Substitutions at positions PB2-389 and -598 were identified with potential effect on virus pathogenicity. PB2-K389R and -V598T/I substitutions enhanced virus growth capacity in human and mammalian cells. The PB2-K389R and -V598T/I increased virus transcription/replication by producing more viral RNAs in human cells. The PB2-K389R and -V598T/I substitutions elevated the polymerase activity in human cells. The PB2-V598T/I substitutions increased virus replication and virulence in mice.