ROS in USUV infection.

(A) Vero cells were mock-infected or infected with USUV (MOI of 1) and then treated with drug vehicle (DMSO), ArsNa (0.5 mM) or BTdCPU (20μM) for 4 hours at 48 h p.i. CellROX green Reagent was added at a final concentration of 5 μM and incubated for 30 minutes at 37°C, and cells were analyzed by immunofluorescence. Green fluorescent signal showed the ROS-mediated oxidation of the reagent. Nuclei were stained with To-Pro-3 (blue). Scale bars, 25 μm. (B) Quantification of CellROX fluorescent puncta in cells treated as in A. Data are means of three independent experiments. Statistically significant differences were considered when P<0.05 and marked with an asterisk. (C and D) Antioxidant activity of USUV infection. (C) Vero and (D) Neuro2a cells were mock-infected or infected with USUV at an MOI of 10 and left untreated. The conversion of DCFH-DA into DCF upon addition of a free radical initiator was measured by recording the fluorescence in a plate reader every 5 min. Antioxidant capacity was inversely proportional to fluorescence intensity. Quercetin (25 μM) was used as control of antioxidant activity. See methods for details.