【摘要】：豬繁殖與呼吸綜合征(Porcine Reproductive and Respiratory Syndrome, PRRS)是由豬繁殖與呼吸綜合征病毒(Porcine Reproductive and Respiratory Syndrome virus, PRRSV)引起的一種危害全球養(yǎng)豬業(yè)的病毒性傳染病。目前,在我國(guó)豬群中經(jīng)典毒株、高致病性毒株和類NADC30毒株等美洲型PRRSV以及歐洲型PRRSV同時(shí)存在,導(dǎo)致國(guó)內(nèi)PRRS流行狀況十分復(fù)雜。為掌握我國(guó)PRRSV的流行狀況及其遺傳變異特點(diǎn),本研究對(duì)2008-2015年間在國(guó)內(nèi)分離的61株P(guān)RRSV毒株進(jìn)行全基因測(cè)序和序列比對(duì),并對(duì)主要易變區(qū)Nsp2、GP5、GP3蛋白進(jìn)行分析。全基因分析結(jié)果顯示,61個(gè)毒株中有5個(gè)歐洲型PRRSV和56個(gè)美洲型PRRSV。56個(gè)美洲型PRRSV中有2個(gè)類NADC30 PRRSV和54個(gè)高致病性PRRSV。54個(gè)高致病性PRRSV分離株之間的同源性為95.2%-99.9%,與高致病性PRRSV代表毒株JXA1的同源性為97.2%～99.3%。2個(gè)類NADC30分離株之間的同源性為99.9%,與類NADC30 PRRSV代表毒株NADC30的同源性為95.4%。5個(gè)歐洲型分離株之間的同源性為84.4%～99.7%,與歐洲代表毒株LV的同源性為87.2%-90.9%。重要蛋白基因分析結(jié)果顯示,PRRSV在不斷變異中,以非結(jié)構(gòu)蛋白Nsp2和結(jié)構(gòu)蛋白GP5的變異最快。61個(gè)分離株在非結(jié)構(gòu)蛋白Nsp2部位均發(fā)生了 1-150aa不等的缺失。大部分分離株在重要結(jié)構(gòu)蛋白GP5發(fā)生氨基酸突變,尤其是糖基化位點(diǎn)的位置和數(shù)量發(fā)生了不同程度的變化。歐洲型分離株GP3高變區(qū)含有多個(gè)缺失及突變。我們還發(fā)現(xiàn)了 4株新的PRRSV基因缺失毒株,這些毒株核苷酸或氨基酸的缺失位置及數(shù)量在中國(guó)尚未見相關(guān)報(bào)道。以上結(jié)果表明,豬群中的PRRSV主要流行毒株仍然是以高致病性PRRSV為主,但隨著歐洲型PRRSV和類NADC30 PRRSV的發(fā)現(xiàn),使得PRRS的防控情況十分嚴(yán)峻,迫切需要我們加強(qiáng)PRRSV監(jiān)測(cè)工作。鑒于我國(guó)PRRSV流行毒株復(fù)雜多變的特點(diǎn),為改進(jìn)現(xiàn)有PRRSV通用熒光RT-PCR檢測(cè)方法,根據(jù)國(guó)內(nèi)PRRSV主要流行毒株全基因序列,設(shè)計(jì)特異性探針與引物,經(jīng)過探針引物篩選及優(yōu)化,建立了新的PRRSV通用型熒光定量RT-PCR檢測(cè)方法。該方法具有較好的特異性、敏感性和重復(fù)性,能特異地檢測(cè)國(guó)內(nèi)所有PRRSV美洲型和歐洲型主要代表毒株,與其他主要豬病病原無(wú)交叉反應(yīng);其美洲型和歐洲型毒株的檢測(cè)下限分別為20copies/μL和22copies/μL,特別在檢測(cè)美洲經(jīng)典型毒株和歐洲毒株時(shí),敏感性優(yōu)于當(dāng)前商用試劑盒;其批內(nèi)、批間重復(fù)試驗(yàn)中變異系數(shù)均在2.5%以內(nèi)。用812份臨床樣品進(jìn)行檢測(cè),符合率為94.2%。結(jié)果表明,該方法可廣泛應(yīng)用于PRRSV臨床診斷以及PRRSV病原普查等工作。總之,本研究摸清了國(guó)內(nèi)PRRSV的流行狀況和遺傳進(jìn)化趨勢(shì)并且建立了 PRRSV通用型熒光定量RT-PCR檢測(cè)方法,對(duì)PRRS預(yù)防、控制和消滅具有重要意義。
[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，

本文編號(hào)：2507460