1株猪源牛病毒性腹泻病毒的分离与鉴定

从BVDV阳性仔猪病料中分离病毒,为开展猪源BVDV病原学研究奠定基础。将处理后BVDV阳性仔猪组织样品接种MDBK细胞,分离到1株猪源BVDV,命名为SD0803株。通过细胞培养、直接免疫荧光、5′-UTR与Npro PCR扩增、电镜观察、TCID50测定及对其分子进化特征加以分析。结果表明,该毒株在MDBK细胞上盲传至13代未出现细胞病变。在直接免疫荧光试验中呈阳性荧光信号。PCR扩增分别获得5′-UTR与Npro预期大小DNA片段。电镜观察,病毒粒子略呈圆形,有囊膜,直径约50nm。病毒滴度为10-6.5 TCID50.0.2 mL-1。SD0803 5′-UTR、Npro序列进化分析显示,该分离株属于BVDV-1,与已知BVDV-1各亚型之间同源性较低,单独成一分支。结果表明,成功分离鉴定1株猪源BVDV SD0803,该毒株为非致病变型BVDV-1,极有可能为BVDV-1新的亚型。

Isolation and Identification of a Pig Bovine Viral Diarrhea Virus

The aim of this study was to isolate virus from bovine viral diarrhea virus(BVDV) positive sample of the piglet and to study BVDV originating from pigs.BVDV positive samples from the piglet were inoculated in MDBK cells and a pig BVDV strain(SD0803) was obtained.In the study,cell culture,direct immunofluorescence assay,PCR amplification of 5′-UTR,electron microscopy and TCID50 were performed,moreover,bioinformatics software were used to analyze the characteristics of molecular evolution.The results showed that the virus strain hadn’t caused cytopathic effect(CPE) until the 13th passage on MDBK cell.SD0803 infected cells showed strong fluorescent signal.DNA fragment of 307 bp and 428 bp were amplified by PCR from 5′-UTR and Npro of SD0803.Under electron microscopy,virus particles were near round and enveloped with size of 50 nm in diameter.Virus titer of SD0803 was 10-6.5 TCID50·0.2 mL-1.The homology comparison and phylogenetic analysis of 5′-UTR and Npro between SD0803 and representative BVDV isolates showed that SD0803 belongs to BVDV-1.However,SD0803 had an obvious distance from the subtypes of BVDV-1 and formed a separate group according to the phylogenetic relationship and this showed that the isolate might be a new subtype of BVDV-1.So a pig originated BVDV SD0803 was successfully isolated and identified,and belongs to NCP BVDV-1 and maybe a probably new subtype of BVDV-1.