Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3-non-coding region were prepared by passage in fetal rhesus lung (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or virus neutralizing antibody. In 2009, analysis of consensus sequences revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu327-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells, as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney (AGMK) cells or Vero cells. The Glu327-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu327-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate (HS), compared to the parental DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu327-Gly mutation in DIII of DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation, since the virus may change during passages in certain cells through adaptive selection and such mutations may affect cell tropism, virulence and vaccine efficacy.