猪繁殖与呼吸综合征病毒四川株的分离鉴定及Nsp2生物信息学分析  

Identification of porcine reproductive and respiratory syndrome virus strains from Sichuan and Nsp2 bioinformatics analysis

作　　者：毛汐语 周远成[1] 赵军[1] 殷玥[1] 吕雯婷[1] 邓益超 樊毅[1] 许思遥 朱玲[1,2] 徐志文[1,2] 

机构地区：[1]四川农业大学动物医学院动物生物技术中心,四川成都611134 [2]四川农业大学动物疫病与人类健康四川省重点实验室,四川成都611134

出　　处：《中国兽医科学》2016年第12期1497-1504,共8页Chinese Veterinary Science

基　　金：四川省科技支撑计划项目(2014NZ0043);科技部国际科技合作项目(2014DFA31260)

摘　　要：为了解四川省猪繁殖与呼吸综合征病毒(PRRSV)的流行情况及分子特征,本研究选用2012—2014年四川省不同地区采集并诊断为PRRSV阳性的病料,以Marc 145细胞进行病毒分离,经蚀斑纯化,病毒基因扩增、测序后与NCBI现有序列进行比对分析并通过构建系统进化树等方法研究分离株的分子特征。共成功分离到10株PRRSV,分析证明所有分离毒株均是高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)株,序列比对显示,其中SC2012、SC2012-2株Nsp2部分基因与众多HP-PRRSV株核苷酸同源性在91%~97%之间,与经典毒株CH-1a株同源性为72.9%,与所有BLAST毒株相比存在新缺失位点,与CH-1a、BJ-4株相比,在Nsp2蛋白485 aa^498 aa处存在14个连续氨基酸缺失,其他8株均含30个不连续氨基酸缺失。氨基酸同源性比对显示,SC2012、SC2012-2株高变区与国内分离株同源性介于61.5%~97.2%之间。通过TCID50法测得SC2012、SCSN2012-1、SCSN2012-2、SCCD2012、SCLS2012、SCMY2012、SCBZ2012、SCXC2012、SCXJ2012和SC2012-2病毒毒价分别为10^(-5.41)/m L、10^(-6.5)/m L、10^(-7.29)/m L、10^(-6.41)/m L、10^(-5.5)/m L、10^(-7.67)/m L、10^(-6.29)/m L、10^(-6.8)/m L、10^(-7.41)/m L和10^(-6)/m L。以上结果表明,本研究所分离的SC2012、SC2012-2株是新变异毒株,为四川省PRRSV分子流行病学和遗传变异研究奠定了一定基础。In order to understand the epidemic situation and molecular characteristics of porcine reproductive and respiratory syndrome virus（PRRSV）in Sichuan Province,the materials used in this study were collected from different areas of Sichuan province in 2012-2014 with diagnosis of PRRSV-positive disease, Marc 145 cells were used for virus isolation.Non-structural protein Nsp2 gene was cloned and sequenced for alignment analysis on NCBI database.Phylogenetic tree was constructed to to study the molecular characteristics of the isolates.Results showed that 10 strains of PRRSV were successfully separated and all isolates were HP-PRRSV strains.Sequence alignment results showed thatNsp2 genes of SC2012 and SC2012-2 strains share high sequence homology of 91%-97% with many HP-PRRSV strains in database,comparison of 72.9% homology with the classical strain CH-1a strain.In addition,Nsp2 of SC2012 and SC2012-2 has a new deletion site compared with the database sequence,and has a deletion of 14 consecutive amino acids between the 485 aa-498 aa compared with CH-1a and BJ-4 strains,whereas,the other 8 strains contained a deletion of 30 discontinuous amino acids.Amino acid homology shows that the hypervariable regions of SC2012,SC2012-2 strains share the homology of 61.5%-97.2% with other domestic isolated strains.The viral titer has been determined by TCID50 method,the SC2012 SCSN2012-1、SCSN2012-2、SCCD2012、SCLS2012、SCMY2012、SCBZ2012、SCXC2012、SCXJ2012 and SC2012-2 strains could cause cytopathic effect in Marc 145 cells with titers of 10^（-5.41）/m L,10^（-6.5）/m L,10^（-7.29）/m L,10^（-6.41）/m L,10^（-5.5）/m L,10^（-7.67）/m L,10^（-6.29）/m L,10^（-6.8）/m L,10^（-7.41）/m L and 10-6/m L,respectively.The results show that SC2012 and SC2012-2 strains are two new mutation strains,our study lays a foundation for the molecular epidemiology and genetic variation of PRRSV research in Sichuan Province.

关 键 词：猪繁殖与呼吸综合征病毒 分离鉴定 NSP2 生物信息学 

分 类 号：S852.659.2[农业科学—基础兽医学]