Figure 2

Intracellular viral genome replication and viral protein translation in NSC-34 cells.

(a) IF assay. NSC-34 cells were infected with S41, C2 and MS strains at MOI 10, or with UV-inactivated EV71 (UV-iEV71). At 72 h.p.i., the infected cells were fixed with ice-cold methanol and processed for immunostaining using antibodies specific to dsRNA (red) and VP0/VP1 capsid protein (green). The images were captured at 40× magnification. Scale bar denotes 30 μm. Representative views are shown. The corrected total cell fluorescence (CTCF) was measured using ImageJ according to the equation: CTCF = Integrated Density – (Area of selected cell × Mean fluorescence of background readings). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test (^*p < 0.1, ^**p < 0.01, ^***p < 0.001). Data are expressed as the mean ± SD of three different microscopic views. (b) Quantitative real-time PCR. NSC-34 cells were infected with S41, C2 and MS strains at MOI 1. At various time points post-infection, the cells were harvested and intracellular viral RNA was extracted for real time PCR analysis using VP1 primers specific to each virus strain. The viral RNA titers were normalized to GAPDH RNA levels in each sample and expressed as fold changes (EV71 expression/GAPDH expression). The data are expressed as the mean ± SD of technical triplicates. Legends: ^*statistical analysis between S41- and C2-infected cells; ^#statistical analysis between S41- and MS-infected cells; ⁺statistical analysis between MS- and C2-infected cells. ^*,# or ⁺p < 0.1, ^{**, ##}or ⁺⁺p < 0.01, ^{***, ###} or ⁺⁺⁺p < 0.001. (c) Western blot analysis of EV71-infected NSC-34 cell lysates using an anti-VP0/VP1 primary antibody. β-actin was used as loading control and the band intensities were analysed using ImageJ software. Gels images were cropped. All these experiments were performed twice independently. One representative set is shown.