Combination of computing algorithms for the design of artificial microRNAs to control the replication of Foot-and-Mouth disease virus

CURRÁ A.; CACCIABUE M.; GIORDANO A.; TOLOSA G.; GISMONDI M.I.

Congreso; GFRA 2021 Scientific Meeting; 2021

Global Foot-and-Mouth Disease Research Alliance

The use of rationally designed artificial microRNAs (amiRs) has been postulated as a safe and efficient tool for silencing an RNA of interest including viral RNAs. A critical step is to select appropriate target sequences within this RNA molecule. There are numerous online tools for RNAi design; however, not all of them are regularly updated. The aim of this work was to combine the results obtained from different RNAi target selection algorithms to design amiRs with the potential to control FMDV replication. We hypothesized that target sequences predicted by two or more programs would be efficiently silenced by amiRs. Target sequences within FMDV A/Arg/01 genome were predicted with DSIR, i-Score, RNAxs and VIRrsiRNApred. The results were integrated using an ad hoc designed algorithm and five 22 bp-sequences located in the PK, IRES, VP4, 2C and 3C regions were selected. At 16 h post-transfection with amiR-expression vectors, cells were infected with FMDV A/Arg/01 (moi=3) and viral replication was assessed at 5 h post-infection by means of virus titration and quantitation of viral genomes. Viral titers were reduced by 36%, 77%, 64% and 65% in cells expressing amiRPK, amiRVP4, amiR2C and amiR3C, respectively, as compared to cells expressing a control amiR (miRneg). This was accompanied by a significant reduction in the amount of viral RNA molecules in infected cells for amiRPK (p