FIG 4.

The C type lectin DC-SIGN enhances infection of human cells by tick cell-derived rUUKV S23. (A) BHK-21 cells were infected (at an MOI of 0.1) with rUUKV S23 derived from IRE/CTVM19 cells for 18 h and immunostained for N, G_N, and G_C proteins prior to flow cytometry analysis. (B) Parental (Raji) and DC-SIGN-expressing Raji cells (Raji DC-SIGN+) were infected with IRE/CTVM19 cell-derived rUUKV S23 and analyzed by flow cytometry 16 h after immunostaining against the viral nucleoprotein. (C) Parental (HeLa) and DC-SIGN-expressing HeLa cells (HeLa DC-SIGN+) were exposed to various MOIs of IRE/CTVM19 cell-derived rUUKV S23. The next day, infected cells were immunostained for the intracellular virus nucleoprotein N using the anti-N primary mouse monoclonal antibody 8B11A3 and an AF488-coupled anti-mouse secondary monoclonal antibody (green). Nuclei were stained with Hoechst (blue), and samples were analyzed by wide-field microscopy. (D) Raji DC-SIGN-expressing cells were exposed to IRE/CTVM19 cell-derived rUUKV S23 (MOI of ∼1) in the presence of inhibitors blocking DC-SIGN, namely, EDTA (5 mM) or the neutralizing mouse monoclonal antibody mAb1621 (25 μg · ml⁻¹). Intracellular viral antigens were detected by immunostaining with an anti-UUKV rabbit polyclonal antibody, followed by incubation with AF647-conjugated secondary antibodies. Infection was analyzed by flow cytometry 18 h later and normalized to infection of DC-SIGN-expressing Raji cells in the absence of inhibitor (as a percentage of the control).