Molecular Surveillance and Genomic Characterization of Influenza A(H3N2) Viruses From a Secondary‐Care Surveillance Site in Pakistan During the 2025–2026 Winter Season

ABSTRACT Respiratory viral infections, particularly Influenza A and SARS‐CoV‐2, remain major public health concerns in endemic settings such as Pakistan, where high influenza‐like illness burden and limited genomic data underscore the need for surveillance to guide vaccine strategies. This study assessed respiratory virus circulation and genetic diversity among ILI cases at a secondary‐care hospital in Islamabad during the 2025–2026 winter season. Among 170 qRT‐PCR‐tested cases, Influenza A was detected in 24.1%, SARS‐CoV‐2 in 1.17%, and Influenza B was not detected. Influenza A positivity was slightly higher in females (25.0%), with highest burden in individuals aged 11–20 years. Whole‐genome sequencing of 21 Influenza A(H3N2) isolates revealed co‐circulation of subclade K (52.4%), J.2.2 (28.6%), and J.2 (9.5%). Phylogenetic analysis showed subclade K clustering with recent isolates from North America, Oceania, Europe, and WHO‐recommended 2026–2027 Northern Hemisphere H3N2 vaccine strains, whereas J.2 and J.2.2 aligned with earlier vaccine‐like strains. NA analysis showed predominance of clade B.4.2.2 associated with K‐lineage and relatedness to recent North American strains. Subclade K exhibited conserved HA substitutions (K2N, S144N, N158D, I160K, Q173R, T328A) and HA2 S49N with limited variation. No antiviral resistance mutations were detected. Overall, findings demonstrate predominance and genetic stability of H3N2 subclade K and support continuous genomic surveillance for vaccine strain selection and influenza preparedness.