猪繁殖与呼吸综合征河南毒株Henan-1的分离及其NSP2、ORF3、ORF5的序列分析

将2007年1月河南省某猪场送检的疑似高热病病料处理后接种于Marc-145细胞,出现细胞聚集、固缩、脱落等特异性细胞病变.根据GenBank中猪繁殖与呼吸综合征病毒(PRRSV)的基因序列设计并合成针对NSP2、ORF3和ORF5基因的引物,RT-PCR扩增目的基因并将其克隆入pMD18-T载体进行测序.并与国内外已发表的毒株的序列进行比较.结果表明:分离毒株Henan-1是美洲型PRRSV,而且NSP2基因发生30个AA的不连续缺失;Henan-1的NSP2、ORF3和ORF5基因与JXA1、JX0612、HZ-X、HB-1(sh)、CH-la、HB-2(sh)、SP、ATCC VR-2332、Henan-HN1、Resp PRRS/Repro MLV、PA8、BJ-4等毒株等位基因的核苷酸同源性分别为83.0%～99.0%、88.3%～99.2%、88.6%～99.5%,推导的氨基酸同源性分别为66.7%～97.9%、84.7%～98.8%、87.1%～99.5%;而ORF3和ORF5基因与LV4.2.1等位基因核苷酸同源性分别为64.7%和64.2%,推导的氨基酸同源性分别为56.7%和61.0%.基因系统发育树分析表明:Henan-1与JXA1、JX0612株的亲缘关系很近,与HB-1(sh)亲缘关系较近,与HZ-X、CH-1a、HB-2(sh)、SP、RespPRRS/Repro MLV等毒株处于不同的分支.

Isolation and Sequence Analysis of NSP2, ORF3 and ORF5 of PRRSV Henan-1 Strain from Henan Province

The samples collected from Henan province were inoculated to Marc-145 cells,the PRRSV specific cytopathogenic effect(CPE) was observed and the isolates named Henan-1 obtained.NSP2,ORF3 and ORF5 genes of PRRSV Henan-1 strain were amplified by RT-PCR and sequenced after being cloned directly into the pMD18-T.The sequences of NSP2,ORF3 and ORF5 genes of Henan-1 strain were compared with sequences of other PRRSV strains in GenBank by Blast and DNAStar software.The results demonstrated that Henan-1 strain could classified into the North American type of PRRSV and its NSP2 had 30 amino acids deletion.The NSP2,ORF3 and ORF5 of Henan-1 shared 83.0%~ 99.0%,88.3%~99.2% and 88.6%~99.5% of nucleotide homology,and 66.7%~97.9%,84.7%~98.8% and 87.1%~99.5% of amino acid identity,with that of JXA1,JX0612,HZ-X,HB-1(sh),CH-1a ,HB-2(sh),SP,ATCC VR-2332,Henan-HN1,PA8,BJ-4 and Resp PRRS/ Repro MLV respectively.The ORF3 and ORF5 of Henan-1 shared only 64.7%,64.2% of nucleotide homology,and 56.7%,61.0% of amino acid homology with that of LV4.2.1 respectively.The phylogenetic tree revealed Henan-1 had closer relationship with JXA1 and JX0612 strains than that of HB-1(sh) strain.