Evaluation of an artificial microRNA-mediated strategy against foot-and-mouth disease virus (FMDV).

GISMONDI MI; ASURMENDI S; TABOGA OA

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

FMDV, an icosahedral (+) ssRNA virus, is the etiological agent of the highly contagious foot-and-mouth disease. Current vaccines require 7 days to induce protection, and new tools of control are needed. Our aim was to determine the antiviral potential of an artificial microRNA (amiR)-mediated strategy against FMDV. A 21-nt sequence within 3D region of FMDV-A (3D1A) was chosen as target. BHK21 cells were stably transfected with a plasmid encoding the precursor of an amiR against 3D1A and transgenic cell lines were selected and cloned. Expression of amiR3D1A in cell lines was demonstrated by RT-real time PCR and + sequencing. Two amiR cell lines were transfected with plasmids pRLUC/3D1A (encoding 3D1A sequence downstream of Renillasequencing. Two amiR cell lines were transfected with plasmids pRLUC/3D1A (encoding 3D1A sequence downstream of Renilla luciferase) and pFLUC (encoding firefly luciferase). Twenty four h post-transfection, normalized luciferase activity was significantly lower in cells transfected with pRLUC/3D1A than in cells transfected with a control plasmid, whereas it did not decrease in cells transfected with pRLUC/3D1O carrying an homologous FMDV-O target sequence. A growth curve of the highly virulent FMDV/A/Arg2001 strain, as determined by quantitation of intracellular FMDV genomes by RT-qPCR and intracellular virus + titration, was similar in amiR and BHK21 cells. Although functional, the sole expression of amiR3D1A does not suffice to control replication of FMDV/A/Arg2001 in transgenic cells. control replication of FMDV/A/Arg2001 in transgenic cells. control replication of FMDV/A/Arg2001 in transgenic cells. control replication of FMDV/A/Arg2001 in transgenic cells. control replication of FMDV/A/Arg2001 in transgenic cells. control replication of FMDV/A/Arg2001 in transgenic cells. control replication of FMDV/A/Arg2001 in transgenic cells. titration, was similar in amiR and BHK21 cells. Although functional, the sole expression of amiR3D1A does not suffice to control replication of FMDV/A/Arg2001 in transgenic cells.