猪繁殖与呼吸综合征病毒分离株遗传进化分析及通用实时荧光RT-PCR检测方法的建立和应用
发布时间：2019-06-28 16:52
【摘要】：猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome, PRRS)是由猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome virus, PRRSV)引起的一种危害全球养猪业的病毒性传染病。目前,在我国猪群中经典毒株、高致病性毒株和类NADC30毒株等美洲型PRRSV以及欧洲型PRRSV同时存在,导致国内PRRS流行状况十分复杂。为掌握我国PRRSV的流行状况及其遗传变异特点,本研究对2008-2015年间在国内分离的61株PRRSV毒株进行全基因测序和序列比对,并对主要易变区Nsp2、GP5、GP3蛋白进行分析。全基因分析结果显示,61个毒株中有5个欧洲型PRRSV和56个美洲型PRRSV。56个美洲型PRRSV中有2个类NADC30 PRRSV和54个高致病性PRRSV。54个高致病性PRRSV分离株之间的同源性为95.2%-99.9%,与高致病性PRRSV代表毒株JXA1的同源性为97.2%～99.3%。2个类NADC30分离株之间的同源性为99.9%,与类NADC30 PRRSV代表毒株NADC30的同源性为95.4%。5个欧洲型分离株之间的同源性为84.4%～99.7%,与欧洲代表毒株LV的同源性为87.2%-90.9%。重要蛋白基因分析结果显示,PRRSV在不断变异中,以非结构蛋白Nsp2和结构蛋白GP5的变异最快。61个分离株在非结构蛋白Nsp2部位均发生了 1-150aa不等的缺失。大部分分离株在重要结构蛋白GP5发生氨基酸突变,尤其是糖基化位点的位置和数量发生了不同程度的变化。欧洲型分离株GP3高变区含有多个缺失及突变。我们还发现了 4株新的PRRSV基因缺失毒株,这些毒株核苷酸或氨基酸的缺失位置及数量在中国尚未见相关报道。以上结果表明,猪群中的PRRSV主要流行毒株仍然是以高致病性PRRSV为主,但随着欧洲型PRRSV和类NADC30 PRRSV的发现,使得PRRS的防控情况十分严峻,迫切需要我们加强PRRSV监测工作。鉴于我国PRRSV流行毒株复杂多变的特点,为改进现有PRRSV通用荧光RT-PCR检测方法,根据国内PRRSV主要流行毒株全基因序列,设计特异性探针与引物,经过探针引物筛选及优化,建立了新的PRRSV通用型荧光定量RT-PCR检测方法。该方法具有较好的特异性、敏感性和重复性,能特异地检测国内所有PRRSV美洲型和欧洲型主要代表毒株,与其他主要猪病病原无交叉反应;其美洲型和欧洲型毒株的检测下限分别为20copies/μL和22copies/μL,特别在检测美洲经典型毒株和欧洲毒株时,敏感性优于当前商用试剂盒;其批内、批间重复试验中变异系数均在2.5%以内。用812份临床样品进行检测,符合率为94.2%。结果表明,该方法可广泛应用于PRRSV临床诊断以及PRRSV病原普查等工作。总之,本研究摸清了国内PRRSV的流行状况和遗传进化趋势并且建立了 PRRSV通用型荧光定量RT-PCR检测方法,对PRRS预防、控制和消灭具有重要意义。
[Abstract]:Porcine Reproductive and Respiratory Syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a viral infectious disease caused by Porcine Reproductive and Respiratory Syndrome virus (Porcine Reproductive and Respiratory Syndrome virus, PRRSV), which endangers the global pig industry. At present, American PRRSV, such as classical strains, highly pathogenic strains and NADC30-like strains, as well as European PRRSV, exist at the same time in pig populations in China, which leads to the complex epidemic situation of PRRS in China. In order to master the epidemic situation and genetic variation characteristics of PRRSV in China, 61 PRRSV strains isolated in China from 2008 to 2015 were sequenced and sequenced, and the Nsp2,GP5,GP3 proteins in the main variable regions were analyzed. The results of whole gene analysis showed that the homology between 5 European PRRSV and 56 American PRRSV.56 American PRRSV and 54 highly pathogenic PRRSV.54 highly pathogenic PRRSV isolates was 95.2% and 99.9%, respectively, and the homology with the highly pathogenic PRRSV representative strain JXA1 was 97.2% 鈮，
本文编号：2507460
[Abstract]:Porcine Reproductive and Respiratory Syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a viral infectious disease caused by Porcine Reproductive and Respiratory Syndrome virus (Porcine Reproductive and Respiratory Syndrome virus, PRRSV), which endangers the global pig industry. At present, American PRRSV, such as classical strains, highly pathogenic strains and NADC30-like strains, as well as European PRRSV, exist at the same time in pig populations in China, which leads to the complex epidemic situation of PRRS in China. In order to master the epidemic situation and genetic variation characteristics of PRRSV in China, 61 PRRSV strains isolated in China from 2008 to 2015 were sequenced and sequenced, and the Nsp2,GP5,GP3 proteins in the main variable regions were analyzed. The results of whole gene analysis showed that the homology between 5 European PRRSV and 56 American PRRSV.56 American PRRSV and 54 highly pathogenic PRRSV.54 highly pathogenic PRRSV isolates was 95.2% and 99.9%, respectively, and the homology with the highly pathogenic PRRSV representative strain JXA1 was 97.2% 鈮，
本文编号：2507460
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