Real-Time Visualization of the Infection and Replication of a Mouse-Lethal Recombinant H9N2 Avian Influenza Virus

H9N2 avian influenza viruses (AIVs) continuously cross the species barrier to infect mammalians and are repeatedly transmitted to humans, posing a significant threat to public health. Importantly, some H9N2 AIVs were found to cause lethal infection in mice, but little is known about the viral infection dynamics . To analyze the real-time infection dynamics, we described the generation of a mouse-lethal recombinant H9N2 AIV, an influenza reporter virus (V -NanoLuc virus) carrying a NanoLuc gene in the non-structural (NS) segment, which was available for imaging. Although attenuated for replication in MDCK cells, V -NanoLuc virus showed similar pathogenicity and replicative capacity in mice to its parental virus. Bioluminescent imaging of the V -NanoLuc virus permitted successive observations of viral infection and replication in infected mice, even following the viral clearance of a sublethal infection. Moreover, V -NanoLuc virus was severely restricted by the K627E mutation in PB2, as infected mice showed little weight loss and a low level of bioluminescence. In summary, we have preliminarily established a visualized tool that enables real-time observation of the infection and replication dynamics of H9N2 AIV in mice, which contributes to further understanding the mechanisms underlying the pathogenic enhancement of H9N2 AIV to mice.