Phylogenetic distribution of PB2 E627K and D701N mutations in global H5N1 influenza A viruses

Objective: This study investigates the phylogenetic distribution of the mammalian adaptation–associated E627K and D701N mutations in the PB2 gene of H5N1 influenza A virus. It aims to determine whether these substitutions cluster within specific viral lineages or arise independently across distinct genetic backgrounds.Materials and Methods: We retrieved 1,870 global PB2 sequences of H5N1 influenza A virus from publicly accessible databases, including 169 human-derived isolates. The sequences were aligned using multiple sequence alignment, and phylogenetic reconstruction was performed using the Maximum Likelihood method under the best-fit nucleotide substitution model. The presence of E627K and D701N amino acid substitutions in the PB2 protein was identified through translation analysis and mapped onto the phylogenetic tree. Host origin (avian vs. human/mammalian) was recorded and incorporated into the analysis to assess mutation–host associations.Results: Phylogenetic analysis revealed that E627K substitutions exhibited lineage-associated clustering, with mutation-enriched subclades containing a considerable proportion of human-derived isolates. This pattern suggests clade-related expansion following acquisition of the adaptive mutation. In contrast, D701N substitutions showed a more scattered distribution across the phylogenetic tree, indicating multiple independent emergence events consistent with parallel evolution. Human-derived isolates were not restricted to a single evolutionary lineage, demonstrating that mammalian-adaptive mutations can arise across diverse viral genetic backgrounds.Conclusions: The evolutionary dynamics of PB2 adaptive mutations in H5N1 reflect both clade-associated expansion and recurrent parallel emergence. These findings highlight the considerable adaptive flexibility of H5N1 and emphasize the importance of continuous genomic surveillance to improve zoonotic risk assessment and early detection of mammalian-adapted variants.