Construction Of EV71 Virus Reverse Genetics System And Identification Of Rescue Viruses

Objectives An efficient reverse genetics system for EV71 virus was constructed and the rescued viruses were quickly obtained and identified by transfection and infection respectively.And the characteristics and advantages of the two different ways to acquire the virus were compared and discussed.Methods 1.The receiving plasmid p HT-ccd B containing human RNA polymerase I promoter(Ppol I),ccd B suicide gene and murine terminator(m Ter)was constructed.Aar I enzyme cut site was designed into two side sequence of ccd B gene.Since the EV71 genome also contains Aar I enzyme cut site,the virus genome was amplified by PCR in two steps and Aar I enzyme cut site was designed into the primers.Then the PCR products were cloned into the target vector to obtain the virus rescued plasmid p HT-EV71 by Golden Gate Clone method.After plasmid p HT-EV71 was transfected into RD cells,the rescued EV71 viruses were obtained and identified by RT-PCR,virus titer,Western blot and transmission electron microscopy(TEM);2.The plasmid p HT-EV71 was transformed into E.coli AcMulti Bac/? asd Sw106.Then 10 large colonies were selected for culture to obtai n bacterial liquid and directly infected into RD cell.The rescued EV71 viruses were obtained and identified by virus titer,Western blot.3.The wild strains and the rescued viruses obtained by transfection a nd infection were compared by virulence,growth curve and Western blot.The characteristics of the two methods were summarized and pr eliminarily discussed.Results 1.The rescued plasmid of EV71 virus(p HT-EV71)was successfully constructed by inserting ccd B lethal gene and Golden Gate Clone me thod.2.After the p HT-EV71 was transfected and infected into RD cells,obvious cytopathic effect(CPE)was observed,and the rescued viruses EV71-Rescue and EV71-Rescue2 were amplified by RT-PCR with EV71 special primers,and the 1900 bp products were observed.The rescued viruses were bound with EV71 vp1 specific antibody by Western blot.20~30 nm spherical virus particles were observed by TEM.The growth curve showed that the viruses were stably proliferated for 8 generations in RD cells,and the virulence of EV71-Rescue and EV71-Rescue2 were higher than that of the maternal strain(6.35 lg TCID50/m1).3.Compared proliferation trend of the rescue viruses obtained by the two methods based on the growth curve,and also compared with that of the wild strains.The results showed that the proliferation of EV71-Rescue and EV71-Rescue2 was stable,and the titer reached more than 8 lg TCID50/ ml after continuous proliferation.EV71-Rescue and EV71-Rescue2 have similar trends of growth and replication with the wild strains.Conclusion 1.A rapid and efficient method to construct the rescued plasmid of EV71 virus was successfully established with the efficiency of 100% through introducing ccd B gene and Golden Gate Clone technology.T his method provides a new strategy for the construction of reverse g enetics system for positive-stranded RNA virus.2.Not only obtained the rescued viruses successfully through the traditional transfection methods,but also obtained the rescued viruses by infecting RD cell as a more efficiently new method.3.Comparing the rescued viruses obtained by the two methods with the wild strains,it is proved that infection is a more efficient wayto acquire and rescue the virus.The study provides a more efficient wa y to obtain the rescued viruses and a technical platform for further r esearch on pathogenesis and vaccine preparation of EV71 virus.