Introduction: Influenza A viruses cause global health concerns due to their high amino acid substitution rates. They are linked to yearly seasonal epidemics and occasional pandemics. This study focused on sequencing influenza A virus strains in Pakistan.

Materials and methods: We analyzed the genetic characteristics of influenza A(H1N1)pdm09 and A(H3N2) viruses circulating in Pakistan from January 2020 to January 2023. Whole genome sequences from influenza A (n = 126) virus isolates were amplified and sequenced by the Oxford Nanopore (MinION) platform.

Results: The HA genes of influenza A(H1N1)pdm09 underwent amino acid substitutions at positions K54Q, A186T, Q189E, E224A, R259K, and K308R in sequenced samples. The HA genes of influenza A(H3N2) had amino acid substitutions at G53D, E83K, D104G, I140M, S205F, A212T, and K276R in the sequenced samples. Furthermore, the HA gene sequences of influenza A(H1N1)pdm09 in this study belonged to subclade 6B.1A.5a.2a. Similarly, the HA gene sequences of influenza A(H3N2) were classified under six subclades (3C.3a.1 and 3C.2a1b.2a [2, 2a.1, 2b, 2c, and 2a.3b]). Notably, amino acid substitutions in other gene segments of influenza A(H1N1)pdm09 and A(H3N2) were also found.

Conclusion: These findings indicate influenza A(H1N1)pdm09 and A(H3N2) viruses co-circulated during the 2020-2023 influenza season in Pakistan. Continued surveillance is crucial for real-time monitoring of possible high-virulence variation and their relevance to existing vaccine strains.