Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify E. coli promoter (ECP) sequences within nucleotide (nt) 1-3000 of the dengue virus type 2 (DENV2) and Japanese Encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1-3000 of DENV2 and JEV genome, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. Full-length DENV2 and JEV cDNAs were obtained by inserting mutations reducing their ECP activity in bacteria without altering amino acid sequences. Severe cytopathic effect occurred when BHK21 cells were transfected with in-vitro transcribed RNAs either from a DENV2 cDNA clone with multiple silent mutations within prM-E-NS1 region of dengue genome or a JEV cDNA clone with an A-to-C mutation at nt 90 of JEV genome. The virions derived from the DENV2 or JEV cDNA clone exhibited similar infectivity as their parental viruses in C6/36 and BHK21 cells. A cis-acting element essential for virus replication was revealed by introducing silent mutations into the central portion (nt 160-243) of core gene of DENV2 infectious cDNA or subgenomic DENV2 replicon clone. This novel strategy constructing DENV2 and JEV infectious clones could be applied to other flaviviruses or pathogenic RNA viruses to facilitate research in virology, viral pathogenesis, and vaccine development.
