Mapping the adaptive landscape of H9N2 hemagglutinin links hotspot substitutions to cross-species potential and thermostability

Abstract H9N2 avian influenza virus poses a persistent threat to poultry and public health because of its widespread circulation and role in generating novel viruses. To investigate the genetic determinants of HA antigenic diversity and adaptation, we constructed a hotspot library targeting 179 substitution hotspots identified through large-scale sequence analysis of 13,984 HA sequences distributed across the HA protein. The highly variable residues 145 and 168 represent prominent substitution hotspots that correlate with host-specific lineages and highlight poultry as a key adaptation reservoir. The library was subjected to replication in multiple host-cell systems, as well as immune and thermal selection. Functional screening revealed that N115H enhances viral replication in mammalian cells, residue 164 is critical for antigenic escape, and I134L, L230S, and V306L collectively increase HA thermostability. These results link natural sequence variation to functional phenotypes, demonstrating that H9N2 HA antigenic diversity and cross-species potential are shaped by mutation hotspots. Our integrated computational and experimental framework provides a platform for probing viral fitness landscapes, with implications for risk assessment and the rational design of next-generation vaccines.