高致病性PRRSV缺失变异株Nsp2基因的克隆与遗传变异分析

Cloning and genetic variation analysis of Nsp2 gene of highly virulent PRRSV mutant strains with deletions

从湖南省内多个出现疑似猪"高热病"症状的猪场采集病料70份,采用RT-PCR方法进行分子流行病学调查.结果检出PRRSV阳性病料54份,阳性率高达77.14%(54/70).对其中8份分别来自娄底、浏阳、永州、双峰、益阳、株洲、宁乡和溆浦的病料中的PRRSV基因组Nsp2高变区部分基因片段进行RT-PCR扩增、克隆和序列测定分析.与国内外美洲型PRRSV分离株比较,序列相似性为72.2%～100%,其中,与公布的高致病性PRRSV缺失变异毒株的同源性最高,序列相似性为98.2%～100%.序列分析表明,这些毒株均是天然存在缺失的变异毒株,其Nsp2均有2个部位出现了缺失,分别为编码1个氨基酸的3个核苷酸的缺失和编码29个氨基酸的连续87个核苷酸的缺失,与高致病性PRRSV缺失变异毒株序列的缺失情况完全一致,且与其具很高的同源性,从而确定了分离的毒株均为引起猪"高热病"的猪繁殖与呼吸综合征病毒变异株,这是国内外少见的PRRSV缺失变异及毒力显著增强的现象.

A total of 70 samples from swine with likely porcine ＂high fever＂ disease symptom, collected in various porcine farms in Hunan province were tested for PRRSV by RT-PCR amplification. PRRSV were detected in 77.14% （54/70） of the samples. Amplification, cloning and sequencing analysis were conducted towards part of Nsp2 genetic fragments in highly variable section ofgenome PRRSV of the 8 samples, from the following area： Loudi, Liuyang, Yongzhou, Shuangfeng, Yiyang, Zhuzhou, Ningxiang and shupu in Hunan province. Comparative analysis with the genomic sequences of domestic and abroad North American isolates showed that the strains shared 72.2%-100% of homology with them, and were genetically closely related to the released highly virulent PRRSV mutant strains with deletion and shared 98.2%-100% of homology with them. The strains were mutants of PRRSV with natural deletions, namely 3 nucleotides and 87 nucleotides deletion in Nsp2 which encoded 1 and 29 amino acids, respectively. The deletions of them were parallel to the highly virulent PRRSV mutant strains. The results confirmed that the strains were PRRSV mutant strains which caused porcine ＂high fever＂ disease. The finding proved that deletion mutation existed in PRRSV and it enhanced its virulence.