Location: Foreign Animal Disease Research2012 Annual Report
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.
3. Progress Report:
This project is targeted to defining important innate immunity functions that play a significant role in infections by Foot-and-Mouth Disease (FMD) and Classical Swine Fever (CSF), with the ultimate objective of developing a new generation of therapeutic control measures to prevent their spread in the U.S. animal industry. During FY 2012, the most significant progress has been achieved in viral therapeutics. Work continued in the development of additional inhibitors of Foot-and-Mouth Disease Virus (FMDV) 3Dpol which is an RNA-dependant polymerase. A combination of molecular modeling tools was used to obtain new compounds, which were validated using in vitro RNA polymerase assays. These new compounds were tested at ARS, PIADC for in vivo antiviral activity and their ability to block FMDV replication. Importantly, we discovered four new compounds that inhibit FMDV growth at low concentrations that are not cytotoxic up to at least 100 units of measure. Hence, similar to our previous compounds for which we have submitted a patent application, these new compounds are also potential leads for treatment of FMDV infections. In addition we have established a 96-well assay for high throughput screening using Bovine Viral Diarrhea Virus (BVDV). We have also discovered a related binding pocket for potential inhibitors of BVDV and Classical Swine Fever Virus (CSFV). Importantly, the pocket residues are identical in CSFV and BVDV, suggesting that BVDV inhibitors are likely to work for CSFV as well. Previous studies have resulted in the discovery of several compounds that inhibit the RNA-dependent RNA polymerase (RdRp) of FMDV. We tested the most active of these compounds for their ability to inhibit the replication rate of fully active virus. Four of the FMDV RdRp inhibitors were sent to ARS, PIADC. From the four compounds, two were found to be effective in restricting the replication of the A-24 Cruzerio strain of virus. Studies regarding the creation of constructs for knockout development – Rag1 Knockout continued in FY 2012. Rag1 is a required gene for immunoglobulin and T-cell receptor gene rearrangement. Disruption of this gene yields animals that cannot produce B- or T-Cells and results in a severe combined immunodeficiency (SCID) phenotype. Production of Rag1 null pigs will allow evaluation of immune responses that are independent of B- and T-cells. This model will facilitate description of pig responses to specific antigens or vaccines. The goal was to knockout Rag1 in porcine fetal fibroblast cells and to regenerate pigs by somatic cell nuclear transfer. The resulting genetic line could be maintained in the heterozygous state without expression of the SCID phenotype. To produce experimental SCID animals, heterozygous pairs could be mated. The resulting litter would average 25% SCID piglets for experimentation. Thus far, we have cloned the porcine Rag1 gene and constructed a targeted vector. A total of 21 independent transfections have been completed. After genotyping the resulting 801 colonies, none were found to have been gene targeted. In the development of pigs resistant to Porcine Reproductive and Respiratory Syndrome (PRRS) virus, all of the necessary constructs were made in previous years, transferred to fetal fibroblast cells, and somatic-cell nuclear transfer (SCNT) had been performed. Sialoadhesin Knockout Pigs – We currently have 14 females and 4 males that are heterozygous for the knockout mutation. CD163 Modified Pigs – We have designed a targeting vector that would result in a CD163 gene from which scavenger receptor cysteine-rich (SRCR) domain 5 was replaced with SRCR8 from CD163L. The major accomplishments over the life of this collaboration include: 1. The identification and characterization of small molecule inhibitors of the FMDV RdRp and the publication of a manuscript describing these molecules and filed patent registration for these compounds. We continue to pursue similar searches for inhibitors of BVDV and CSFV polymerases. 2. The establishment of cell lines and infection conditions to distinguish signaling pathways for cp and ncp BVDV infections. We anticipated that these experiments will provide useful information about Pestivirus pathogenicity applicable to CSFV, providing we can identify new sources of support for this project. 3. The development of swine models for viral innate immunity by targeting for genetic ablation key genes in the antiviral response. These models if successful will be of significant benefit to the investigation of viral pathogenesis in many virus-host interactions. In a related project funded from previous USDA ARS funds supporting this program, we have developed pig models for investigating PRRSV pathogenesis. This collaborative research project terminated in FY 2012. Technologies which have been transferred include: 1. Candidate small molecule inhibitors have been transferred to ARS, PIADC for in vivo analysis of inhibition of viral replication. One of these inhibitors has proven effective and is the subject of a recent publication. Patent disclosures have been filed through both MU and the USDA. 2. It is anticipated that the porcine lines with targeted gene knockouts will be transferred to ARS, PIADC once they have been constructed and verified. At PIADC, the lines will serve as important models for FMDV and CSFV infection experiments. 3. The putative PRRSV-resistant pigs are being transferred to Kansas State University scientists for viral infection studies. They will be made available to other investigators if appropriate. Publications resulting from this collaborative research agreement include: Durk, R.C., Singh, K., Cornelison, C.A., Rai, D., Matzek, K., Leslie, M., Schafer, E., Marchand, B., Adedeji, A., Michailidis, E., Moran, J., Pautler, C., Rodriguez, L., McIntosh, M,, Rieder, E., & Sarafianos, S.G. (2010). Inhibitors of Foot and Mouth Disease Virus Targeting a Novel Pocket of the RNA-Dependent RNA Polymerase. PLoS ONE, 5(12):e15049. Rai, D. K., Cornelison, K., Dorst, C. A., McIntosh, A., Rodriguez, L. L., Schafer, E., Rieder, E., Singh, K. and Sarafianos, S. G. (2012). Structure-Based Discovery of Foot-and-Mouth Disease Inhibitors that Target the 3Dpol RNA-Dependent RNA polymerase. Asian Chem. Letts. (In Press). Rai et al. Repeated exposure to 5D9; an inhibitor of 3Dpolymerase, effectively limits the replication of Foot and Mouth Disease Virus in cell culture. 2nd Antiviral Conference – From Bench to Bedside, November 11-13, 2012, Cambridge, MA.