Identification of mutations that occurred on the genome of Japanese encephalitis virus during the attenuation process

The total nucleotide sequences of the genomes of two Japanese encephalitis virus (JEV) strains, the attenuated vaccine strain SA14-14-2 and its parental virulent strain SA14, were determined by using the molecular cloning technique. The sequence analysis revealed that both virion RNA molecules were 10,976 nucleotides long with 95 and 585 flanking bases at the 5' and 3' untranslated sequences, respectively. A single, long open reading frame spanning 10,296 nucleotides was observed to encode a polyprotein of 3432 amino acid residues. When these sequences were compared with each other, 57 nucleotide substitutions were found to be scattered all over the genome. Of these, 24 resulted in amino acid changes within viral proteins. Structural proteins C and E contain one and eight amino acid changes, respectively. Of the nonstructural proteins, NS1 contains three, NS2a two, NS2b two, NS3 four, NS4a one, NS4b one, and NS5 two amino acid substitutions. The 5'- and 3'-terminal untranslated regions contain one- and two- point mutations, respectively. These data and comparative studies with other JEV strain genomes provide a molecular basis for investigating attenuation mechanisms of JEV.