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.
FY 2009 was the first year of a newly refocused cooperative agreement between MU and ARS, PIADC to investigate immunity to the viral pathogens FMDV and CSFV and to identify potential antiviral compounds which inhibit the viral polymerases. The most significant accomplishment was the identification and characterization of effective antiviral compounds directed against viral RNA-dependent RNA polymerases (RdRp) of FMDV. Two established compounds were shown to inhibit the purified enzyme activity. We identified and evaluated 9 compounds cytotoxicity in BHK-21 cells. Two compounds were chosen for further study (C5D9 and C7F8). Kinetic and biochemical inhibition studies are currently underway with these compounds. One of the compounds (C5D9) was shown to suppress virus production in FMDV-infected cells. This and additional compounds uncovered in these studies merit further exploration as potential therapeutic agents to contain FMDV outbreaks. A manuscript describing this work will be submitted soon and patent disclosures are being filed.
Other significant progress includes: 1. Initiation of characterization studies against CSFV, 2.Initiation of creation of mutant swine lines for creating a KO mutation in IFN alpha/beta receptor genes, 3. Initiation of identification of innate viral sensors in BVDV infection studies.
Activities in this project were monitored through email and telephone exchange.