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