Importance: Understanding the molecular mechanisms driving H5N1 clade 2.3.4.4b is critical for pandemic preparedness.

Objective: To characterize the molecular drivers of viral fitness and mammalian adaptability in recent H5N1 viruses by integrating evolutionary dynamics with structural simulations.

Methods: This study analyzed 2,398 H5Nx genomes (2000-2024) through phylogenetic and selective pressure analyses. HA/NA structures were predicted with AlphaFold 3 and evaluated by AutoDock4 docking, whereas polymerase-ANP32A/B complexes were modeled using template-based methods and their binding free energies were estimated using MM/GBSA. Polymerase-ANP32E complexes were predicted with AlphaFold 3 and similarly evaluated by MM/GBSA. The binding affinities (ΔG) for the sialic acid (SA) receptors and human ANP32 proteins were quantified through molecular mechanics/generalized born surface area calculations.

Results: Clade 2.3.4.4b showed significant antigenic drift in the HA receptor binding site, reducing affinity for α2,3-SA and α2,6-SA receptors. On the other hand, the emergence of a full-length stalk N1 NA with second sialic acid-binding site mutations (e.g., N366S) compensated for reduced HA affinity by enhancing the NA binding stability. In the polymerase complex, both the PB2-627E/631L variant (-144.00 kcal/mol; unadjusted p = 0.0058) and the known mammalian-adaptive 627K/631M variant (-144.67 kcal/mol; unadjusted p = 0.0165) showed more favorable predicted human ANP32B binding free energies than the ancestral 627E/631M state (-136.46 kcal/mol).

Conclusions and relevance: The co-occurrence of HA, NA, PB1, and PB2 signatures was associated with clade expansion and produced structural predictions consistent with altered receptor or ANP32 interactions; experimental validation is required before inferring effects on fitness or zoonotic risk.