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

RNA interference (RNAi) is a well-conserved mechanism in eukaryotic cells that directs post-transcriptional gene silencing through small RNA molecules. RNAi has been proposed as an alternative approach for rapid and specific control of viruses including foot-and-mouth disease virus (FMDV), the causative agent of a devastating animal disease with high economic impact. The aim of this work was to assess the antiviral activity of different small RNA shuttles targeting the FMDV RNA-dependent RNA polymerase coding sequence (3D). Three target sequences were predicted within 3D considering RNA accessibility as a major criterion. The silencing efficacy of short-hairpin RNAs (shRNAs) and artificial microRNAs (amiRNAs) targeting the selected sequences was confirmed in fluorescent reporter assays. Furthermore, BHK-21 cells transiently expressing shRNAs or amiRNAs proved 70 to >95% inhibition of FMDV growth. Interestingly, dual expression of amiRNAs did not improve FMDV silencing. Lastly, stable cell lines constitutively expressing amiRNAs were established and characterized in terms of antiviral activity against FMDV. As expected, viral replication in these cell lines was delayed. These results show that the target RNA-accessibility-guided approach for RNAi design rendered efficient amiRNAs that constrain FMDV replication. The application of amiRNAs to complement FMDV vaccination in specific epidemiological scenarios shall be explored further.

Instituto de Biotecnología

Fil: Curra, Anabella Paola. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina

Fil: Curra, Anabella Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Cacciabue, Marco Polo Domingo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina

Fil: Cacciabue, Marco Polo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Gravisaco, María José. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina

Fil: Gravisaco, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina

Fil: Asurmendi, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina

Fil: Taboga, Oscar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Gismondi, Maria Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina

Fil: Gismondi, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina