Construction and identification of a chimeric cDNA infectious clone of enterovirus 71 strain

Objective To construct a chimeric infectious clone of the fatal virulent strain SDLY 107, containing the gene fragments encoding 2A and 3B proteins of the mild virulent strain SDLY 1, and to establish a reverse genetic system platform for further investigation on virulence of enterovirus 71 strains. Methods The overlap PCR analysis was performed to obtain the gene fragments encoding 2A and 3B proteins of the mild virulent strain SDLY 1. The obtained gene fragments were digested and then cloned into a plasmid pMD19-T containing the full-length gene of SDLY 107 strain by using gene replacement strategy. The recombinant RNA was transfected into Vero cells for the preparation of recombinant virus particles. Several assays including the PCR, indirect immunofluorescence (IFA), Western blot and sequencing were performed for virus identification. Virus titers were measured by 50% cell culture infective dose (CCID50) and plaque assay. Results The infectious clones of SDLY 107-2A-1 and SDLY 107-3B-1 chimeric virus strains were constructed successfully. Typical cytopathic effect was observed in Vero cells after viral transfection. Identification of the rescued viruses by PCR, IFA, Western blot and sequencing further confirmed the successful construction of infectious virus strains. The virus titers of SDLY 107-2A-1 and SDLY 107-3B-1 strains detected by CCID50 and plaque assay were 1.25×105 PFU/ml and 0.7×105 PFU/ml, respectively. Conclusion The chimeric viruses SDLY 107-2A-1 and SDLY 107-3B-1 were rescued successfully, causing cytopathic effects similar to those by using the parental virus strain SDLY 107. This study might pave the way for further investigation on in vitro and in vivo virulence of enterovirus 71 strains. Key words: EV71; Chimeric virus; Virulence