5 INHIBITION OF FOOT AND MOUTH DISEASE VIRUS IN VITRO USING RNA INTERFERENCE

Abstract

The use of short-hairpin RNA (shRNA) targeting viral genomes has shown great promise in human medicine and in vitro research in animal agriculture. However, this research has not been extrapolated into livestock applications. Foot and mouth disease virus (FMDV) is a world-wide disease resulting in decreased production and export limitations in countries with endemic FMDV, as well as severe economical impacts if an outbreak occurs in an FMDV-free country. The long-term goal for this project is to produce transgenic cattle that express shRNA targeting the FMDV genome resulting in resistance to infection. As a starting point, five siRNA and one non-targeting control siRNA (Null) were developed targeting different highly conserved regions of a FMDV type-A based replicon. The siRNA were transfected into BHK cells 48 h before viral RNA challenge. Eighteen hours post challenge the cells were lysed and analyzed. Three siRNA targeting the non-structural polymerase protein exhibited severe knockdown of 87, 90, and 92% when compared with the Null siRNA transfected control. The siRNA targeting the VPG3 cap protein reduced activity by 59%, and the siRNA targeting the internal ribosomal entry site had a minimal effect of 15% reduction. Based upon these results, we produced recombinant lentiviral particles designed to deliver the shRNA sequence targeting the FMDV genome and the fluorescent marker, dsRed, into a bovine fetal fibroblast cell line. This transgenic cell line expressing the most effective shRNA (based on initial siRNA screening) was used for somatic cell nuclear transfer to create bovine embryos. One hundred and sixty oocytes were enucleated, of which 149 had successful fusion resulting in 35 blastocysts after in vitro culture. Two embryos per recipient were transferred into five recipients. At Day 40 of pregnancy three of the five recipients had a fetus, but no heart beat could be detected. We are currently in the process of creating another cell line and repeating this experiment. If successful, transgenic calves will be visually and genetically analyzed for expression of dsRed and shRNA targeting FMDV. Transgenic and control animals/tissues will then be analyzed for resistance to infection with FMDV.