Development and characterization of a stable eGFP enterovirus 71 for antiviral screening

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5' untranslated region and VP4 gene of the EV71 genome. Vero cells transfected with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of the eGFP reporter virus. The reporter virus was infectious to Vero cells, producing robust eGFP fluorescence signals. Compared with the wild type virus, the reporter virus replicated slower in cell culture. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. The passaged viruses maintained the eGFP gene, demonstrating the stability of the reporter virus. Using a known EV71 inhibitor, we demonstrate that the reporter virus could be used for antiviral screening. The infectious cDNA clones of the wild type virus and the eGFP reporter viruses will be useful for antiviral research as well as for studying viral replication and pathogenesis of EV71.