猪繁殖与呼吸综合征病毒类NADC30株CHsx1401感染性克隆的构建

摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) remains the most economically important pathogen impacting swine industry worldwide.Recently,the novel NADC30-like strains of PRRSV resulting in the re-emerging of clinically PRRS outbreaks has been characterized in China.In order to provide a basis for research on the mechanisms in relation to the genetic variation and evolution of PRRSV NADC30-like strain,CHsx1401,as well as the differential pathogenesis between this virus and other PRRSV strains,a full-length cDNA clone of NADC30-like CHsx1401 was generated.Four fragments of the whole genome of CHsx1401 were amplified by RT-PCR,and each was cloned into pJET1.2 blunt,and each clone was then ligated into the modified low-copy-number vector pWSK29 to generate the full-length cDNA clone,plasmid pWSK-CHsx1401.To distinguish the rescued virus from its parental virus,a restriction enzyme site Asc Ⅰ was introduced between fragment C and D through synonymous mutation (T11582G) as a genetic marker.Subsequently,the full-length eDNA clone was transfected into MARC-145 cells using Lipofectamine LTX and plus reagents to rescue the virus.As the results showed,the full-length eDNA clone (plasmid pWSK-CHsx1401) was constructed successfully,cytopathic effect (CPE) typical of PRRSV in MARC-145 cells could be observed at the second passage.The virus could be rescued from the plasmid pWSK-CHsx1401 by IFA,RT-PCR and sequencing.The rescued virus could be passaged stably in MARC-145 cells,and showed a similar growth curve to its parental isolate in vitro,with slightly lower titers at some time points than CHsx1401 in MARC-145 cells.Our results indicate that the full-length cDNA clone of the PRRSV NADC30-like CHsx1401 is infectious and viable virus can be rescued.The rescued virus (RvCHsx1401) exhibited similar growth ability to its parental virus in vitro.Our present study provides a platform for further research on the molecular mechanisms in relation to the variation and evolution of PRRSV NADC30-like and its differential pathogenesis from other strains of PRRSV.%猪繁殖与呼吸综合征病毒(PRRSV)是影响全球养猪业的一种重要病原.近年来,国内新出现的类NADC30毒株导致临床PRRS疫情再次出现.本研究旨在构建PRRSV类NADC30毒株CHsx1401的感染性克隆,以期为研究该类毒株遗传变异与演化以及与其他毒株致病性差异的分子机制奠定基础.将类NADC30毒株CHsx1401全长基因组分为4个片段进行RT-PCR扩增,分别将扩增片段克隆到pJET1.2载体构建中间质粒,将4个克隆依次连接并克隆到改造后的低拷贝质粒载体pWSK29中,构建全长cDNA克隆pWSK-CHsx1401,并在全长cDNA克隆的C与D片段之间通过同义突变(T11582G)引入Asc Ⅰ酶切位点,作为遗传标记,用于区分拯救病毒与亲本病毒.采用脂质体LTX将全长cDNA克隆质粒转染至MARC-145细胞拯救病毒.结果表明,成功构建出全长cDNA克隆pWSK-CHsx1401,转染MARC-145细胞后传至第二代可观察到明显的细胞病变(CPE).经间接免疫荧光、RT-PCR和序列测定鉴定结果表明,从全长cDNA克隆可拯救出病毒.拯救病毒(RvCHsx1401)在MARC-145细胞上可稳定传代,增殖动态与亲本病毒相似,在MARC-145细胞上各时相的病毒滴度略低于亲本病毒.本研究构建的类NADC30毒株CHsx1401的全长cDNA克隆具有感染性,可拯救出病毒;拯救病毒与亲本病毒的体外增殖特性相似,为研究该毒株的变异与演化以及与其他毒株致病性差异的分子机制提供了技术平台.