Figure 1

JEV upregulates miR-146a, and miR-146a enhances viral replication. JEV infection upregulates miR-146a in CHME3 cells. (A) Human brain microglial cell line CHME3 was infected with JEV (MOI-5), and cells were harvested at 12, 24, and 48 h post infection. qRT-PCR with Taqman probes were used to determine miR-146a levels. RNU24 was used to normalize the fold change in miR-146a levels as compared to uninfected control. (B,C) CHME3 cells were transfected with scramble sequence or miR-146a mimic sequence (B), Cy3-labeled scrambled anti-miR or anti-miR-146a (C) and infected by JEV 24 h post transfection. The cells were harvested at 12, 24, and 48 h post infection for RNA isolation. Viral RNA level was determined by RT-PCR using JEV NS3 specific primers. The fold change was normalized by GAPDH RNA levels. Fold change was determined by 2^−∆∆C _T method. For statistical analysis, scrambled miR and Cy3-labeled scrambled anti-miR group was used as control. (D) Western blots showing upregulation of viral NS1 protein in miR-146a overexpressing cells. A 100 pmol of scrambled sequence and miR-146a mimic was used. Scramble + JEV group was used as control for comparison. The cells were infected by JEV (MOI-5) and harvested 24 and 48 h post infection. All the experiments were done three times independently. The data are shown as mean ± SE from three independent experiments. The fold change is statistically significant. The fold change is significant where *denotes P < 0.05, **denotes P < 0.005, and ***denotes P < 0.001.