H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a shorter neuraminidase (NA) stalk have become predominant in China. The A316S was shown to increase HA cleavage efficiency when combining with short stalk NA, and the short stalk NA improved NA enzyme activity and release of virus from erythrocytes. Single or combination of these mutations strengthened the virulence of H9N2 virus in chickens and mice.