Enhancing the stability of influenza a reporter viruses by recoding the gfp gene

Influenza A virus (IAV) is one of the most important zoonotic pathogens and can cause global influenza pandemics and seasonal influenza outbreaks. Generation of recombinant IAV expressing a fluorescent protein will allow the infection to be easily monitored. In this study, we initially constructed a replication-defective H1N1/ΔPB2-GFP and a replication-competent H1N1/NS-GFP. However, these two reporter IAVs exhibited genetic instability. To stabilize the recombinant viral genome, we recoded the gfp sequence (rGFP) using synonymous codons to mimic the high-NP-binding regions involved in NP-vRNA interaction. This approach resulted in the development of replication-defective H1N1/ΔPB2(300)-rGFP and replication-competent H1N1/NS-rGFP, both of which exhibited enhanced stability in GFP expression. By replacing the HA segment from strain A/mink/China/CY 2017 (H5N1), we also generated a replication-defective H5N1/ΔPB2(300)-rGFP, which showed excellent genetic stability. Using these reporter IAVs, the blocking of virus infection by neutralizing antibodies and antivirals can be rapidly detected by the loss of fluorescent reporter expression. Replication-defective reporter IAVs constructed in this study can only infect and replicate in cells expressing PB2, allowing the possibility of manipulation of highly pathogenic IAV and their related reassortant strains in biosafety level-2 laboratories. Our data highlight the importance of NP-vRNA interaction for the stability of IAV genome, and the reporter IAVs generated using this strategy could be powerful tools for both basic and applied influenza virus research.