FIG. 1.

Differential staining of NS1 on DENV- and WNV-infected cells. (A) Confocal microscopy. BHK21 cells were mock infected or infected with DENV (MOI of 1) or WNV (MOI of 0.1) and harvested 24 h later. A higher MOI was used for DENV to obtain the same relative level of infection as it replicates more slowly. Cells were cooled on ice and stained for NS1 on the surface using 2G6 (DENV) or 17NS1 (WNV) mouse MAbs. Subsequently, cells were fixed, permeabilized, and then stained for intracellular E protein using the cross-reactive humanized WNV E18 MAb. Nuclei were counterstained with TO-PRO-3 (blue). The images were analyzed using a LSM 510 confocal microscope. Scale bar, 20 μm. (B) Flow cytometry. BHK21 cells were infected with DENV or WNV as detailed above. Total NS1 or E was determined after cell permeabilization and indirect immunofluorescence staining with the cross-reactive MAb 9NS1 or WNV E18. Surface levels of NS1 were assessed by staining live cells at 4°C with the cross-reactive MAb 9NS1. The y axis indicates the number of cells, and the x axis shows relative NS1 or E levels. Shaded histograms are isotype controls. For microscopic and flow cytometric analyses, data are representative of at least three independent experiments.