免疫压力下HP-PRRSV田间流行毒株的遗传进化分析

Evolution of highly pathogenic porcine reproductive and respiratory syndrome virus field strains under immune selective pressure

为研究在免疫压力下HP-PRRSV田间流行毒株的遗传进化情况,选取2家疫苗免疫猪场、2家未免疫猪场,连续2年每季度采集血清利用ELISA检测PRRSV抗体;采集血清、组织病料及精液通过RT-PCR检测PRRSV病原,并对阳性样品进行扩增、克隆、测序分析。结果,共获得Nsp2基因序列91个,ORF5、ORF6、ORF7基因序列各98个,均属于美洲型高致病性变异毒株(HP-PRRSV);Nsp2、ORF5、ORF6、ORF7基因核苷酸及其推导氨基酸序列的同源性分别为90.1%~100%和91.6%~100%、83.3%~100%和81.1%~100%、96.6%~100%和96.0%~100%、91.1%~100%和91.4%~100%,表明ORF6基因的同源性最高、ORF5基因的同源性最低;Nsp2、ORF5、ORF6、ORF7基因的平均进化速率分别为4.30×10-4、4.09×10-5、2.65×10-6、4.89×10-6 substitutions/site/day(s/s/d),表明Nsp2基因最容易发生变异,而ORF6基因最稳定;Nsp2、ORF5、ORF6、ORF7基因的平均进化速率在免疫猪场分别为4.42×10-4、4.20×10-5、2.71×10-6和4.93×10-6 s/s/d,在非免疫猪场分别为4.18×10-4、3.98×10-5、2.59×10-6和4.84×10-6 s/s/d,表明免疫猪场中HP-PRRSV流行毒株的进化速率高于非免疫猪场中HPPRRSV流行毒株的进化速率。本研究结果为掌握HP-PRRSV的分子流行病学规律提供了详细的基础数据。

To investigate the molecular evolution of highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)field strains under immune selective pressure,serum,semen and tissue samples were collected quarterly from four scale pig farms during 2011-2012,of which two farms were vaccinated and the other two farms were not vaccinated with PRRSV vaccines,to detect PRRSV antibody by ELISA and pathogen by RT-PCR.The pathogen positive samples were amplified,cloned,sequenced and analyzed.As a result,all the four farms were positive for PRRSV antibody and pathogen,and 91Nsp2,98ORF5,98ORF6 and 98ORF7gene sequences were obtained.Sequence analysis revealed that all the sequences belonged to HP-PRRSV of North American(NA)type and the nucleotide and amino acid homology of Nsp2,ORF5,ORF6 and ORF7gene sequences were 90.1%-100% and 91.6%-100%,83.3%-100% and 81.1%-100%,96.6%-100%and 96.0%-100%,91.1%-100% and 91.4%-100%,respectively,indicating that ORF6 gene was the most conservative and ORF5 gene was the most variant.The mean substitution rates of the Nsp2,ORF5,ORF6 and ORF7genes of the epidemic strains in the four farms were 4.30×10-4,4.09×10-5,2.65×10-6 and 4.89×10-6 substitutions/site/day(s/s/d),respectively,indicating that Nsp2 gene is the most easily mutated and ORF6 gene is the most stable among the four genes.The mean substitution rates of Nsp2,ORF5,ORF6 and ORF7genes were 4.42×10-4,4.20×10-5,2.71×10-6 and 4.93×10-6 s/s/d in the vaccinated farms,respectively,and 4.18×10-4,3.98×10-5,2.59×10-6 and 4.84×10-6 s/s/d in the non-vaccinated farms,respectively,indicating that the evolutionary rates of these genes in the vaccinated herds were higher than those in the non-vaccinated herds.The results of this study provided the fundamental data for HP-PRRSV molecular epidemiology.