Construction of reverse genetics system for recombinant entervirus 71

Objective To recombine 3C and 3CD region of virulent strain SDLY107 and low virulent strain SDLY1 of entervirus 71(EV71)with reverse genetics,and to rescue recombinant viruses.Methods Recombinant fragments,3C(1)-3D-3'UTR(107)and 3CD(1)-3'UTR(107),were obtained with PCR,and then cloned to pMD19-T.3CD-3'UTR of pMD19-T-107 constructed previously was replaced by recombinant 3CD-3'UTR through double digestion and ligase T4 to construct recombinant pMD19-T-107.Then the in vitro synthesized RNA transcripts were transfected into rhabdomyosarcoma (RD) cells to produce the rescued recombinant virus,SDLY 107(1-3C)and SDLY 107(1-3CD).DNA sequences of the recombinant viruses were analyzed.Results The full length cDNA clone was constructed successfully.After 36 hours of third passages,cytopathic effect(CPE)was observed,and the CPE was observed obviously after 48 hours,suggesting the recombinant viruses were rescued successfully.There were a few differences between the CPE induced by recombinant viruses and SDLY107.Conclusion Reverse genetics system for recombinant EV71 was constructed successfully,and recombinant viruses were rescued successfully.The study lays a foundation for further research on pathogenesis of EV71.