Comprehensive Evolutionary and Structural Analysis of the H5N1 Clade 2.4.3.4b Influenza a Virus Based on the Sequences and Data Mining of the Hemagglutinin, Nucleoprotein, and Neuraminidase Genes Across Multiple Hosts

H5N1 Influenza A virus continues to pose a significant zoonotic threat, with increasing evidence of interspecies transmission and genetic adaptation. Previous studies primarily focused on avian or human isolates, with limited comprehensive analysis of H5N1 evolution across multiple mammalian hosts. Existing molecular surveillance often lags behind viral evolution; this study underscores the necessity for real-time monitoring of ongoing mutations affecting pathogenicity and transmissibility. Our goals are (1) to retrieve and analyse HA, NP, and NA gene sequences of H5N1 Influenza A virus from diverse hosts, including humans, poultry, and multiple mammalian species, to assess genetic diversity and evolutionary patterns. (2) To evaluate positive selection sites across the three major genes (HA, NP, NA) to determine adaptive mutations linked to host adaptation and viral survival. To achieve these goals, in this study, we analysed 78 HA, 62 NP, and 61 NA gene sequences from diverse hosts, including humans, poultry, and multiple mammalian species, retrieved from the NCBI database. The phylogenetic analysis revealed distinct clade formations, indicating regional spread and cross-species transmission events, particularly from avian sources to mammals and humans. Selection pressure analysis identified positive selection across all three genes, suggesting adaptive mutations contributing to host adaptation and viral survival. Homology modeling and molecular dynamics simulations were performed to generate high-quality structural models of HA, NP, and NA proteins, which were subsequently validated using multiple stereochemical parameters. Domain analysis confirmed conserved functional motifs, while protein-ligand docking demonstrated stable interactions at conserved binding sites, despite observed residue substitutions in recent isolates. Earlier research concentrated on HA alone; this study integrates HA, NP, and NA genes for a broader understanding of viral evolution and adaptation. These findings highlight ongoing evolutionary changes in H5N1 genes that may enhance viral adaptability and pathogenicity, underscoring the need for continuous molecular surveillance and updated antiviral strategies.