Background: Seasonal influenza A viruses (IAVs) remain a major global health concern, causing up to 650,000 deaths annually. Over the past century, four influenza pandemics have occurred, with H3N2 and H1N1 subtypes becoming endemic in humans. The hemagglutinin (HA) glycoprotein, essential for viral entry and a key vaccine target, contains critical antigenic sites. While antigenic drift enables immune evasion, certain substitutions can affect protein stability and intraprotein interactions, influencing viral fitness.

Methods: This study employed a Bayesian approach to investigate the phylogenetic origins of full-length HA genes from seasonal IAVs circulating in Izmir, Türkiye (2017-2023). Publicly available HA sequences from Türkiye were incorporated to assess selection pressures using four models available on Datamonkey and to examine antigenic mismatches between circulating viruses and vaccine strains. The structural impact of positively selected substitution was analyzed via molecular dynamics simulations.

Results: Phylogenetic analysis identified four and six subclades for H1N1 and H3N2, respectively, revealing cocirculation of genetically distinct strains within the same season. Both subtypes were under negative selection, but the N260D substitution in H1N1 was consistently detected under positive selection across all models. Molecular dynamics simulations suggested that this substitution may influence intraprotein dynamics with the vestigial esterase domain, introducing a transient electrostatic bond. Furthermore, H3N2 exhibited more antigenic mismatches than H1N1, including a novel mismatch in 2022-2023.

Conclusions: This is the first comprehensive study documenting the genetic evolution of IAVs in Türkiye over 6 years. Regional surveillance of antigenic changes can improve vaccine strain selection and vaccination strategies.