H9N2 avian influenza virus (AIV) has an extended host range but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64-B71), and formed a distinct antigenic subgroup. Three isolates belonging to the genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses in parallel tested in other genotypes replicated poorly although they have a same leucine at position 226 in the hemagglutinin (HA) receptor binding site critical for binding human-like sialic acid receptors as these three viruses. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity, but enhances binding affinity to human cells and lung tissues from mouse and human. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insight on understanding host range expansion of H9N2 AIVs.

IMPORTANCE:

Influenza hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring an amino acid 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that amino acid 190V in the HA does not change virus receptor binding specificity, but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from human and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance, which have a potential threat to mammals including humans.