Recovery Of Recombinant Human Respiratory Syncytial Virus From The Constructed Antigenomic CDNA Clone

Objective:Using methods of reverse genetics and molecular biology, the antigenomic cDNA of the derived recombinant RSV(rRSV/EGFP) containing the gene of enhanced green flurorescent protein (EGFP) is constructed. The plasmid, pBRAT-RSV-EGFP, of rRSV/EGFP antigenome cDNA is under the control of T7promoter. The rRSV/EGFP will be recovered by T7RNA polymerase (T7RNP) provided by engineering cells such as BSR T7/5or by the infectied or transfected cells with modified vaccinia virus encoding T7RNP (MVA-T7) or plsmids encoding T7RNP, and the rescue system of rRSV/EGFP will be established upon T7RNP driven expression of antigenomic RNA.Methods:We construct an antigenomic cDNA clone of rRSV/EGFP, respectively, based on multiple steps of clone manipulation. The resulting antigenomic cDNAs are flanked by T7promoter, HDV ribozyme, and T7transcritption terminal signal in5’to3’ order. The rRSV/EGFP is rescued by three different ways, co-transfecting with four helper plasmids expressing the RSV N, P, M2-1and L proteins (pcDNA3.1-N-OPT、 pcDNA3.1-P-OPT、pcDNA3.1-M2-1-OPT、and pcDNA3.1-L-OPT respectively) into MVA-T7infected HEp-2cells; co-transfecting with four helper plasmids into BSR T7/5cells; co-transfecting with four helper plasmids and pcDNA3.1-T7RNP plasmid into HEp-2cells. Then the recovery efficiency is evaluated by the expressed fluorescence under fluorescent microscope.Results:The cloned antigenomic cDNA was confirmed by assays of restriction endonuclease and nucleic acid sequencing. Comparing to the other two methods, stronger fluorecence was observed in MVA-T7infected HEp-2cells.Conclusion:We successfully constructed a plasmid expressing rRSV/EGFP antigenomic cDNA, respectively. The rescue system based on MVA-T7is effective for RSV recovery. All these works have paved the way for the successful recovery of RSV.