Identification of thermostability-enhancing mutations in H9N2 avian influenza virus hemagglutinin.

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 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.