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Several studies have demonstrated that neutralizing epitopes in the genus <jats:italic>Flavivirus<\/jats:italic> are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM\u2010E) gene\u2010based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H\/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM\u2010E gene backbone showed high neutralizing antibody titers with less DENV infection\u2010enhancing activity. 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