Figure 1
Rescue of EV71 viruses by hPolI-driven reverse genetics (RG) system. (A) Amplification of the full-length genomic cDNA of EV71 (~ 7.5 Kb). RNA of wild-type EV71 strains belonging to subgenogroup A, B2, B4, B5, C1, C2, C4 or C5 and 5 unknown EV71 strains (#363 to #577) from Malaysia was extracted and amplified using two universal primers EV71-Uni-F and EV71-Uni-R in a fast RT-PCR reaction. L represented 2log DNA ladder (Bio-rad). (B) Strategy for the construction of EV71 RG plasmid. The linear pJET-hPolI/mTer vector and EV71 cDNA amplicon had 15 identical nucleotides at both 5′ and 3′ ends. They were joined together so that EV71 cDNA was directly flanked by hPolI promoter and murine terminator (mTer) using In-Fusion cloning method. The recombinant plasmid produced authentic and infectious viral genomic RNA upon transfection into Vero cell. (C) CPE and IFA identification of the rescued EV71-B5 RG virus. Vero cells infected by the EV71-B5 RG or wild-type viruses were observed for CPE at 5 days after infection. The mock and pJET-hPolI/mTer-EV71-B5 plasmid transfected cells did not show CPE. IFA signals were detected at 24 h in the tranfected cells and cells infected with RG or wild-type B5 virus; while mock cells were negative after treating with guinea pig anti EV71 serum.