Construction of EV71 virus reverse genetics system and identification of rescued viruses

Abstract:

Objective：To construct an efficient reverse genetics system for EV71 virus，and to rapidly obtain the rescued viruses and identify their activity. Methods：A recipient plasmid pHM-ccdB containing human RNA polymerase Ⅰ promoter，ccdB suicide gene，and murine terminator（mTer） was constructed，and the AarⅠ enzyme cut site was introduced into both sides of the ccdB gene. The virus genome was amplified by PCR in two steps to avoid the AarⅠ enzyme cut site contained in the EV71 genome. The necessary AarⅠ enzyme cut site was introduced into the primer；then the products were connected with the target vector by Golden Gate Clone technique to obtain the rescued plasmid of EV71 virus（pHT-EV71）；after the pHT-EV71 was transfected into RD cells，the rescued EV71 viruses were obtained. The progeny viruses were identified by RT-PCR，virus titer，Western blot，and electron microscopy. Results：The pHT-EV71 was successfully constructed by introducing ccdB lethal gene and Golden Gate Clone technique. After the pHT-EV71 was transfected into RD cells，obvious cytopathic effect was observed. The rescued progeny viruses obtained were ampli－fied by RT-PCR with EV71-specific primers，and specific bands of approximately 1 900 bp were observed. The viruses can be bound with EV71 VP1 specific antibody as showed by Western blot. Spherical virus particles of 20 to 30 nm in size were observed by transmission electron microscopy. After eight generations of progeny viruses in RD cells，the virulence of the progeny viruses was stronger than that of the parental viruses（6.35 lgTCID50/mL）. Conclusion：A rapid and efficient method to construct the pHT-EV71 with a 100% efficiency was successfully established through introducing the ccdB gene and Golden Gate Clone tech-nique. This method provides a new strategy for the construction of reverse genetics system for positive-strand RNA viruses and a technical platform for further study on the pathogenesis and vaccine preparation of EV71 virus.