We investigated the tropism, host responses and virulence of two variants of A/Quail/Hong Kong/G1/1997 (H9N2) (H9N2/G1) with D253N and Q591K in the PB2 protein in primary human macrophages and bronchial epithelium in vitro and in mice in vivo. Virus with PB2 D253N and Q591K had greater polymerase activity in mini-replicon assays, induced more TNF-α in human macrophages, replicated better in differentiated normal human bronchial epithelial (NHBE) cells and was more pathogenic for mice. Taken together, our studies help define the viral genetic determinants that contribute to pathogenicity of H9N2 viruses.