A Synthetic Virology Platform for Rapid Prototyping of Influenza A Virus mutants

Influenza A virus (IAV) remains a significant public health threat, with the need for effective vaccines and antiviral therapies remaining a high priority. In vitro testing is paramount in studying novel IAV viruses and their immunogenicity. To address this need, we developed a synthetic virology approach to rapidly prototype and test IAV mutants with altered antigenic sites. By strategically inserting restriction sites within an ambisense plasmid, we generated a tool where we can seamlessly replace either 1) the full hemagglutinin (HA), 2) the HA globular head domain to generate head/stalk chimeras or 3) the five canonical antigenic sites. The cloning inserts can be computationally designed and artificially synthesized, enabling rescue of synthetic Influenza A viruses. Using this platform we rescued IAV H1N1, H3N2 & H5N1, and went on to prototype their respective antigenic site mutants & chimeric IAVs using viral genome sequence alone. This platform could be useful for generating vaccine seed strains or to study anti-viral immunity.