Figure 2.

The late epidemic signature NS5_357E exhibits enhanced virus replication in both mosquito and mammalian cells

(A) Illustration of the amino acid variations among the eight infectious clone-derived DENV-2 viruses, covering all possible combinations of the three amino acid signatures (E₄₆, NS5₂₇₁, and NS5₃₅₇). We marked the II-LLE strain as II-LLE∗ to reflect its mixed population.

(B) Plaque morphology (upper panel) and focus morphology (lower panel) of viruses carrying various combinations of mutations were assessed in BHK-21 and Vero cells, respectively.

(C and D) For quantitative analysis, we selected at least 15 plaques or foci, and measured their size using ImageJ software. The results are presented in the bar chart showing plaque size (C) and focus size (D), with plaque/focus sizes represented as mean ± SD.

(E and F) We evaluated the growth of DENV-2 with different combinations of mutations at MOI 0.1 in C6/36 (E) and Vero (F) cells. Virus titers at 2 days post-infection (d.p.i.) are presented as the mean and SD in the jitter plots. Statistical analysis was performed using one-way ANOVA to determine the differences between Ia and the mutant viruses, with significance levels denoted as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.