Toward Predicting Pandemic Potential: A Comparative Analysis of Virus‐Host Interactions Between Diverse Influenza A Viruses and the Human Innate Immune System

Zoonotic infections caused by influenza A viruses (IAVs) of animal origin are a major public health concern, as they may represent the initial step toward the emergence of a pandemic strain. The host range of IAVs is shaped by species-specific virus-host interactions, and several host barriers have been identified that restrict the adaptation of avian viruses to humans. We used a comparative interaction screen by mammalian cell-based Nanoluciferase two-hybrid (mN2H) assay to investigate whether differences in the interaction networks between viral proteins from human, avian, and zoonotic IAVs and a curated panel of human innate immune factors could serve as indicators of the zoonotic potential of avian IAVs. The results revealed both conserved and strain-specific interactions, while underscoring limitations in comparing interactions across independent screens. Complementary functional analyses by siRNA knockdowns identified cellular factors and pathways potentially involved in host restriction, as well as pro-viral factors that could represent potential antiviral targets. The comparative interactomic mN2H approach holds promise for evaluating avian IAV zoonotic potential, but our findings highlighted the need for further studies using broader cellular libraries to fully validate its predictive value.