1株绵羊边界病病毒的分离鉴定与序列分析  

Isolation and Sequence Analysis of a Border Disease Virus from Sheep

作　　者：刘霞[1,2] 毛立[2] 李文良[2] 杨蕾蕾[2] 张纹纹[2] 魏建忠[1] 江杰元[2] 

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,南京210014

出　　处：《畜牧兽医学报》2014年第3期434-442,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏省农业科技自主创新资金项目(CX(12)3064)

摘　　要：对华东地区某羊场采集定点监测的绵羊血液样品进行边界病的感染情况调查。通过RT-PCR方法检测瘟病毒5′-UTR基因,发现1只绵羊的血清样品在不同时间检测均为阳性,证实其为边界病病毒(BDV)持续性感染。将该阳性血清样品接种MDBK细胞,进行病毒分离鉴定。通过Npro基因RT-PCR扩增、电镜观察、病毒全基因组序列分析,并对分离毒株5′-UTR和Npro基因进行遗传进化分析。结果显示,该毒株在MDBK细胞上盲传至第8代,不发生细胞病变。RT-PCR扩增获得Npro基因预期大小片段,电镜观察以及测序分析表明成功分离出1株BDV,将其命名为JSLS12-01。设计覆盖BDV全基因组的6对引物,RT-PCR扩增并测序,该分离株全基因组序列大小为12 227bp(GenBank登录号为KC963426)。序列比对结果显示,该毒株基因组序列和氨基酸序列与已有BDV分离株之间相似性分别为72.3%~80.4%和80.1%~89.7%。5′-UTR和Npro基因系统进化分析显示,该分离株与BDV 3参考毒株亲缘关系最近,相似性最高分别为87.7%和75.8%。结果证实从绵羊血液样品中鉴定并分离到1株ncp型BDV分离株,经序列分析表明该毒株属于BDV 3亚型。In order to investigate the prevalence status of border disease,serum samples were collected from one sheep flock in east China and tested through RT-PCR method with the 5′-UTR generic primers of pestivirus.One sheep was detected as positive infection of border disease virus(BDV)at different sampling times.Positive serum sample was inoculated onto MDBK cells for viral isolation and identification.Then RT-PCR amplification of Npro gene,electron microscopy detection of isolated virus,analysis of the complete genome sequences and phylogenetic analysis of the 5′-UTR and Npro gene were performed.The results showed that no cytopathy effect(CPE) was observed until the eighth generation by serial passages.BDV was detected from cell cultures by RT-PCR using Npro gene primers;and confirmed with sequence analysis as well as electron microscopy detection.The isolate was named as JSLS12-01.Amplification of complete genomic sequence was performed with six pairs of primers covering the near-full genome.The viral genomewas about 12 227nucleotides in size and had been submitted to the GenBank with registered number KC963426.Based on comparative sequence analysis of full genome and the deduced amino acids between this isolate and BDV reference strains,there were 72.3%-80.4% and 80.1%-89.7%identities,respectively.Phylogenetic analysis using partial 5′-UTR nucleotide sequence and Npro sequence identified that the virus clustered within BDV 3viruses,with the highest homology of 87.7%and 75.8%,respectively.Above all,we successfully isolated and identified one BDV 3strain from sheep of the non-cytopathogenic biotype.

关 键 词：边界病病毒 分离 鉴定 序列分析 

分 类 号：S852.659.6[农业科学—基础兽医学]