Emergence and Genomic Characterisation of Influenza Virus A(H3N2) Subclade K in Saudi Arabia: Dominant Circulation with Fixed HA1 Substitutions and High-Frequency HA-NA Clade Association, 2025-2026.

The 2025-2026 Northern Hemisphere influenza season was characterised globally by rapid expansion of A(H3N2) subclade K. Saudi Arabia reported an A(H3N2)-dominant epidemic peaking in epidemiological Weeks 43-46 (20 October-16 November 2025), with national test positivity reaching 36.8%. To characterise circulating H3N2 viruses and assess divergence from the vaccine reference strain, we performed amplicon-based whole-genome Oxford Nanopore sequencing of 149 residual A(H3N2)-positive respiratory specimens collected in Riyadh between August and December 2025 (one specimen in August and the remainder during October-December, reflecting the epidemic curve). We recovered 81 haemagglutinin (HA) and 71 neuraminidase (NA) high-quality consensus sequences. Nextclade classified 64 HA sequences (79.0%) as subclade K and 17 (21.0%) as the parental clade J.2.4; NA sequences segregated into B.4.2.2 (61, 85.9%) and B.4.2 (10, 14.1%). Among 69 isolates with paired HA and NA, the two clade assignments were strongly non-independent (Fisher exact two-sided p = 8.4 × 10-4; odds ratio 0.077, 95% CI: 0.011-0.436), with the K + B.4.2.2 constellation predominating (50/69, 72.5%). Twelve of 69 isolates (17.4%) showed an HA-NA clade mismatch (nine J.2.4-HA + B.4.2.2-NA; three K-HA + B.4.2-NA), consistent with within-season reassortment. Whole-genome maximum-likelihood phylogenies of all eight gene segments showed that the K and J.2.4 lineages remained largely coherent across the genome, with the internal segments broadly co-segregating (pairwise topological concordance, Spearman ρ 0.18-0.71); the reassortment signal was therefore concentrated at the HA-NA surface-gene pairing. Six HA1 substitutions (K2N, S144N, N158D, I160K, Q173R, T328A) discriminated subclade K from J.2.4 at high statistical significance and mapped to antigenic sites A, B, C, and D and the protein N-terminus. No neuraminidase-inhibitor or baloxavir resistance markers were detected. These data document a K-clade-predominant epidemic with persistent J.2.4 co-circulation, within-season HA-NA reassortment, and marked HA1 divergence from the vaccine reference, against a background of fully susceptible antiviral genotypes.