基因2型牛病毒性腹泻病毒河南株分离鉴定及全基因序列分析

Identifi cation and the Whole Genome Sequence Analysis of Henan Isolate of Genotype 2 Bovine Viral Diarrhea Virus

河南某地区牛场部分牛出现呼吸道症状,伴随有怀孕母牛的流产,本研究使用细胞培养的方法于病死牛脾脏中分离得到1株BVDV病毒,命名为BVDV-HEN01。该病毒在MDBK细胞上生长,未发生固缩、拉网等病变,可确定该毒株为1株非致细胞病变毒株;下载若干条BVDV全基因序列,经MegaAlign软件比对在同源性较高的区域设计全基因组扩增引物10对,经鉴定该毒株为BVDV-2型毒株,并扩增出该毒株的全基因组;直接免疫荧光法测得病毒TCID_(50)为10^(-4.1)/0.1 mL。使用电镜观察病毒颗粒,颗粒的直径为50 nm左右。对分离株进行同源性分析并绘制系统进化树,发现HEN01株与我国SD1301株进化关系密切。本实验所分离的毒株为1株新的BVDV-2b亚型毒株,全基因组序列测定的完成对于反向遗传工程与疫苗的研发有重大意义。

Some cattle in a certain farm in Henan have respiratory symptoms accompanied by the miscarriage of pregnant cows.In this experiment,a BVDV virus was isolated from the spleens of aborted fetuses using the RT-PCR method and named BVDV-HEN01.The virus grows on MDBK cells without specific antigens,and the cells have not contracted and other lesions.The strain can be identified as a non-cytopathic strain;according to the design of the SD1303 strain in NCBI GenBank,a pair of primers and the whole genome Amplified10 pairs of primers,and the strain was identified as a BVDV-2 strain,and the whole genome of the strain was amplified;The virus titer measured by direct immunofluorescence was 10~(-4.1)/0.1 mL;the virus particles were observed using an electron microscope.The diameter is about 50 nm.The phylogenetic tree showed that the phylogenetic relationship between the HEN01 strain and the SD1301 strain is close.The strain isolated in this experiment is a new strain of BVDV-2b and the completion of the whole genome sequence determination is of great significance for reverse genetic engineering and vaccine research.