Hemagglutinin and neuraminidase of an H7N7 non-pathogenic avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens

Abstract Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named Vac2sub-P0, which is non-pathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs and a virus with high intravenous pathogenicity, namely, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 is only partially lethal in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the passaged virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages are responsible for increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with the parental virus in vitro; the virus growth at 40°C was higher, the binding affinity to a sialic acid receptor was lower, and the release activity by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study critically demonstrated that additional adaptations were required for the highly pathogenic virus via intravenous administration in chickens for increased pathogenicity via intranasal administration.