FIG 7.

The wing domain of DENV NS1 and residues 91 to 93 within the wing domain confer NS1-induced localized vascular leak in vivo. (A, B) Wild-type C57BL/6J mice were shaved by removing hair from their backs 3 days before the experiment. On the day of experiment, PBS, WT, and chimeric NS1 proteins (15 μg) were administered intradermally into discrete spots on the shaved dorsal dermis. Immediately following NS1 injections, 25 μg of Dextran conjugated to Alexa Fluor 680 was administered retro-orbitally. Two hours postinjection, the dermis of each mouse was collected and visualized using a fluorescent scanner. Representative images of the dorsal dermis from at least 4 mice are shown. Chimeric proteins were analyzed in different mice with each mouse containing both positive and negative controls (WT DENV and WNV NS1, respectively). (C) Mean fluorescent intensity (MFI) quantification of (A) and (B), normalized to PBS injection. (D) Same as (A) and (B), but using site-directed NS1 mutants that exchanged residues 91 to 93 between DENV and WNV NS1. Representative image of the dorsal dermis from at least 4 mice backs are shown. (E) MFI quantification of (D), normalized to PBS injection. Data plotted as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 by unpaired Mann-Whitney U test. Star colors indicate the respective control to which each construct was compared.