PRRSV国内流行毒株的分离鉴定及结构基因的分析  

Islolation and Characterization of Chinese PRRSV Isolates and Sequence Analysis of Structural Genes

作　　者：马平[1] 石文达[1] 刘永刚[1] 蔡雪辉[1] 王洪峰[1] 王淑杰[1] 李成君[1] 张琪[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,黑龙江哈尔滨150001

出　　处：《动物医学进展》2008年第6期43-50,共8页Progress In Veterinary Medicine

摘　　要：从国内发病猪场采集的猪繁殖与呼吸综合征（PRRS）病料中,分离到8株猪繁殖与呼吸综合征病毒（PRRSV）。病毒分离培养表明,JS1-JS5第1代即可适应Marc-145细胞。GD1-GD3经Marc-145细胞3次-4次传代后出现明显的细胞病变。间接免疫荧光试验表明,8个分离毒株与抗PRRSV抗体均可出现特异性的胞浆荧光。它们对氯仿、甲醛、酸和碱均敏感。5-溴-2′-脱氧尿核苷对其无抑制作用。参照GenBank中已发表的PRRSV的序列设计了两对特异性引物,分别对所分离毒株进行部分nsp2基因和结构基因的扩增。发现JS1-JS5nsp2基因发生了部分缺失。选择JS1、JS5、GD3进行nsp2基因和结构基因的扩增和测序。序列分析结果表明,JS1、JS5和2006年以后国内分离到的高致病性PRRSV高度同源。GD3的Nsp2基因虽然没有发生第534位-第562位氨基酸的缺失,但与2006年以后分离的毒株一样,发生了第482位氨基酸的缺失。通过对3个分离毒株与其他国内分离毒株的结构基因序列进行系统进化分析发现,我国的PRRSV流行毒株可大致划分为两个亚型,JS1、JS5、GD3、CH-1a、JXA1、HUN4、SHH等属于一个亚型,BJ-4、CC-1属于另一个亚型。亚型间核苷酸序列同源性为91.2%-91.8%。Eight strains of PRRSV were isolated from pig farms which seemed suffering PRRS. JS1 to JS5 strains could deduce cytopathogenic effects（CPE） at the first generation. GD1 to GD3 strains produced cytopathogenic effects （CPE） after three to four passages. Indirect immunofluorescent assay （IFA） tests showed that specific fluorescence occurred in cytoplasm when reacted with antibody against PRRSV. The virus did not resist treatments with chloroform, pH 3.0 and decreased significantly in infectivity when exposed to 0.2% formalin for 2h at 37℃ and could not be inhibited by BUDR. Partial Nsp2 gene and structural gene were amplified by RT-PCR with primers designed by the reported sequence in GenBank. The results indicated that JS1 to JS5 had deletions in Nsp2 region. JS1 ,JS5 and GD3 strains were selected for the Nsp2 and structural gene sequencing. Nucleotide analysis by the DNASTAR（6.0） software showed that JS1 and JS5 strains shared a high homology with other strains that were isolated in 2006, such as JXAland HUN4 strains. GD3 had only one acid deletions in Nsp2 gene. Homology analysis of structural gene showed that two subtype existed in China, JS1,JSS, GD3, Ch-1a,JXA1, HUN4, SHH belong to one and BJ-4 and CC-1 belong to another. The two subgroups shared 91.2 %-91.8% homologies.

关 键 词：猪繁殖与呼吸综合征病毒 流行毒株 分离 鉴定 结构基因 

分 类 号：S852.659.6[农业科学—基础兽医学]