Nath H, Arndt A, Mann JT, Baker SF. 0. Tandem split-GFP influenza A viruses for sensitive and accurate replication analyses. Microbiol Spectr 0:e02772-25
Reporter influenza A viruses (IAVs) are valuable tools for studying virus fitness, screening antivirals, and assessing host–virus interactions. However, the compact and segmented nature of the IAV genome presents major challenges for engineering genetically stable reporter viruses with minimal fitness defects. To address this, we developed a replication-competent IAV incorporating a tandem split-GFP reporter strategy. Specifically, we appended seven tandem repeats of the GFP11 peptide (GFP11×7) to the C-terminus of the PB2 or PA polymerase subunit. Viruses either contained or lacked a self-cleaving 2A peptide between the viral and reporter genes, thereby creating polymerase fusion proteins or released GFP, respectively. These viruses are complemented in trans by cells expressing GFP1-10, allowing bright fluorescence with minimal disruption to viral function. The tandem GFP11 reporter viruses exhibit delayed replication kinetics but are genetically stable over serial passages with a strong concordance between GFP signal and viral gene expression. We demonstrate their utility in high-throughput applications including fluorescence-based quantification of infection, neutralizing antibody titration, antiviral drug screening, and host factor identification, with results matching traditional assays but on an accelerated timeline. Furthermore, we engineered a battery of GFP1-10-expressing cell lines from multiple vertebrate species, illustrating the broad compatibility of this platform for comparative host studies. This system enables sensitive, scalable, and quantitative evaluation of influenza virus replication across diverse experimental contexts. The GFP11×7 reporter platform offers a versatile and robust tool for virology research and therapeutic screening, with potential for rapid adaptation to emerging IAV strains.
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