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为了监测猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)流行毒株的基因变异情况,对四川省2014-2015年蓝耳病发病猪场分离到的6株PRRSV毒株ORF5基因进行了克隆测序及序列分析。同源性分析表明,6株PRRSV分离株ORF5基因核苷酸(氨基酸)与VR2332株的同源性为88.9%~89.4%(86.5%~88.7%),与CH-1a株同源性为93.7%~94.7%(90%~92%),与JXA1株同源性为97.2%~98.5%(95%~97%),与美国新出现的变异株NADC30同源性为85.9%~86.7%(85.5%~87.5%),与欧洲型代表株Lelystad virus同源性为62.7%~63.7%(56.5%~57.5%)。遗传进化树分析表明,6株PRRSV与JXA1、HuN4等高致病毒株亲缘关系近,并位于同一分支。氨基酸序列分析表明,6株PRRSV ORF5基因编码氨基酸在PRRSV毒力相关位点aa13、aa151和区分野毒与疫苗毒的位点aa137均与JXA1、HUN4等强毒株相同,表明6个分离株均为较强的野毒株。在中和表位(aa37~aa45)和非中和表位(aa27~aa30,aa180~aa197)等区域与国内外参考毒株VR2332、CH-1a、JXA1、HUN4、NADC30、HENAN-XINX、JL580等相比,也出现了不同程度的变异。抗原性分析结果表明,6株PRRSV ORF5基因编码产物的抗原表位主要位于aa30~aa39,aa50~aa60,aa128~aa132,aa136~aa141,aa146~aa155,aa161~aa183,aa191~aa200,与JXA1具有相似的抗原性特征,而与VR2332差异较大,主要表现在aa30~aa39相较于VR2332株抗原区域明显变窄。而在6个分离株中,SN9的抗原表位明显低于其他任何毒株。本研究结果表明,四川省PRRSV流行毒株仍然为JXA1变异株,但在当前高频度活疫苗免疫下,其基因的变异和抗原表位的改变在加剧,需加强对PRRSV基因变异的监控。
In order to monitor the genetic variation of the porcine reproductive and respiratory syndrome virus (PRRSV) epidemic strains, 6 PRRSV isolates of PRRSV isolates were isolated from pigs infected with blue ear disease in 2014-2015 in Sichuan Province Clone sequencing and sequence analysis were performed. Homology analysis showed that the homology of ORF5 nucleotide and amino acid sequence of VR2332 was 88.9% ~ 89.4% (86.5% ~ 88.7%) in 6 PRRSV isolates and 93.7% in CH-1a strain % To 94.7% (90% -92%). The homologies with JXA1 strains ranged from 97.2% to 98.5% (95% -97%). The homology with the newly emerged mutant strain NADC30 in USA was 85.9% -86.7% 85.5% -87.5%). The homology was 62.7% -63.7% (56.5% -57.5%) with Lelystad virus of European representative strain. Phylogenetic tree analysis showed that 6 strains of PRRSV were closely related to JXA1 and HuN4 strains and were located in the same branch. Amino acid sequence analysis showed that the 6 ORFs of PRRSV ORF5 encoded the same amino acid sequences as aa13 and aa151 of virulence-associated sites of PRRSV and aa137 of virulent and virulent vaccines, which were similar to those of JXA1 and HUN4. For the stronger wild strains. In the regions of neutralizing epitope (aa37 ~ aa45) and non-neutralizing epitopes (aa27 ~ aa30, aa180 ~ aa197) and other domestic reference strains VR2332, CH-1a, JXA1, HUN4, NADC30, HENAN-XINX, JL580 Compared to other, there have been varying degrees of variation. The results of antigenicity analysis indicated that the epitopes of ORF5 gene of six PRRSV genes were located in aa30 ~ aa39, aa50 ~ aa60, aa128 ~ aa132, aa136 ~ aa141, aa146 ~ aa155, aa161 ~ aa183, aa191 ~ aa200, Similar to the antigenic characteristics, and VR2332 differences are larger, mainly in the aa30 ~ aa39 VR2332 strain compared to the antigen region was significantly narrowed. In the six isolates, SN9 epitopes were significantly lower than any other strains. The results of this study showed that the epidemic strains of PRRSV in Sichuan Province were still JXA1 mutant strains. However, under the current immunization with high-frequency live vaccines, the variation of their genes and antigenic epitopes are aggravating. The surveillance of PRRSV gene mutation needs to be strengthened.