Multiple amino acid substitutions involved in the virulence enhancement of an H3N2 avian influenza A virus isolated from wild waterfowl in mice

Frequent emergence of low pathogenic avian influenza H3N2 viruses in the wild birds has caused concern for human health. Here, we generated mouse-adapted strains of a wild waterfowl-origin low pathogenic avian influenza H3N2 virus to identify adaptive mutations that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the adapted strains were reduced >562-fold compared to the parental virus. Mouse-adapted strains displayed enhanced replication in vitro and in vivo, and acquired the ability to replicate in extrapulmonary tissues. These observations suggest that enhanced growth characteristics and modified cell tropism may increase the virulence of H3N2 AIVs in mice. Genomic analysis revealed mutations in the PB2 (E192 K and D701N), PB1 (F269S, I475 V, and L598P), HA (V242E), NA (G170R), and M1 (M192 V) proteins. Our results suggest that these amino acid substitutions collaboratively enhance the ability of H3N2 avian influenza A virus to replicate and cause severe disease in mammals.