Time course of DENV penetration.

<p>A. 100–200 PFU/well of DENV-1 HW, DENV-2 NGC, DENV-2 16681 or JUNV were bound to cells at 4°C and then allowed to internalize after rapid warming to 37°C. Ammonium chloride was added at different time points to inhibit penetration and infection. Cells were further incubated at 37°C for 3 h, then extracellular virus was inactivated and cultures were overlaid with plaquing medium. The infection levels observed were normalized to the level in control cells without ammonium chloride. Each point is the mean±SD of three independent experiments. B. Cells were treated or not with 50 mM ammonium chloride and then stained with acridine orange. C. DENV-2 NGC was adsorbed to Vero cells during 1 h at 4°C in the presence or absence of 50 mM ammonium chloride and then cultures were shifted to 37°C. At different time intervals cultures were fixed and processed to reveal C and E protein by immunofluorescence using mouse anti-C and mouse anti-E antibodies followed of FITC-labelled anti-mouse IgG, respectively.