A D200N hemagglutinin substitution contributes to antigenic changes and increased replication of avian H9N2 influenza virus.

• The amino acid position 200 in HA1 of H9N2 AIVs is a newly identified multi-functional site. • The D200N substitution of HA1 changed antigenicity of H9N2 AIVs. • D200N increased the HA cleavage efficiency and reduced acid and thermal stability of HA protein. • Residue 200N increased the replication of H9N2 viruses in both chicken embryo fibroblast cells and chicken embryonated eggs. Influenza virus hemagglutinin (HA) plays an important role in viral antigenicity, replication and host range. However, few amino acid positions in HA were reported to play multiple functions in both viral antigenicity and replication. In the present study, through analyzing the amino acid sequences of H9N2 avian influenza viruses (AIVs) isolated from China, we identified a multi-functional substitution of D200N in HA1 protein. Firstly, the substitution of D200N changed the antigenicity of H9N2 AIVs. Secondly, the D200N increased the HA cleavage efficiency and reduced acid and thermal stability of HA protein, which triggered viral-endosomal membrane fusion whereby promoted the release of viral genome into the host cytoplasm. Finally, residue 200-N increased the replication of H9N2 viruses in both chicken embryo fibroblast (CEF) cells and chicken embryonated eggs. In summary, the D200N substitution is a newly identified antigenicity and replication determinant of H9N2 AIVs, which should be paid more attention during surveillance. [ABSTRACT FROM AUTHOR]