Location: Foreign Animal Disease Research2007 Annual Report
1a. Objectives (from AD-416)
1. Develop a defined model to study early (pre-viremic) events in the pathogenesis of FMDV in its natural hosts. 2. Identify critical FMDV genetic virulence determinants associated to mechanisms of pathogenesis, persistence and transmission in-vivo. 3. Assess the role of insect vector transmission on the pathogenesis of VSV.
1b. Approach (from AD-416)
First, we will establish an infection model in cattle that allows close examination of the first few hours post-exposure. Identification of key tissues(s) and cell types responsible for primary virus replication and virus-host interactions that lead to control of local infection or generalized disease can be accomplished utilizing molecular tools such as confocal and laser-capture micro-dissection microscopy, in situ RT-PCR hybridization, and microarray analysis of host and viral genes. Second, we will utilize transposon insertion mutagenesis of an infectious FMD clone, to create a series of mutant viruses to be tested in the model developed under objective 1 to identify virulence determinants of FMDV. Viable mutants will be tested for their ability to invade and colonize primary replication site(s), generalize, cause clinical disease, and persist. Third, we will utilize VSV inoculation by insect bite and treatment with insect salivary components, combined with skin sampling and molecular tools described in the first objective, to determine the effect of insect bite on VSV pathogenesis. Information derived from the detailed understanding of early events in FMDV and VSV infections will provide a basis for disease control strategies that target essential steps in viral infection.
3. Progress Report
1940-32000-047-01S - Specific Cooperative Agreement with the University of Georgia entitled, "Molecular Pathogenesis of Vesicular Stomatitis in Cattle." This project was monitored through email and telephone exchange, and site visits to ARS, PIADC. For a complete report on the progress of this agreement, see the report for 1940-32000-047-01S. 1940-32000-047-02S - Specific Cooperative Agreement with the Univeristy of Missouri entitled, "Vesicular Stomatitis Outbreak Field Investigations and Epidemiological Studies of Endemic Areas." This project was monitored through email and telephone exchange. For a complete report on the progress of this agreement, see the report for 1940-32000-047-02S. 1940-32000-047-03S - Specific Cooperative Agreement with the University of Georgia entitled, "The Role of Insect Vector Transmission in the Pathogenesis of Vesicular Stomatitis Virus." This project is monitored through email and telephone exchange as well as site visits to ARS, PIADC and joint field visits to endemic areas. For a complete report on the progress of this agreement, see the report for 1940-32000-047-03S. 1940-32000-047-04S - Specific Cooperative Agreement with the University of Connecticut entitled, "Assessment of the Role of Foot-and-Mouth Disease Virus (FMDV) Protein in Viral Pathogenesis." This project is monitored through email and telephone exchange as well as site visits to ARS, PIADC and UConn. For a complete report on the progress of this agreement, see the report for 1940-32000-047-04S.
Designed a bovine whole genome microarray Little is known about the host response to FMDV infection in cattle. There is a lack of tools allowing a comprehensive analysis of the effect of infection on host gene expression. A bovine whole genome microarry was designed at FADRU in PIADC containing 42,034 gene probes. Microarrays, based on this design were utilized in initial pilot studies to identify differentially expressed genes between FMDV wilt-type and leaderless mutant (LLV2) in infected embryonic bovine kidney (EBK) primary cells. We found 43 genes with a significant expression difference. Most of the differentially expressed genes have anti-viral and transcription regulatory effects and are inducible by interferon cytokines based on reports in scientific literature. Impact: These results demonstrated the utility of our bovine whole genome micro array in studying molecular mechanisms of FMDV pathogenesis. This project address component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health. Generation of FMDV mutants by random transposon insertion mutagenesis There is limited information on virulence factors for FMDV. Tools to systematically analyze the role of specific regions of the FMDV genome in pathogenesis are lacking. In this project we attempted for the first time to generate FMDV mutants utilizing a novel approach. We used our infectious cDNA clones of FMDV to generate plasmid libraries containing randomly inserted transposons. From these we were able to derive a number of viable viruses carrying single insertions in specific regions of the genome. These viruses are currently being analyzed for virulence both in-vitro and in-vivo. Impact: These viral mutants will be utilized to gain a better understanding of the FMDV life cycle and its interaction with the bovine host. This project address component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health.
5. Significant Activities that Support Special Target Populations
Nothing to report.