H9N2 avian influenza virus in Bangladesh: three genetically distinct co-circulating subgroups retain a largely conserved antigenic phenotype

Low pathogenic avian influenza (LPAI) H9N2 viruses are endemic in poultry across Bangladesh, causing substantial economic losses and posing a persistent zoonotic risk. Although genetic surveillance has documented ongoing viral evolution, the relationship between genetic diversification and antigenic change remains poorly understood. This study investigated the genetic diversity, reassortment, and antigenic characteristics of contemporary H9N2 viruses circulating in Bangladesh. Phylogenetic analysis of 219 sampled sequences revealed three genetically distinct monophyletic haemagglutinin (HA) G-lineage sub-groups, each characterised by unique amino acid substitutions. In contrast, neuraminidase (NA) displayed similar genetic diversity without distinct phylogenetic clusters. A dual-metric analysis of paired HA and NA phylogenies identified 20 putative reassortant H9N2 viruses. Further analysis showed that genetic diversity was geographically structured; however, all three HA subgroups co-circulated across multiple regions, and some live bird markets contained representatives of all three groups simultaneously, suggesting that live bird markets may facilitate mixing between genetically distinct viruses, and create opportunities for reassortment among otherwise geographically separated strains. Despite substantial genetic diversity and reassortment, all viruses remained antigenically similar based on haemagglutination inhibition and virus neutralisation assays. However, a representative strain from one subgroup induced significantly lower homologous and heterologous antibody titres, suggesting reduced immunogenicity. Our findings demonstrate a notable disconnect between genetic evolution and antigenic change in Bangladeshi H9N2 viruses. While reassortment and genetic diversification continue to shape viral populations, antigenic properties remain largely conserved, supporting the continued relevance of current vaccine strategies while highlighting the importance of ongoing genetic and antigenic surveillance.