Infectious Subgenomic Amplicon Strategies for Japanese Encephalitis and West Nile Viruses

Area of Science:

• Virology
• Molecular Biology
• Synthetic Biology

Background:

• Classical flavivirus infectious cDNA clone construction faces challenges with DNA instability and toxicity.
• The Infectious-Subgenomic-Amplicons (ISA) method uses in-cell recombination but can be limited by toxic DNA fragments during bacterial synthesis.

Purpose of the Study:

• To validate modified ISA strategies for producing toxic Japanese encephalitis virus (JEV) and West Nile virus (WNV) DNA fragments.
• To develop efficient, bacteria-free methods for synthetic flavivirus production.

Main Methods:

• Explored strategies including fragment subdivision, low-copy plasmids, and a novel approach using four short overlapping sub-fragments (<1.8 kb).
• Synthesized bacteria-free ISA DNA fragments using the optimized subdivision method.
• Compared ISA efficacy in zinc finger antiviral protein 1 (ZAP) wild-type and knockout cells.

Main Results:

• Subdividing toxic ISA DNA fragments into smaller sub-fragments (<1.8 kb) enabled efficient, bacteria-free synthesis.
• This novel approach circumvents bacterial plasmid requirements and accelerates synthetic flavivirus production.
• Zinc finger antiviral protein 1 (ZAP) knockout cells showed potential for enhanced ISA rescue of flaviviruses, including CpG-rich strains.

Conclusions:

• Modified ISA strategies, particularly fragment subdivision, provide an efficient method for producing synthetic JEV and WNV.
• These advancements facilitate rapid research on emerging flaviviruses by enabling quick generation of viral variants.
• The bacteria-free approach shortens production timelines and enhances the generation of live attenuated vaccine candidates.