ABSTRACT H9N2 avian influenza viruses (AIVs) remain a significant economic burden on poultry production and a persistent zoonotic threat. Hemagglutinin (HA), a surface glycoprotein mediating viral entry and pathogenesis, critically depends on thermostability for its function. Our previous study indicated that recent H9N2 AIVs have experienced a reduction in hemagglutination activity and exhibit low HA thermostability; however, the underlying molecular determinants for this instability remain poorly defined. To address this gap, we employed an in vitro -directed evolution approach to identify HA mutations that enhance thermostability. By subjecting a diverse HA mutant library to iterative heat selection at 48°C, we isolated several HA-stabilizing mutations, including L29I, N133S, N210D, G266R, D387N, A423T, and E509G, and confirmed their effect by site-directed mutagenesis. Further characterization revealed a complex interplay between HA stability, receptor binding specificity, and acid tolerance. Our findings demonstrate that enhancing HA stability can exert pleiotropic effects on key viral properties, highlighting the importance of understanding these relationships for developing effective mitigation strategies against H9N2 AIVs. IMPORTANCE H9N2 avian influenza viruses pose a persistent threat to poultry production and human health, demanding improved control strategies. This study addresses a key knowledge gap by uncovering the molecular determinants that modulate the stability of the hemagglutinin (HA) protein in H9N2 viruses. We identify specific HA mutations that increase thermostability, a property potentially linked to environmental persistence. Furthermore, our findings demonstrate a critical interplay between HA stability and essential viral functions, including receptor binding, hemagglutination activity, replication kinetics, and overall acid tolerance. By disentangling these properties, we provide insights into the mechanisms underlying HA-mediated viral entry and infectivity, which could inform the development of more effective vaccines and therapeutics.
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