Genetic Characterization of Influenza A (A/H3N2) Viruses Reveals Antigenic Drift in Receptor Binding Domain and Possible Vaccine Mismatch in Strains Circulating in Riyadh, Saudi Arabia, 2024-2025

Introduction: Influenza A/H3N2 viruses undergo continuous antigenic evolution, necessitating ongoing surveillance for informed vaccine strain selection. This study characterized the molecular epidemiology of H3N2 viruses circulating in Riyadh, Saudi Arabia, during the winter season of 2024-2025 and assessed their compatibility with current vaccine strains. Methods: Nasopharyngeal samples (NPAs) (n=363) were collected from patients presenting with influenza-like illness at King Khalid University Hospital in Riyadh. Influenza A/H3N2 detection and subtyping were performed using RT-PCR. Complete hemagglutinin (HA) and neuraminidase (NA) gene sequencing was conducted on confirmed A/H3N2 strains (n=7), followed by phylogenetic analysis, amino acid substitution mapping, and glycosylation site prediction. Results: Of 363 samples tested, 110 (30.3%) were positive for influenza A, with 42 (38.2%) identified as A/H3N2 and 68 (61.8%) as A/H1N1pdm09. Phylogenetic analysis revealed that all seven sequenced A/H3N2 strains belonged to clade 2a.3a.1, which is identical to the current vaccine strain clade. However, molecular analysis identified six amino acid substitutions in the HA gene and four in the NA gene that distinguished circulating strains from the A/H3N2 vaccine strain A/Thailand/8/2022. Notably, all study strains showed deletion of an N-glycosylation site (N122S) that is present in the vaccine strain. Conclusions: While phylogenetic clade compatibility suggests potential vaccine effectiveness, the observed amino acid differences and glycosylation site deletion highlight the importance of continued molecular surveillance to monitor antigenic drift and assess vaccine performance in the Saudi Arabian population.