The origin of internal genes contributes to the replication and transmission fitness of H7N9 avian influenza virus

H9N2 avian influenza viruses (AIVs) have donated internal gene segments during the emergence of zoonotic AIVs, including H7N9. We used reverse genetics to generate three reassortant viruses (2:6 H7N9) which contained the Haemagglutinin and Neuraminidase from Anhui/13 (H7N9) and the six internal gene segments from H9N2 AIVs of G1-like or BJ94-like lineages enzootic in different geographic regions in Asia. Infection of chickens with the 2:6 H7N9 containing internal gene segments from G1-like H9N2 conferred attenuation in vivo, with lower shedding and reduced transmission to contact chickens. However, possession of BJ94-like H9N2 internal gene segments resulted in more rapid transmission and significantly elevated cloacal shedding compared to the parental Anhui/13 H7N9. In vitro analysis also showed that the 2:6 H7N9 having BJ94-like internal genes had significantly increased replication compared to the Anhui/13 H7N9 in chicken cells. In vivo co-infection experiments followed, where chickens were co-infected with pairs of Anhui/13 H7N9 and one of each of the three 2:6 H7N9 reassortants. During ensuing transmission events, the Anhui/13 H7N9 virus outcompeted 2:6 H7N9 with internal gene segments of BJ94-like or G1-like H9N2 viruses. Co-infection did lead to the emergence of novel reassortant genotypes that were transmitted to contact chickens. Some of the reassortant viruses had a greater replication in chicken and human cells compared to the progenitors. We demonstrated that the internal gene cassette determines the transmission fitness of H7N9 viruses in chickens and the reassortment events can generate novel H7N9 genotypes with increased virulence in chickens and enhanced zoonotic potential.