Investigation of H9N2 avian influenza immune escape mutant that lacks haemagglutination activity

H9N2 avian influenza viruses pose a global threat to animal and human health. While vaccination is essential for mitigating disease impact, these viruses evolve to evade vaccine immunity through changes in the haemagglutinin (HA) glycoprotein. In this study, we identified immune escape mutation in an H9N2 virus resulting from pressure exerted by homologous chicken antisera. The immune-escape variant acquired an amino acid substitution, replacing glycine (G) with glutamic acid (E) at position 149 in the HA protein. The G149E mutant virus lost the ability to agglutinate chicken erythrocytes, while still maintaining replication comparable to the wild-type virus in chicken embryos and cells. This led to the hypothesis that the G149E substitution, leading to a shift from a neutral to a negative charge polarity at HA position 149, might be crucial for the optimal interaction between the virus and receptors on erythrocytes. Investigation indicated that agglutination could be restored by substituting E to positively charged amino acids histidine (H), arginine (R) or lysine (K). These findings suggest that the H9N2 virus may be likely acquire the G149E mutation under immune pressure in nature. This mutation poses challenges to vaccination and surveillance efforts as it partially evades immune protection and is not easily detectable by conventional haemagglutination assays. This underscores the intricate interplay between antigenic variation and viral traits, emphasising the critical need for ongoing surveillance and research to effectively mitigate the risks associated with avian influenza H9N2 viruses. IMPORTANCEUnderstanding how avian influenza viruses evolve to persist in nature is crucial for enhancing disease mitigation tools such as vaccines, diagnostics, and risk assessment. In this study, we identified an H9N2 virus antibody escape mutant with G149E mutation in the haemagglutinin that had lost the ability to agglutinate chicken erythrocytes, while retaining infectivity and replication fitness. The lack of haemagglutination activity potentially negatively impacts routine surveillance and commonly used diagnostics such as haemagglutination assay or haemagglutination inhibition assay. Therefore, it is urgent to develop and adopt alternative methods for viral detection. Difficult to detect variants potentially that are not compatible with common surveillance techniques could circulate remain silent while reassort with other influenza viruses, which posing unpredictable risks to animal and human health. This research helps us better understand avian influenza, leading to improved disease control, diagnostics, and risk assessment to protect both animals and humans.