Inability of the reporter DENV2 to replicate in mosquito host.

A) Fluorescent microscopy (top panel) and flow cytometry (bottom panel) of C6/36 cells infected with DENV2-16681 (WT) and DENV2-GFPs with 1x 2A and 2x 2A designs. C6/36 cells were infected with a DENV2 virus at MOI = 0.1 and cultured for 3 days before analyses. Infected cells were identified by immunostaining with 4G2 antibody. The number in each quadrant of the flow cytometry scatter plot represents the percentage of cells in the quadrant. B) Fluorescent microscopy of Aedes aegypti mosquitoes that received DENV2-16681 (WT), DENV2-Clover2-2x 2A, and DENV2-eGFP-2x 2A by intrathoracic injection. The mosquitoes were imaged seven days after injection. C) Propagation of viruses in the mosquitoes injected with DENV2-16681 (WT), DENV2-Clover2-2x 2A, and DENV2-eGFP-2x 2A. The infectious titer (FFU/mosquito) was quantified from the whole-body homogenate of a mosquito by immunostaining foci assay at 4, 7, and 10 days post intrathoracic injection (dpi). The table underneath the plot reports the descriptive statistics of the infectious-titer measurements that include the number of the mosquitoes (n), the minimum (Min), the maximum (Max), the median, the mean, and the standard deviation (SD) of the infectious titers for each condition.