NS5-V372A and NS5-H386Y variations are responsible for differences in interferon α/β induction and co-contribute to the replication advantage of Japanese encephalitis virus genotype I over genotype III in ducklings

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings asin vitroandin vivomodels of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-alpha and beta production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-alpha and beta induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-alpha and beta production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-alpha and beta induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-alpha and beta production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-alpha and beta production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift. Author summary The Japanese encephalitis virus (JEV) transmission cycle is maintained by mosquitoes and amplification hosts (pigs and birds). In areas without large pig populations, birds play a major role in the maintenance of the JEV transmission cycle. The shift in the dominant JEV genotype from genotype III (GIII) to genotype I (GI) is occurring in most countries in Asia. GI strains replicates more efficiently than GIII strains in birds, and this difference has been considered one of the reasons for the JEV genotype shift. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we demonstrated that NS5 is the viral determinant of the differences in replication efficiencies between the GI and III strains in birds. Furthermore, the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in type I interferon (IFN-I) induction and replication efficiency between the strains. Our findings suggested that the NS5-V372A and NS5-H386Y variations enable GI strains to inhibit IFN-I production more efficiently than GIII strains, thus resulting in antagonism of the IFN-I mediated antiviral response and consequently conferring a replication and host adaption advantage to GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.