Antiviral efficacy of short-hairpin RNAs and artificial microRNAs targeting foot-and-mouth disease virus

Figure 1. RNAxs results of target prediction within FMDV 3D region. Data are represented as the number of predicted target sequences (coverage) per 3D nucleotide. Total number of predicted sequences (blue areas) and sequences corresponding to the top 50 hits in the RNAxs ranking (red areas) are shown. Target sequences selected for further study are indicated at the top of the corresponding peaks.

Figure 2. Silencing activity of small RNAs directed against FMDV 3D sequences. Co-transfected cells were trypsinized and EGFP expression was analyzed by flow cytometry at 24 hpi as described in Material and Methods. Cells were co-transfected with pEGFP. 3D and shRNAFMDV (A), pre-amiRNAFMDV (B) or dual pre-amiRNAFMDV (C) expressing plasmids. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. Antiviral effect of amiRNAsFMDV stably expressed in BHK-21 cells. (A) Cytopathic effect observed in BHK-21 cell lines expressing amiRNAFMDV and control group (cell lines expressing miRneg) at 48 h post-infection. Cellular detachment, rounding, and destruction were more severe in the control group than in the experimental group (Magnification 100 ×). (B) Morphology and (C) dimensions of lysis plaques produced by FMDV in amiRNAFMDV-expressing cell lines and control cells. FMDV infection produced less plaques of smaller dimensions in cell lines expressing amiRNA than in control cells. *** p < 0.0001. (D–F) Viral titers in supernatants of infected cells were determined by the TCID50 method at 18 hpi (D), 24 hpi (E) or 48 hpi (F). ** p < 0.01.

Figure 4. Antiviral activity of cloned amiRNA290 (A–C) and amiRNA1055 (D–F) cell lines. Cell lines were obtained from polyclonal cell lines by limiting dilution as described in Material and Methods. Cells were infected with FMDV A/Arg/01 at a low moi and viral titers in supernatants at 18 hpi (A, D), 24 hpi (B, E) or 48 hpi (C, F) were determined by end-point dilution. * p < 0.05 (as compared to miRneg cells, Student’s t-test).