Infectious Subgenomic Amplicon Strategies for Japanese Encephalitis and West Nile Viruses.

Classical methods for constructing infectious cDNA clones of flaviviruses are often hindered by instability and toxicity. The Infectious-Subgenomic-Amplicons (ISA) method is an advancement which utilizes overlapping DNA fragments representing viral genomic sequence and in-cell recombination to bypass bacterial plasmid assembly. However, the ISA method has limitations due to the toxicity of some ISA DNA fragments in bacteria during synthetic production. We validated modified ISA strategies for producing toxic ISA Japanese encephalitis virus (JEV) and West Nile virus (WNV) DNA fragments. Three approaches were explored, including subdividing toxic DNA fragments into two sub-fragments for synthetic clonal production, using a low-copy bacterial plasmid, and subdividing the toxic DNA fragments into four short overlapping sub-fragments, each up to 1.8 kb. The latter novel approach in ISA applications enabled the synthesis of entirely bacteria-free ISA DNA fragments. Our results demonstrate that subdividing toxic fragments into sub-fragments smaller than 1.8 kb for synthesis is the efficient strategy, circumventing the need for bacterial plasmids and ensuring rapid production of synthetic flaviviruses. This method also shortens the production timeline. We also compared the efficacy of JEV and WNV ISA in zinc finger antiviral protein 1 (ZAP) wild-type and knockout cells and found that knockout cells may be more effective for ISA rescue of flaviviruses, including CpG-enriched strains for live attenuated vaccines. The validated modified ISA strategies provide an efficient approach for producing synthetic JEV and WNV. This will enable rapid research during outbreaks of emerging flaviviruses by facilitating the quick generation of new virus variants.