Fig. 1.

High-content assay workflow. (A) Life cycle of DENV in host cells. (B) Design schematic of DENV infection assay. HEK293 cells (4,000 cells per well) are treated with compound, followed by DENV-2 inoculation at an MOI of 0.5, with antiviral activity and cytotoxicity evaluated at 48 h postinfection. DENV is detected by fluorescent antibody–based detection of the DENV-2 structural glycoprotein envelope (α-E). Compounds that inhibit viral replication show little or no green florescent signal. Cytotoxicity of compounds is evaluated by measuring a reduction in number of nuclei per well. Plates were fixed and stained as described in the Materials and Methods section. Images were acquired using the INCA3000, with green channel for DENV E and blue channel for detection of Hoechst-stained nuclei. DENV, dengue virus; DENV E, Dengue virus glycoprotein envelope; INCA3000, IN CELL Analyzer 3000; MOI, multiplicity of infection.