Background: The Influenza B virus (IBV), which accounts for nearly a quarter of annual global influenza-associated morbidity, represents a major human respiratory pathogen. Since late 2019, IBV transmission patterns have shifted markedly following the emergence of COVID-19, yet systematic studies on IBV prevalence before and after the pandemic remain limited.

Methods: We conducted systematic surveillance of 66 IBV isolates collected from Guangzhou during 2022-2023. Phylogenetic analysis of HA/NA genes was complemented with evolutionary rate estimation, hemagglutination inhibition assays, antigenic cartography, and structural modeling of hemagglutinin variants.

Results: We revealed the distinct epidemic trends of IBVs in Southern China: clade V1A.3a.1/V1A.3a.2 co-circulated in 2022-2023 influenza season, while complete replacement of V1A.3a.1 by V1A.3a.2 in 2023-2024 season, which was driven by a 1.36-fold increase in the evolutionary rate of the HA gene (1.632 × 10-3, P < 0.05) post-NPIs. Spatially, discrete Bayesian phylogeographic analysis confirmed that Jiangxi Province acted as the core upstream hub of the nationwide transmission of V1A.3a.1, which persisted endemically from Jiangxi in 2020 to six adjacent provinces in 2021 before its final extinction in China. Notably, cross-reactive HI and quantitative antigenic cartography elucidated antigenic divergence between V1A.3a.1 and V1A.3a.2 subclades, and revealed that the complete displacement of V1A.3a.1 by V1A.3a.2 was mediated by the broadened antigenic recognition of 2023 V1A.3a.2 isolates. Structural modeling revealed L144P in the 150-loop may be the primary driver of antigenic divergence, inducing conformational rearrangement in residues 146-148.

Conclusion: This study elucidates the unique evolutionary and epidemic patterns of IBV in the subtropical region of southern China after the relaxation of COVID-19 NPIs, deciphers the molecular mechanism of clade replacement and antigenic diversification of the Victoria lineage V1A.3a subclade, and validates the sustained effectiveness of the WHO 2022 influenza B vaccine strain update in southern China. Our findings fill the gap in systematic surveillance of IBV in subtropical sentinel regions, identify Guangzhou as a key monitoring site for IBV evolutionary dynamics, and provide critical molecular and antigenic evidence for the optimization of regional influenza surveillance systems and vaccine strain selection strategies.