A reverse genetics system for enterovirus D68 using human RNA polymerase I.

Human enterovirus D68 (EV-D68) is a highly contagious virus, which causes respiratory tract infections. However, no effective vaccines are currently available for controlling EV-D68 infection. Here, we developed a reverse genetics system to recover EV-D68 minireplicons and infectious EV-D68 from transfected plasmids using the RNA polymerase I (Pol I) promoter. The EV-D68 minireplicons contained the luciferase reporter gene, which flanked by the non-coding regions of the EV-D68 RNA. The luciferase signals could be detected in cells after transfection and Pol I promoter-mediated luciferase signal was significantly stronger than that mediated by the T7 promoter. Furthermore, recombinant viruses were generated by transfecting plasmids that contained the genomic RNA segments of EV-D68, under the control of Pol I promoter into 293T cells or RD cells. On plaque morphology and growth kinetics, the rescued virus and parental virus were indistinguishable. In addition, we showed that the G394C mutation disrupts the viral 5'-UTR structure and suppresses the viral cap-independent translation. This reverse genetics system for EV-D68 recovery can greatly facilitate research into EV-D68 biology. Moreover, this system could accelerate the development of EV-D68 vaccines and anti-EV-D68 drugs.