Figure 3.

Cell attachment and uptake of recombinant sNS1^WNV by different cell types. Aliquots of extracellular, H₆-tagged sNS1^WNV protein, released by pNS1^WNV-IS98-transfected HEK-293T cells and purified by affinity chromatography, were added to the cell culture media of Vero E6, SH-SY5Y, and U-87MG cells, at a final concentration of 10 μg/mL. In one series of samples, cells were collected after 1 h incubation on ice (gray bars) and cell-bound NS1^WNV was detected and quantified by flow cytometry. In another series (black bars), cells were first incubated on ice for 1 h, then transferred to 37 °C for 6 h, permeabilized, and intracellular NS1^WNV was evaluated by flow cytometry. Control mock-treated cells were incubated with samples from the culture medium of HEK-293T cells transfected with empty plasmid pcDNA3.1, subjected to the same chromatographic process as the culture supernatant of pNS1^WNV-IS98-transfected cells. The mean fluorescence intensity (MFI) values shown in the bar graph (expressed in arbitrary units; AU), represented the mean values (m ± SD) of duplicate samples from two separate experiments, corrected by subtraction of the values of the signal obtained with mock-treated cells. Statistical analyses were performed using the Mann–Whitney test. Note: ** = P ≤ 0.01.