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

Accurate antigenic characterization of influenza viruses is critical for vaccine strain selection but is often confounded by intrahost genetic diversity and culture-induced adaptations. We analyzed 60 A(H3N2)-positive nasopharyngeal specimens collected during the 2017-2018 influenza season to determine how virus propagation in MDCK cells affects viral genetic and antigenic properties. Deep sequencing revealed frequent genome-wide intrahost polymorphisms, including amino acid variants within major hemagglutinin (HA) antibody-binding sites. Virus propagation imposed rapid purifying selection, markedly reducing intrahost genetic diversity. Serological analyses demonstrated that these selective events altered antigenic properties, indicating that culture adaptation can alter antigenic profiles. To assess the functional impact of HA polymorphisms, we generated mixed viral populations containing defined ratios of HA-160K, HA-160T, and HA-160I variants identified in clinical samples. These reconstituted populations exhibited a continuum of antigenic reactivities, confirming that amino acid polymorphisms drive antigenic heterogeneity among clinical samples. Together, our findings demonstrate that intrahost variation contributes to antigenic diversity in human A(H3N2) clinical specimens and that virus isolation procedures can bias antigenic characterization, underscoring the importance of direct profiling of uncultured clinical material to improve vaccine strain selection accuracy for seasonal human influenza. IMPORTANCE: 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.