1a.Objectives (from AD-416)
1. Determine protective immune responses in genetically defined swine to better understand innate resistance mechanisms against FMD and CSF viral infections.
2. Determine variations in the molecular pathogenesis of endemic and exotic viral diseases in the same taxonomic family that will lead to the discovery of effective biotherapeutics to prevent viral transmission and viral persistence.
3. Analyze the structural features of polymerase-drug interactions to design effective anti-viral therapeutics against FMD and CSF.
1b.Approach (from AD-416)
1. Knockout pigs will be generated in swine genetics center containing specific deletions of genes of interest for pathogenesis and innate response. Target genes include the receptor for IFN alpha and beta; B2M or CD8alpha gene and IFN gamma receptor. Swine will be assessed for their ability to respond to FMDV and CSF infections or FMDV and CSF vaccines or biotherapeutics.
2. Utilizing cytopathic and non-sytopathic strains of bovine viral diarrhea virus as a model, will determine type I interferon activation pathways in cattle such as PKR and toll-like receptor 3. The effects of Si RNAs targeting PKR and TLR3 on IFN induction will be determined. The role of suppressors of cytokine signaling in blocking IFN mediated IFN production will also be determined. Viral genes that target innate response will be identified. These results will be contrasted with those obtained with CSF virus in swine.
3. Utilizing cloned and expressed RNA-dependent-RNA polymerases (RdRps) from FMDV, BVDV and CSFV will identify structural similarities and potential active sites. Utilizing the structural information will identify potential compounds that can block enzyme activity and viral replication. Candidate inhibitors will be validated utilizing gel-based biochemical assays and high throughput surface plasmon resonance analysis, mass spectrometry and proteomics approaches.
Discovery of several non-competitive inhibitors of FMDV viral polymerase, 3Dpol, that target a novel binding pocket. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. This inhibitor binding site will now be used for future structure-based drug design studies. These studies can lead to the discovery of even more potent antivirals. This project was monitored through email and telephone exchanges as well as by site visits.