Nucleotide variations in 3’ untranslated region contribute to the enhanced virulence and replication efficiency of reemerging Japanese encephalitis virus genotype V

Japanese encephalitis virus, a major causative agent of viral encephalitis in Asia, is classified into five genotypes. Genotype V (GV) has reemerged after more than 50 years of apparent absence, following identification of the sole GV isolate – the Muar strain – in 1952. Newly reemerged GV strains are genetically distinct from the ancestral Muar strain. In this study, we compared virulence and replication efficiency between the newly reemerged XZ0934 strain and the ancestral Muar strain. Viral titers in cultured cells, as well as viremia and mortality in mice infected with the XZ0934 strain, were significantly higher than those observed with the Muar strain, indicating enhanced virulence and replication efficiency of the XZ0934 strain. Multiple nucleotide variations in the 3’ untranslated region (3’UTR) were identified between the XZ0934 and Muar strains; such variations contributed to the increased virulence and replication efficiency of the XZ0934 strain. These nucleotide variations resulted in substantial differences in the stem – loop (SL) I, SLII, and 3’SL structures of the 3’UTR and led to differential production of subgenomic flaviviral RNA (sfRNA). The variations also were responsible for differential inhibition of type I interferon production in vitro and in vivo between the two strains. These effects collectively contributed to the enhanced virulence and replication efficiency of the XZ0934 strain relative to the Muar strain. Our findings provide insight into the role of genetic variation in determining virulence and replication efficiency between newly reemerged and ancestral GV strains, as well as the molecular basis underlying GV virus reemergence.