Molecular Characterization of Influenza A(H3N2) Hemagglutinin Variants Circulating in Western Mexico, 2022

Loss of hemagglutination ability by H3N2 influenza A virus, subclade K

Seasonal human H3N2 influenza viruses, subclade K (J.2.4.1), have been the predominant influenza A viruses in the Northern hemisphere influenza season of 2025/2026. Since 2024, the vaccine virus A/Darwin/6/21 has emerged in different antigenic variants. Antigenic changes are frequently caused by amino acid substitutions near the hemagglutinin (HA) receptor-binding pocket, which can also affect receptor binding properties, such as hemagglutination. Hemagglutination is crucial for assessing antigenicity using the hemagglutination inhibition (HAI) assay, and a loss of binding to turkey erythrocytes could significantly hamper this process. In this study, we explored how substitutions in or around the HA receptor-binding site affect binding to glycans at the molecular level. We employed ELISA, glycan array, flow cytometry, hemagglutination assays, and tissue staining. Substitutions at positions 140, 192, and 223 establish clade J viruses that emerged in 2024. Computational analysis of HA in complex with an elongated glycan reveals that mutation F192 forms a CH-Pi interaction to stabilize the binding. Based on this background, substitutions in antigenic sites A and B within subclade K viruses exhibit a binding preference for elongated glycans, which are not displayed on turkey erythrocytes. Conversely, our previously established glyco-remodeled erythrocytes are efficiently bound by these subclade K H3N2 viruses and could support influenza surveillance and vaccine development.

Identification of two novel B-cell epitopes targeting the hemagglutinin protein of H9N2 avian influenza virus

H9N2 avian influenza virus (AIV) remains a global threat to poultry health and has zoonotic potential. Antigenic drift in the hemagglutinin (HA) protein complicates vaccine efficacy and diagnostic accuracy, highlighting the need for precise epitope characterization. In this study, the HA protein of H9N2 AIV was expressed in a eukaryotic system, and two monoclonal antibodies (mAbs), 9C12 and 9F4, were generated. Both mAbs specifically bound HA, as shown by ELISA, Western blot, and immunofluorescence, but lacked hemagglutination inhibition activity. Epitope mapping revealed two minimal linear epitopes: 123FSSSRSYQ130 within the vestigial esterase domain and 201NLYTRTDTT209 within the receptor-binding domain. Alanine scanning revealed key residues required for antibody binding, whereas structural modeling confirmed that both epitopes are surface exposed. Sequence analysis demonstrated strong conservation across H9N2 strains, with the 9F4 epitope showing near-complete invariance, whereas both epitopes exhibited low conservation among other influenza A virus subtypes. These findings define two novel, nonneutralizing epitopes on H9N2 HA that expand the antigenic map and represent promising targets for subtype-specific diagnostic assays.

Genetic Diversity, Reassortment Patterns, and Antigenic Characterization of Two H9N2 Avian Influenza Viruses Isolated from Quails in China

H9N2 avian influenza viruses pose a persistent zoonotic risk owing to their broad host adaptability. Between 2024 and 2025, two H9N2 isolates (ZHY1022B2 and ZHY0417A11) were recovered from quail flocks in Hebei, China. Phylogenetic analysis clustered their HA genes within the dominant B4.7.2 subclade, but diverged into two subgroups (B4.7.2.2 and B4.7.2.1). Of particular interest was ZHY0417A11, which displayed a multigenic reassortment pattern—its PB1 originated from an H3N8 virus, PA and PB2 from H3N3, NS from H10N3, and the M gene was nearly identical (99.32%) to a human H3N8 isolate, whereas the remaining HA, NA and NP segments maintained the H9N2 backbone. Despite the presence of the HA mammalian-adaptive markers (H191N, A198V and Q234L), both strains showed marked antigenic drift from the vaccine strain SS (BJ/94-like; R < 0.5), while retaining reactivity with currently circulating field strains. These findings argue for heightened vigilance in quail populations, given their role as mixing vessels, and highlight the limitations of current vaccine matching in light of ongoing H9N2 evolution.

Mutations in severe human H5N1 cases facilitate evasion from human mucus and antivirals

In late 2024, two individuals in Canada and the United States were treated in intensive care for acute respiratory distress caused by infection with the avian Influenza A Virus H5N1 2.3.4.4b genotype D1.1. Viral sequence data obtained from sampling these patients indicated mixed alleles at haemagglutinin (HA) positions 190 and 226. Mutations at these positions are key determinants of HA usage of α2,6-linked sialic acids (SA), the most abundant influenza receptors in human upper respiratory tracts. Thus, these mutations raised concerns about human adaptation and pandemic potential of the H5N1 virus. In this study, we investigated the impact of the mutations at residues 190 and 226 in H5 HA. We studied the receptor binding properties, cell entry phenotypes and fitness impacts of the mutations using recombinant proteins, pseudotyped lentiviruses, and in the context of influenza viruses using reverse genetics. The mutations did not confer any detectable α2,6-linked sialic acid receptor usage either alone or in combination. Rather, viruses carrying these mutations exhibit weakened binding towards α2,3-linked sialic acid receptors. This correlated with an enhanced capacity to evade human airway mucus, and a reduced susceptibility to oseltamivir and zanamivir. This research underscores that in addition to the way HA interacts with SA as entry receptors, other factors that impact the HA/NA balance might influence the evolutionary trajectory of a zoonotic virus in the human respiratory tract. This study presents a new paradigm for the evolutionary drivers of HA, where reduced sialic acid binding can serve as an advantage for escape from host barriers and antivirals.

Identification and computational analysis of B-cell epitopes on the hemagglutinin protein of the H3N2 influenza virus

Emerging Haemagglutinin Mutations in Bovine-origin H5N1 Influenza Viruses from Humans and Cattle Retain Avian Receptor Binding with Increased Stability

The current H5N1 panzootic has seen an unprecedented host range expansion, including sustained circulation in US dairy cattle, detected in March 2024. By July 2026, infections had been reported on more than 1,150 dairy farms across 19 states. Although the outbreak initially centred in Texas, California has emerged as the principal focus of transmission and accounts for most human infections associated with exposure to infected dairy cattle. Continued transmission in cattle and repeated spillover into humans increase opportunities for acquisition of mammalian-adaptive mutations that could elevate zoonotic and pandemic risk. The haemagglutinin (HA) protein plays a central role in modulating virus receptor binding and airborne transmission. Here, we characterised the receptor-binding and stability phenotypes of HA mutations identified in viruses circulating in Californian dairy cattle. Receptor-binding specificity was assessed using bio-layer interferometry and pseudotype virus entry assays. All tested HA variants maintained a preference for avian-type α2,3-linked sialic acid receptors. We evaluated HA stability using fusion and thermostability assays. All mutants exhibited fusion pH values >5.5, outside the range associated with efficient airborne transmission in humans (pH 5.0-5.5). However, mutations D88G and S94N increased pH stability, with fusion pH values of 5.6 and 5.7, respectively, compared with 5.9 for wild-type HA. Viruses harbouring both mutations displayed increased thermostability. These findings demonstrate that cattle-origin H5N1 viruses retain avian-like receptor specificity despite acquiring mutations that modestly enhance HA stability. Evolution of H5N1 viruses in dairy cattle underscores the importance of genomic and phenotypic surveillance to identify mutations that may increase zoonotic risk.