FIG. 4.

BKV binding to NA- and ST-treated Vero cells. (A) Vero cells were treated with PBS (bold line and shaded histogram) or 0.25 U/ml NA (dotted line) at 37°C for 1 h. Cells were then incubated with 10 FFU/μl AlexaFluor-488-labeled BKV at 4°C for 30 min (bold and dotted lines). The shaded histogram represents cells that were not treated with labeled virus. (B) To determine if labeling BKV reduced the viability of the virus, Vero cells were infected with 8.9 FFU/μl BKV before label was added or with the same volume of AlexaFluor-488-labeled BKV at 37°C for 2 h. After unbound virus was washed away, the cells were incubated for 3 days, fixed, and stained exactly as JCV was, as described in the legend for Fig. 2. Representative images of BKV before and after labeling are shown (magnification, ×100). The bright spots are cells positive for VP1 expression. (C) Vero cells were treated with PBS (shaded histograms), NA followed by NeuAc-CMP (bold lines), or NA followed by NeuAc-CMP and ST (dotted lines), as described for JCV in the legend for Fig. 2. After the treatments, the Vero cells were washed and incubated with 10 FFU/μl AlexaFluor-488-labeled BKV at 4°C for 30 min. BKV binding was evaluated using flow cytometry. FL1-H, fluorescence 1 height.