Figure 3

SM inhibits early steps of IVA infection. (A) and (B) Influence of the time of addition of SM on the virus production in cells. MDCK cells were infected with IVA at MOI 0.01 (A) or 0.001 (B) for 1 h (from -1 to 0 h). SM (1 µM) was added to cells at the indicate time points (•). In (B) after each incubation period (shown by arrows) the medium containing SM was replaced by the fresh infection medium. At the time points 24 h p.i. (A) and 8 h p.i. (B), the viral supernatants were collected and the viral yields were evaluated by FFA. Black columns – the mean viral yield for control cells; white columns – the mean viral yield for cells treated with SM. Data shown represent the mean ± SD of the results of three independent experiments. *p < 0.05, **p < 0.01. (C) SM inhibits binding of IVA to MDCK cells. MDCK cells were pre-chilled at 4 °C for 1 h and infected with IVA at MOI 0.01 followed by supplementation with SM (1 or 1.5 μM). After 3 h of incubation at 4 °C, cells were washed with PBS and overlaid with fresh medium. At 24 h p.i., viral supernatants were collected and the virus yields were determined by FFA. (D) SM inhibits penetration of IVA into MDCK cells. MDCK cells were pre-chilled at 4 °C for 1 h and incubated with virus (MOI 0.1) at 4 °C for 3 h. SM (1 or 1.5 μM) was then added to the cells and incubated for 60 minutes at 37 °C. Following inactivation and the neutralization of unpenetrated virus using acidic and alkaline PBS, respectively, cells were washed with PBS and overlaid with fresh medium. At 24 h p.i. viral supernatants were collected and the virus yields were determined by FFA. For (C) and (D): Control – untreated infected MDCK cells. Error bars represent the standard deviation of three independent experiments performed in triplicate. P-value: *<0.05; ***<0.001.