An amino acid substitution (V3I) in the Japanese encephalitis virus NS4A protein increases its virulence in mice, but not its growth rate in vitro

Our previous studies have shown that the Japanese encephalitis virus (JEV) strain Mie/40/2004 is the most virulent of the strains isolated by us in Japan from 2002 to 2004. Comparison of the amino acid sequence of Mie/40/2004 with those of low-virulence strains revealed that an isoleucine residue at position 3 of the Mie/40/2004 NS4A protein may increase viral pathogenicity. A recombinant virus with a single valine-to-isoleucine substitution (V3I) at position 3 in the low-virulence Mie/41/2002 background (rJEV-Mie41-NS4A(V3I)) exhibited increased virulence in mice compared with the Mie/41/2002 parent strain. The V3I mutation did not affect virus growth in several cell lines. These results demonstrate that the isoleucine at position 3 in the NS4A protein of Mie/40/2004 is responsible for its high virulence in vivo. This is the first report to show that an amino acid substitution in a flavivirus NS4A protein alters viral pathogenicity in mice.