CHA structure and its effect on RD cell viability.

<p>(A) The structure of CHA. (B) Control: control ; DMSO: 0.1% DMSO was added in RD cells. CHA was serially diluted as different concentrations (160,80,40,20,10,5 and 2.5 µg/mL) in triplicate. After 2 days of incubation, the cytotoxicity of CHA was determined by MTT assay. Data were presented as mean ± S.D. of three independent experiments.</p

CHA inhibits EV71 replication in RD cells.

<p>RD cells (5×10⁶) were infected with EV71 at a MOI of 5 in the presence or absence of CHA (20 µg/ml) and ribavirin (40 µg/ml). CHA and ribavirin were added with EV71 at the same time or absorbation for 1 h. Then cell supernatants were collected at 0, 4, 8,12, 16, 20, 24,28, 32 and 36 h p.i. and the viral titers were determined by a plaque forming assay. Each point represents the mean ± S.D. of three independent experiments (*** <i>p</i> < 0.001).</p

Effects of CHA on EV71 replication.

<p>Ribavirin was used as positive control and 0.1% DMSO as negative control. Inhibitory effects of ribavirin and indicated concentration of CHA on EV71 replication were determined by a plaque reduction assay. Data were represented as mean ± S.D. of three independent experiments.</p

Effects of CHA on EV71 replication before or after viral infection.

<p>DMSO: EV71 infection with DMSO. CHA: EV71 infection with DMSO and CHA RD cells (5 × 10⁶) were infected with EV71 (5 MOI) and CHA (20 µg/ml) was added at the indicated time. Cell supernatants were collected at 25 h p.i. and EV71 titers were determined by a plaque forming assay. Each bar represents the mean ± S.D. of three independent experiments（** <i>p</i> < 0.01, *** <i>p</i> < 0.001).</p

EV71 infection stimulates cytokine secretion.

<p>Control: Uninfected RD cells; EV71: RD cell-infected with EV71 (MOI = 5); CHA: EV71 infection in the presence of CHA (20 µg/ml). RD cells were absorbed with EV71 for 1h, and then treated with or without CHA(20 µg/ml). Culture supernatants were collected at 8h, 12h, and 20h. The concentrations of IL-2, IL-6, IL-10, TNF-α, IFN-γ and MCP-1 were detected by luminex fluorescent technique. The data were expressed as mean ± SE from 3 independent experiments. ** <i>P</i> < 0.01 and ***<i>P</i> < 0.001 by two-way ANOVA.</p

Effects of CHA on EV71 protein expression.

<p>EV71 infection: RD cell-infected with EV71 (MOI = 5). CHA treatment: EV71 infection in the presence of CHA (20 µg/ml). DMSO: EV71 infection treated with 0.1% DMSO. At 4 and 8h, the cell lysates were subjected to 10% SDS-PAGE and transferred to PVDF membrane to determine the level of EV71 VP1, 2A, 3C, and 3D.</p