2009 Annual Report
1a.Objectives (from AD-416)
ARS objectives: The following objectives will be conducted and reported though Research projects 1940-32000-052, "Foot-and-Mouth Disease Virus (FMDV) Host-Pathogen Interactions" and 1940-32000-050-00D, "Development of Methods for Rapid Control of Classical Swine Fever" respectively:
1. Determine the mechanisms of FMDV interference with host response.
2. Develop effective CSFV marker vaccines and companion diagnostic tests capable of differentiating vaccinated from infected animals.
University of Connecticut objectives: 1. Develop novel approaches to inducing mucosal immune responses to FMDV Vaccines with the capacity to cross-neutralize a broader array of virus sub-types. 2. Determination of classical swine fever virus genetic determinants of virulence, immunogenicity and antigenicity.
3. Develop and validate grating coupled surface plasmon resonance imaging multiplexed microarray biosensor platform for the rapid detection of FMDV and CSFV, and the characterization of host responses to each pathogen.
University of Missouri Objectives: 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 antiviral therapeutics against FMD and CSF.
1b.Approach (from AD-416)
ARS:1.Determination of FMDV interference with host responses will be completed by a)examining the interference with the innate response in FMDV-infected cells, b)examining the mechanisms of FMDV immune evasion.2.Development of a novel live-attenuated CSF marker vaccine requires knowledge of the genetic basis of viral virulence.We will systematically evaluate the role of specific viral proteins or virus virulence & use this information to engineer recombinant LAVs.Introduction of modifications in CSFV infectious clone.UofMO:1.Knockout pigs will be generated containing specific deletions of genes of interest for pathogenesis & innate response.Target genes include the receptor for IFN alpha & beta;B2M or CD8alpha gene & IFN gamma receptor.Swine will be assessed for ability to respond to FMDV & CSF infections and vaccines or biotherapeutics.2.Utilizing cytopathic & non-sytopathic strains of bovine viral diarrhea virus as a model,will determine type I interferon activation pathways in cattle.3.Effects of Si RNAs targeting PKR & TLR3 on IFN induction will be determined.The role of suppressors of cytokine signaling in blocking IFN mediated IFN production.Identify viral genes that target innate response.Results will be contrasted with those obtained with CSF virus in swine.3.Utilizing cloned & expressed RNA-dependent-RNA polymerases(RdRps)from FMDV,BVDV & CSFV identify structural similarities & potential active sites.Identify potential compounds that can block enzyme activity & viral replication.Candidate inhibitors will be validated utilizing gel-based biochemical assays & high throughput surface plasmon resonance analysis,mass spectrometry &proteomics approaches.UofCT:1.Evaluation of mucosal adjuvants efficacy delivered through the ad5 platform,to induce mucosal immune responses to FMDV.Evaluate in vivo mucosal adjuvants alone or in combination with FMDV vaccine for induction of rapid protection in swine.Determine cross-neutralization & cross-protection provided by capsid-based vaccines engineered with chimeric VP1 G-H loops containing immunogenic or toleragenic epitopes.Epitope map will be created using anti-sera from murine bearing cross-reactive immune responses between FMDV types O & SAT3.Testing chimeric GH loop bearing hAd5 vectors in swine will be conducted to assess cross-neutralization.Challenge studies will be performed utilizing homo-typic & heterotypic virus.2.Evaluate the role of non-structural proteins in CSFV virulence & protection against infection will be performed through;complete cloning of CSFV structural proteins into Baculovirus transfer vectors, completing the production of recombinant Baculovirus expressing parental CSFV structural proteins & of autonomous replication CSFV defective genomes, & by completing the immunogenicity studies in naïve swine with sera from infected swine.3.Development & validation of GCSPRI device will be done to use as rapid detection of FMDV & CSFV.Identify diagnostic reagents & develop host immune response charactacterization.Assay conditions & sensor chip configurations will be optimized to capture host leukocyte populations.In vivo virus detection will be tested through immune response by GCSPRI.
Advanced Vaccines for Foot-and-Mouth Disease and Classical Swine Fever is a congressionally mandated agreement between the USDA, ARS, University of Connecticut and the University of Missouri. Please refer to the annual report for 1940-32000-055-03S (Univ. of Missouri), 1940-32000-055-04S (Univ. of Connecticut) and 1940-32000-052-00D for the USDA, ARS for progress details.
Identified and characterized of effective antiviral compounds directed against viral RNA-depedenent RNA polymerases (RdRp). (U. Missouri) We have expressed in large-scale the FMDV RNA-dependent RNA polymerase (RdRp) using metal affinity and anion exchange chromatography and optimized protocols to measure its enzymatic activity. We also established assays for high-throughput screening of the Maybridge HitFinder library and screened over 1000 compounds for those that inhibit FMDV RdRp by >50%. Using this assay we identified 9 compounds that at low concentration inhibited the RpRd enzime activity in vitro. Two of these compounds that showed low toxicity for BHK-21 cells and were tested in cell culture for antiviral activity. One of these 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 has been prepared and will be submitted after patent disclosures/provisional patent are filed by Univ. of Missouri.