异源5′非翻译区在猪繁殖与呼吸综合征病毒感染性拯救过程中的功能解析  

THE RECOVERY OF CHIMERIC TYPE Ⅱ PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS WITH REPLACEMENT OF TYPE I 5′ UNTRANSLATED REGION

作　　者：高飞[1] 陆嘉琦[1] 姚火春[2] 杨倩[2] 袁世山[1] 童光志[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]南京农业大学动物医学院,南京210095

出　　处：《中国动物传染病学报》2011年第6期7-14,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金面上项目(30972204);欧盟"第七框架"项目(245141)

摘　　要：猪繁殖与呼吸综合征病毒(Porcine reporoductive and respiratory syndrome virus,PRRSV)基因组5′非翻译区(untranslated region,UTR)对病毒复制转录等过程至关重要,但其发挥作用的机制尚不清楚。本实验室将欧洲型PRRSV 5′UTR替换入北美型PRRSV弱毒株感染性克隆pAPRRS的骨架中,构建pAPLV5。经过DNA转染入MARC-145细胞系中,两次传代后,拯救出嵌合病毒vAPLV5。通过对嵌合病毒的全长测序发现,嵌合病毒基因组较亲本病毒发生了碱基突变,突变主要集中于Nsp2和Nsp9位置。将Nsp9位置(病毒的RNA依赖的RNA聚合酶,RNA-dependent KNA povymerase,RdRp)的4处突变位点引入嵌合病毒的感染性克隆pAPLV5,所构建的4个突变嵌合克隆转染MARC-145细胞后无需传代即产生细胞病变(cytopathic effect,CPE),且P0代病毒滴度较原嵌合病毒vAPLV5高。通过半定量负链和亚基因组检测发现,突变嵌合病毒的RNA合成水平也较亲本嵌合病毒高。由此推测PRRSV 5′UTR功能可能与RdRp相关,异源的5′UTR通过突变RdRp来发挥其调控作用,完成病毒拯救过程。The 5＇ untranslated region （UTR） of the genomic RNA is believed to be vital for the replication and transcription of Porcine reproductive and respiratory syndrome virus （PRRSV）, but its functional mechanism remains unclear. In the present study, a chimeric clone pAPLV5 was generated by substituting type I 5＇ UTR into the backbone of infectious full-length cDNA clone pAPRRS of type II attenuated strain. After two passages post transfection in MARC-145 cells, the chimeric virus vAPLV5 was rescued. Sequence analysis of full-length genome of the chimeric virus vAPLV5 identified several nucleotide mutations, which mainly occurred in Nsp2 and Nsp9. Subsequently, four mutations that were generated in Nsp9 （RNA dependent RNA polymerase, RdRp） were introduced into the chimeric clone pAPLV5 to create four different mutants. As a result, transfected MARC-145 cells developed CPE without any passage. Viral titers of these four mutants were higher than original chimeric virus pAPLV5. Semi- quantitative RT-PCR was conducted for negative-strand genomic RNAs and sg mRNAs analysis. The results showed that the RNA synthetic level of the four mutant viruses were higher than vAPLV5. In conclusion, PRRSV 5＇ UTR was closely associated with theregulatory function of RdRp, and heterologous 5＇ UTR might play a role in virus rescuing processes through alteration in RdRp.

关 键 词：非翻译区 RNA依赖的RNA聚合酶 嵌合病毒 转染 

分 类 号：S852.659.6[农业科学—基础兽医学]