型内非编码区嵌合口蹄疫病毒的拯救及生物学特性分析

Recovery and biological characterization of inter-genotypic chimeric foot-and-mouth disease virus by substituting untranslated region

为探寻口蹄疫病毒(FMDV)非编码区对病毒生命周期乃至生物学特性的影响,以O/HN/93现用疫苗株的感染性克隆为骨架,利用融合PCR分别构建了含持续感染毒株O/CHN/Mya98/33-P 3′非编码区、5′非编码区及3′非编码区和5′非编码区嵌合的3株全长cDNA克隆。3个全长克隆经线性化后分别在脂质体介导下转染表达T7RNA聚合酶的BHK-21细胞,转染后第60小时均出现明显的致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光分析,结果表明成功拯救到3个嵌合的FMDV。拯救的病毒对乳鼠致病力和细胞感染力的比较分析结果显示,发现分别替换两端非编码区会导致病毒细胞感染力和乳鼠致病力下降,而同时替换非编码区的病毒则无明显变化。这些嵌合病毒的成功拯救为进一步阐述非编码区的功能奠定了基础。

In order to study the influence of the substituting untranslated region（UTR） of foot-and- mouth disease virus（FMDV） on its activity and biological characteristics, three full-length eDNA clones were constructed based on O/HN/93 strain eDNA clone via replacing its two UTRs,respectively,or both by the corresponding part of persistent infection virus strain O/CHN/Mya98/33-P. Each eDNA clone was transfected into BHK-21 cells by lipofectamine 2000,which can express T7 RNA polymerase. Cytopatho- genie effects were found in the transfected BHK-21 cells after 60 h and three chimeric virus strains could replicate. The three chimeric virus strains were respectively confirmed by RT-PCR and IFAT. Analyzing TCID50 growth curves and LD50 showed that the infectivity to cells and pathogenicity for suckling mice of chimeric virus was attenuated if either of the UTR was replaced, but not obvious different if two UTRs were replaced together, compared with its parental virus, indicating that the UTR of FMDV plays an important role in the virus biological characteristics.