Endogenous miRNAs alter the expression of GFP fused to the 3′ UTR of DENV RNA - Since our bioinformatics analyses suggested that the DENV 3′ UTR might be targeted by cellular miRNAs, it was important to determine whether the DENV 3′ UTR actually functions as a miRNA target. Based on our findings, the first approach that we used to validate our results was to follow a previously reported strategy (Castillo et al. 2016). The 3′ UTRs of DENV-1, -2, and -4 were individually cloned into the plasmid pEGFP-C1 and the respective constructs, pGUD1, pGUD2, and pGUD4 were obtained. These constructs were subsequently transfected into Vero cells; the empty pEGFP-C1 vector served as the control. Transfection efficiency was determined by fluorescence microscopy and all three plasmids had comparable transfection efficiencies (higher than 50%). We assumed that if endogenous cellular miRNAs that recognise the 3′ UTR of the viral genome exist, a decrease in the GFP expression level compared to pEGFP-C1 should be observed. The GFP expression levels were then assessed by western blotting. Reduced expression of GFP-fused to the 3′ UTR of DENV-1 (pGUD1), DENV-2 (pGUD2), and DENV-4 (pGUD4) versus the control was also observed using the monoclonal anti-GFP antibody (Fig. 2A). Fig. 2B shows the fold change measured by densitometry. A statistically significant inhibitory effect was observed in the expression of GFP fused to the 3′ UTR of DENV-1, DENV-2, and DENV-4 compared with that in the control (pEGFP-C1). To confirm whether among these endogenous miRNAs, miR-484 and miR-744 might have a target site in the DENV 3′ UTR we performed a co-transfection assay using each generated pGUD construct and the plasmids expressing miR-484 and miR-744. Co-transfection of pEGFP-C1 and the miRNA plasmids served as controls. As shown in Fig. 2C-D, significantly reduced GFP expression was observed using miR-484 but unexpectedly with miR-744, GFP reduction was only observed with pGUD1. This might be explained by the difference of the total free energy and opening energy found for miR-744 using the RNAup webserver, which is higher than that for miR-484 and at a lower total energy, and the possibility of an RNA-RNA interaction is greater, as was observed for miR-484. However, the fact that overexpression of miR-744 does not reduce the levels of GFP (Fig. 2D) demands further investigation. In any case, since we observed that Vero cells possess endogenous miRNAs capable of reducing GFP expression (Fig. 2A-B), we wondered whether miR-744 and miR-484 are conserved in humans and monkeys. As a first approach to answer this question, both miR-484 and miR-744 were amplified from HeLa and Vero cells using the same probe. We observed that miR-484 and miR-744 were amplified (data not shown) and although this does not explain the results observed in Fig. 2B, it suggests that the nucleotide sequence of these two miRNAs is conserved in monkeys and humans.