猪繁殖与呼吸综合症病毒分离株ORF5和Nsp2基因的序列分析

根据美洲型猪繁殖与呼吸综合症病毒(PRRSV)基因组序列设计2对引物,对疑似PRRSV感染猪的病料进行RT-PCR检测,核酸检测阳性病料进行PRRSV分离,获得了1株美洲型PRRSV,命名为GD08-2.对PRRSV GD08-2株、GD-XH株、GD08-1株的ORF5基因和部分Nsp2基因进行序列测定与分析.结果表明,GD08-2株的ORF5基因与PRRSV疫苗株pMLV、经典株VR-2332和GD-XH株的相似性较高,而与GD08-1株的相似性较低.GD08-2株的Nsp2基因推导的氨基酸序列出现12个氨基酸的不连续缺失,分别位于66~74位和192~194位,由此推测,GD08-2株可能为PRRSV的突变株.GD08-1株的ORF5基因与国内分离的高致病性PRRSV相似性较高,其Nsp2基因推导的氨基酸序列存在30个氨基酸的缺失,与国内高致病性PRRSV分离株JXA1的缺失位置完全一致,由此推测,GD08-1株属于高致病性PRRSV;GD-XH株的ORF5基因与疫苗株pMLV的相似性较高,其Nsp2基因存在个别核苷酸的点缺失.遗传进化分析表明,GD08-1株与国内分离的高致病性PRRSV的遗传进化关系较近,属同一分支;GD-XH株和GD08-2株与PRRSV经典株VR-2332、疫苗株pMLV的遗传关系较近,属另一分支.

Isolation of Porcine Reproductive and Respiratory Syndrome Virus and Sequence Analysis of ORF5 and Nsp2 Genes

Two pairs of primers were designed and synthesized according to genome sequences of porcine reproductive and respiratory syndrome virus (PRR_SV) available.The tissues of swine were detected by a RT-PCR method.Virus had been isolated and identified from the tissue whose detection turned to be positive.All the results indicated that the virus isolated was American PRR_SV,which was designed GD08-2.ORF5 and Nsp2 genes of GD-XH,GD08-1 and the isolated strain GD08-2 were amplified,cloned and sequenced respectively.The results showed that ORF5 gene of GD08-2 had high similarity with PRR_SV pMLV,PRR_SV VR-2332 and GD-XH strain,but a little low with GD08-1.The discontinuous deletions of 12 amino acids were found at sites 66-74 and 192-194 in the peptide deduced from the Nsp2 gene,The results showed that the GD08-2 strain might be the deletion mutant isolate of PRR_SV.The ORF5 gene of GD08-1 shared highly similarity with highly pathogenic PRR_SV strains.The deletions of 30 amino acids in GD08-1 strain was consistent with the highly pathogenic PRR_SV JXA1 strain.The results showed that the GD08-1 strain might be highly pathogenic PRR_SV.The ORF5 gene of GD-XH strain shared highly similarity with PRR_SV pMLV,and there were some deletions in the Nsp2 gene of GD-XH.Phylogenetic analysis showed that GD08-1 strain had the closest relationship with highly pathogenic PRR_SV strains,and belonged to the same group,while GD08-2 strain and GD-XH strain were attributed to another group with PRR_SV pMLV and VR-2332.