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

ABSTRACT

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.