FIG 2.

Growth kinetics and maturation status of DENVs on Vero and VF1 cells. (A) Schematic of the sleeping beauty-based transposon cassette for ectopic expression of human furin (hFurin). A bidirectional EF1a promoter was used to drive the expression of hFurin and red fluorescent protein (RFP) with a puromycin resistance gene (Puro) linked by a 2A self-cleaving peptide (P2A). (B and C) Western blot (B) and immunofluorescence (C) images of polyclonal and clonal VF1 using anti-furin antibodies. (D to G) Growth kinetics and degree of maturation of DENV1-WT (D), DENV2-WT (E), DENV3-WT (F), and DENV4-WT (G) in unmodified Vero cells (black circles) and VF1 cells (blue triangles). Cells were infected with DENV at an MOI of 0.01 to 0.05 for 120 h. Supernatants were harvested every 24 h for 120 h, and the 120-h supernatants were analyzed by Western blotting for DENV maturation using anti-Env and anti-prM antibodies. All assays were performed with at least two biological repeats with two technical replicates. Growth kinetics of DENV variants were compared to their corresponding wild type using two-way ANOVA multiple comparisons.