Rapid emergence of a PB2-E627K substitution confers a virulent phenotype to an H9N2 avian influenza virus during adaption in mice

The worldwide circulation of H9N2 avian influenza virus in poultry, the greater than 2.3 % positive rate for anti-H9 antibodies in poultry-exposed workers, and several reports of human infection indicate that H9N2 virus is a potential threat to human health. Here, we found three mutations that conferred high virulence to H9N2 virus in mice after four passages. The PB2-E627K substitution rapidly appeared at the second passage and played a decisive role in virulence. Polymerase complexes possessing PB2-E627K displayed 16.1-fold higher viral polymerase activity when compared to the wild-type virus, which may account for enhanced virulence of this virus. The other two substitutions (HA-N313D and HA-N496S) enhanced binding to both α2,3-linked and α2,6-linked sialic acid receptors; however, the HA-N313D and N496S substitutions alone decreased the virulence of mouse-adapted virus. Furthermore, this mouse-adapted virus was still not transmissible among guinea pigs by direct contact (0/3 pairs). Our findings show that adaption in mice enhanced the viral polymerase activity and receptor-binding ability, which resulted in a virulent phenotype in mice but not a transmissible phenotype in guinea pigs, indicating that host factors play an important role in adaptive evolution of influenza in new hosts.