Iterative genetic optimization of mature DENV3 and DENV4. (A) Sequence composition of prM cleavage sites of DV3-WT and DV3-prM-D89K. (B) Growth kinetics of DV3-WT and DV3-prM-D89K in C6/36 and Vero cells. (C) Representative Western blot image (bottom) of DV3-WT and DV3-prM-D89K viral supernatants blotted with anti-Env and anti-prM antibodies and quantification (top) of viral maturation (prM/Env) normalized to DV3-WT. All titers were determined on C6/36 cells. (D) Sequence composition of prM cleavage sites of DV4-WT, DV4-prM-E89K, and DV4-prM-E89N. (E and F) Growth kinetics of DV4-WT, DV4-prM-E89K, and DV4-prM-E89N in C6/36 (E) and Vero (F) cells. (G) Representative Western blot image (bottom) of DV4-WT, DV4-prM-E89K, and DV4-prM-E89N viral supernatants blotted with anti-Env and anti-prM antibodies and quantification (top) of viral maturation (prM/Env) normalized to DV4-WT. Growth kinetics and maturation of DENV variants were compared to their corresponding wild type using two-way ANOVA multiple comparisons.