The Single E627K Amino Acid Substitution in PB2 Enhances the Pathogenicity of Wild-Bird-Origin H6N6 Subtype Avian Influenza Virus in Mice

Abstract

Avian Influenza Viruses (AIVs) are harbored by wild waterfowl as a natural host, and there is a species barrier restricting virus transmission from birds to mammals, including humans. However, it has been reported that, through genetic mutations, AIVs occasionally infect mammals and acquire high pathogenicity. The Amino Acid (aa) substitution of glutamic acid to lysine at position 627 (E627K) in polymerase basic protein 2 (PB2) is one of the wellknown factors underlying mammalian adaptation. Although this substitution was previously observed in mammalian-adapted H5, H7, and H9 AIV subtypes, the impact of this mutation on the mammalian adaptation of other AIV subtypes is not fully verified. Here, we isolated the low pathogenic AIV subtype H6N6 from a wild bird fecal sample in Tokachi Subprefecture, Hokkaido, Japan. We passaged this H6N6 subtype in BALB/c mice four times and acquired the mouse-adapted virus. Whole-genome sequence analysis showed that the adapted virus had only one aa substitution (E627K) in PB2. The adapted virus-inoculated mice tended to show increased weight loss and mortality compared with the original virus-inoculated mice. The viral titer in the lungs of the adapted virus-inoculated mice was significantly higher than that of the original virus-inoculated mice. Additionally, the virus isolated from the lung of the original virus-inoculated mice with serious symptoms harbored the E627K substitution. Our findings indicate the possibility that the PB2 E627K substitution in H6N6 subtype AIV rapidly appears in mammalian hosts and contributes to the enhanced pathogenicity of this virus.