牛病毒性腹泻病毒(BVDV)流行株情况,本研究采集延边州规模化养牛场疑似感染BVDV牛的粪便样品,经常规处理后接种MDBK细胞,盲传5代后观察细胞病变并经PCR鉴定,结果显示未出现BVDV典型的空泡病变,PCR扩增得到约300 bp与预期相符的目的条带,结合患牛临床症状初步判定为非细胞病变型(NCP型)BVDV。对5’UTR基因序列(OM622422)的扩增产物测序并进行遗传进化分析,结果显示分离株为BVDV-1b亚型,为NCP型,命名为YBHC株BVDV-1b。对鉴定为BVDV阳性的细胞培养并传至7代,收集细胞经磷钨酸负染后电镜观察,可见典型BVDV病毒粒子,大小为40 nm~60 nm。本研究首次对延边地区进行BVDV流行株的分离与鉴定,并得到病毒粒子,为BVD疫情防控和疫苗研制提供了物质基础和参考依据。

Yanbian Korean Autonomous Prefecture has a particular geographical location,and the trade of border livestock products is frequent.To understand the epidemic strains of bovine viral diarrhea virus(BVDV)in Yanbian,feces samples from cattle suspected of being infected with BVDV were collected from large-scale cattle farms in Yanbian Prefecture.MDBK cells were inoculated with the samples after routine treatment,and the cell cultures were used for blind passages.Although one of the fifth passage samples was positive by PCR,MDBK cells infected with the fifth passage showed no cytopathic effect.Typical BVDV virions with a size of 40nm-60nm were observed under a phosphotungstic acid negative staining electron microscope.The results of genetic evolution analysis showed that the BVDV isolate,which was a non-cytopathic type(NCP),belongs to subtype 1b and was named BVDV-1b strain YBHC.This study isolated and identified a BVDV epidemic strain in the Yanbian area for the first time.The results of this study provide the material and theoretical basis for BVDV epidemic prevention and control as well as vaccine development.