The premembrane and envelope (E) genes of a full-length cDNA clone of the dengue type-4 (DEN4) virus 814669 strain were replaced with those of the Japanese encephalitis (JE) virus JaOH0566 strain. The in vitro-synthesized RNA transcripts prepared from chimeric cDNA were used to transfect mosquito C6/36 cells. A viable chimeric virus (designated DEN4/JE) was recovered. Unexpectedly, DEN4/JE exhibited restricted growth in Vero cells. After a serial passage in Vero cells, the Vero-adapted chimeras were obtained (two clones, designated Strain I and Strain II, respectively). The entire genomes of DEN4/JE, Strain I, and Strain II were sequenced and compared. There were multiple mutations, but amino acid substitutions occurred only in E and nonstructural (NS) protein NS4B. Our findings in this study indicate that the 5' nontranslated region, E, and NS4B may be involved in Vero cell adaptation in this chimeric system.