细胞内转录拯救Asia1型口蹄疫病毒

 【目的】建立口蹄疫病毒（FMDV）感染性分子克隆的体内拯救技术平台，为深入研究该病毒基因组的结构与功能奠定基础。【方法】将置于T7启动子下的FMDV Asia1/JS/China/2005株全长cDNA的真核重组质粒pFMDV-A经Not I线化后和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞，进行病毒体内拯救的研究。【结果】转染细胞盲传第3代48 h后出现致细胞病变（CPE），第4代10 h出现典型的CPE。RT-PCR、间接免疫荧光、电镜等均检测到了拯救的FMDV，而且存在分子标签，表明构建的FMDV全长cDNA分子克隆在BHK-21细胞内成功包装出FMDV。拯救毒与亲本毒对乳鼠的致病力（LD50）差异不显著，具有相似的生物学特征。共转染试验重复多次，均拯救到FMDV病毒。【结论】成功建立了基于质粒为基础的FMDV的体内拯救方法。

Rescue of Infectious Foot-and-Mouth Disease Virus in vivo from Full-Length cDNA Clone of Asia1 Type

【Objective】The aim of the study was to develop a reverse genetics platform of infectious foot-and-mouth disease (FMDV) in vivo, which is a base for the construction and function researches of viruses. 【Method】 The full-length genome of FMDV Asia1/JS/China/2005 strain was assembled into a mammalian expression vector downstream of a T7 promoter. The recombinant plasmids, pFMDV-A were linearized with NotI enzyme and cotransfected into BHK-21 cells with pcDNAT7P plasmids that could express T7 RNA polymerase to research reverse genetics of FMDV. 【Result】 The transfected cells were serially passaged, and the third passage showed apparent cytopathogenicity effect (CPE) within 48 h, the CPE were observed after 10 h in fourth passage. The results of the RT-PCR, indirect immunofluorescence, and electron microscope showed that FMDV was rescued successfully in vivo from the full-length cDNA clone of FMDV. The recovered virus contained genetic tags. Repeat experiments also obtained recovered virus with the established method. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. 【Conclusion】 Plasmid-based reverse genetics system was developed in authors’ laboratory for efficient and stable generation of infectious FMDV.