Overexpression of miR-484 or of miR-744 suppresses DENV infection - Since the DENV 3’UTR contains target sites for miR-484 and miR-744 and since the expression level of GFP fused to the DENV 3’UTR decreased in Vero cells, we hypothesized that both miRNAs might affect DENV infection. Therefore, the effect of miR-484 and miR-744 overexpression on the DENV life cycle was investigated. The lentiviral plasmids pEZX-MR03-miR-484, pEZX-MR03-miR-744, and the pEZX-MR01-control (scrambled) were transfected into Vero cells, followed by challenge with each DENV serotype at an MOI of 3. Alternatively, the cells were first infected with each DENV serotype and then transfected with each plasmid carrying the gene encoding the corresponding miRNA. The percentage of Vero-infected cells, the DENV RNA copy number, and NS1 expression were evaluated 72 hpi under both conditions. As shown in Fig. 3A, when challenge with DENV occurred 24 h after miR-484 or miR-744 overexpression, the percentage of infected cells was significantly reduced for each of the four DENV serotypes compared with that in the scrambled control. Interestingly, the strongest effect was observed with miR-484 overexpression that reduced the infection of DENV-3 and DENV-4 by up to 60%. For DENV-2, a similar effect was observed with either miRNAs. However, when DENV RNA was quantified by RT-qPCR in the Vero culture supernatants, no difference was observed (Fig. 3B). Similar results were observed when the viral RNA within the infected cells was quantified (data not shown). To confirm our finding that miR-484 and miR-744 play a role in the modulation of DENV infection, the expression of DENV-2 NS1 in infected cells was evaluated by western blotting using anti-NS1 antibodies. Overexpression of miR-484 or miR-744 strongly reduced the amount of NS1 in DENV-2-infected cells compared with that in control infected cells (Fig. 3C). Fig. 3D shows the fold change measured by densitometry, indicating a statistically significant decrease in the expression of DENV-2 NS1 upon overexpression of either miRNA.