Genomic Characterization and Structural Insights Into the Evolution of Influenza A(H1N1)pdm09 in Northeast India From 2009 to 2024

Influenza A(H1N1)pdm09 continues to evolve through antigenic drift, posing challenges to vaccine effectiveness. The present study investigated the evolutionary dynamics, glycosylation patterns, and immunological features of the hemagglutinin (HA) gene of Influenza A(H1N1)pdm09 from the region. A total of 120 positive samples collected between 2022 and 2024 were sequenced using the Illumina MiSeq platform alongside previously generated HA sequences from the laboratory during 2009-2019. Mutational analysis was performed against the reference strain (A/California/07/2009) and the vaccine strain (A/Wisconsin/67/2022), followed by phylogenetic, structural, N-glycosylation, and epitope analysis using bioinformatics tools. Several persistent mutations (P100S, S220T, R240Q, I338V, E391K) were observed across study years, while substitutions such as S101N, D114N, A273T, K300E, S468N, E516K, and K180Q were consistently detected after 2015. Recent circulating strains (2022-2024) acquired additional mutations (K71Q, N146D, K147N, L178I, N173K, and A203T) and predominantly exhibited S202I rather than the earlier S202T substitution. Phylogenetically, these strains clustered within clade 6B.1 A.5a.2a. The presence of an additional N-glycosylation site at residue 179 after 2017 may suggest immune-driven selection. Notably, T137A, K186Q, and E277D substitutions identified in 2024 Influenza A(H1N1)pdm09 strains were associated with the loss of predicted epitope-binding regions relative to the vaccine strain A/Wisconsin/67/2022. These amino acid changes suggest potential alterations in antigenic structure that may influence immune recognition. No differences in mutation distribution were observed between the indoor and outdoor cases. Overall, the study represents the first long-term genomic surveillance of Influenza A(H1N1)pdm09 from Northeast India and demonstrates continuous genetic diversification and evolutionary dynamics of circulating Influenza A(H1N1)pdm09 strains.