Intrahost HA polymorphisms and culture adaptation shape antigenic profiles of H3N2 influenza viruses

Accurate antigenic characterization of influenza viruses is essential for vaccine strain selection, yet routine isolation of viruses in cell culture can introduce genetic changes that obscure the properties of circulating strains. By combining deep sequencing with serological analysis of clinical specimens and cultured isolates, we demonstrate that virus propagation of human seasonal A(H3N2) in MDCK cells imposes strong purifying selection and alters antigenic profiles. Furthermore, we show that minor amino acid polymorphisms present in clinical samples can generate measurable antigenic diversity, emphasizing that natural intrahost variation shapes antigenic outcomes. These findings reveal a critical source of bias in antigenic characterization workflows and underscore the importance of directly assessing uncultured clinical material. Improved understanding of how culture adaptation and intrahost genetic diversity influence antigenic data will advance knowledge of antigenic evolution in circulating influenza viruses and improve the accuracy of vaccine strain selection for human seasonal influenza.