Pre-pandemic contraction, phase-specific rate variation, and site-specific antigenic adaptation shape influenza A(H3N2) evolutionary dynamics in Hubei, China.

Influenza A(H3N2) is a contributor to seasonal epidemics, yet its evolution during and after the COVID-19 pandemic is not fully understood. We analyzed surveillance data and hemagglutinin (HA) and neuraminidase (NA) sequences from Hubei, China, 2017-2024. The epidemic followed regular seasonality before 2020, nearly disappeared during 2020-2022, and resurged with strong peaks after mid-2022. Multiple HA lineages co-circulated pre-pandemic. Several localized reassortment signals were observed before the pandemic, though overall evidence for intra-subtype reassortment remained limited. After resurgence, single dominant clades expanded, genetic diversity dropped, and clade turnover became clear. Bayesian coalescent analyses showed that effective population size had already declined by late 2019, before widespread SARS-CoV-2 transmission and interventions. Relaxed-clock and selection pressure analyses revealed heterogeneous HA substitution rates among lineages, likely reflecting differences in baseline substitution rates and the effects of transmission bottlenecks, rather than detectable shifts in selection intensity. Overall, HA and NA evolution was dominated by purifying selection, with a few sites in antigenic regions showing evidence of positive selection, coinciding with recurrent amino acid changes. Pre-pandemic population contraction, phase-specific rate variation, and site-specific antigenic adaptation shaped lineage turnover and epidemic patterns in Hubei. Monitoring population shifts, lineage-specific rates, and key antigenic substitutions can guide vaccine updates and strengthen preparedness.IMPORTANCEThe COVID-19 pandemic disrupted influenza activity worldwide, raising questions about its long-term effects on viral evolution. Our analysis of H3N2 in Hubei, China, shows that lineage contraction began before the pandemic and that post-pandemic evolutionary dynamics were shaped primarily by demographic processes, while purifying selection continued to dominate, with adaptive changes restricted to a limited number of antigenic sites. These findings suggest that intrinsic viral processes, not just pandemic restrictions, played a central role in shaping epidemic patterns and highlight the importance of continuous genomic surveillance to anticipate future outbreaks.