关键词: 口蹄疫病毒, 感染性分子克隆, 病毒拯救, T7 RNA聚合酶

Abstract:

【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.

Key words: foot-and-mouth disease virus, infectious molecular clones, virus rescue, T7 RNA polymerase