Effect of 3b on DENV-2 entry, RNA synthesis and protein expression. (A) Vero cells were infected with DENV-2 in the absence or presence of 100 μM 3b. After 15 min adsorption at 4°C indirect immunofluorescence staining of E glycoprotein was performed by using mouse monoclonal antibody against DENV E glycoprotein and fluorescein isothiocyanate-labeled goat anti-mouse IgG. Cells were stained with Evans blue. Magnification: 1000X. (B) DENV-2 was adsorbed to Vero cells for 1 h at 4°C adsorption. Then, cells were incubated at 37°C for 1 h in medium containing or not 100 μM 3b, and thereafter cells were stained for indirect immunofluorescence as above. Magnification: 400X. (C) Vero cells were infected with DENV-2 and incubated for 8, 24 and 48 h in the absence or presence of 100 μM 3b. At each time point, total RNA was extracted and cDNA was synthesized with random primers. These cDNAs were amplified by real time PCR using specific primers to amplify the ns1 gene, and cellular actin was used for normalization. Results are expressed as % inhibition DENV-2 RNA level respect to viral control. (D) Vero cells were infected with DENV-2 in the absence (virus control VC) or presence (3b) of 100 μM 3b. CC: cell control. At 48 h p.i. indirect immunofluorescence staining of E glycoprotein was performed as in (A). Magnification: 400X.