Tandem split-GFP influenza A viruses for sensitive and accurate replication analyses

Abstract

This study presents a refined approach for tracking influenza A virus infections using a bright fluorescent signal, minimizing the effects on viral function or stability. Influenza A virus has a compact genome that makes it difficult to insert reporter tags without disrupting its replication. By using a small, split fluorescent protein tag and a self-cleaving linker, we developed a virus that retains replication ability and accurately reports infection through fluorescence. This allows for monitoring of viral infection in living cells or fixed cells using standard laboratory tools. The system enables rapid screening of antiviral drugs, neutralizing antibodies, and host factors that contribute to infection, and it works in cells from multiple animal species. As a result, this tool can be used to study how influenza A virus replicates and spreads, supporting efforts in antiviral development, cross-species transmission research, and pandemic preparedness.