2008 Annual Report
1a.Objectives (from AD-416)
Animal diseases foreign to the United States are potential choices for agro-bioterrorists. They are easy to acquire, disseminate and are readily transmissible. Deliberate induction of foreign animal diseases to the United States livestock population would result in a devastating economic impact. Rapid identification of causative agents are critical to stop the migration of an outbreak; endemic or deliberately induced as an act of agro-bioterrorism. To combat this threat, ARS, PIADC is developing a system of rapid real-time PCR assays for selected OIE class A animal pathogens that will allow rapid detection and identification of diseased animals and effective management of disease outbreaks.
This project focuses on the validation of rapid diagnostic tests for Rinderpest, Capripox and real-time RT-PCR detection of Vesicular Stomatitis Virus (VSV). Rinderpest assays will be transferred to USDA, APHIS. Capripoxvirus validation will be completed and transferred to USDA, APHIS. Evaluation of a modified VSV real-time RT-PCR for detection of VSV in insects for use as a surveillance tool.
1b.Approach (from AD-416)
Real-time PCR tests for Rinderpest and Capripoxviruses were developed under the previous project (CRIS 1940-32000-041-00X). Under this project we will complete the bench validation of these tests utilizing laboratory generated samples as well as those from experimentally infected animals. Tests will be evaluated for clinical sensitivity and specificity. Test performance will be evaluated and final reports prepared and submitted to APHIS for field validation. A real-time RT-CR for rapid detection of VSV has been previously developed that is capable of detecting all currently known genetic lineages of VSV. This test has been thoroughly evaluated in experimental and clinical samples and has been extensively utilized by APHIS during recent outbreaks in the southwestern USA. Since VSV is insect transmitted it is now necessary to modify and evaluate this test for detection of VSV in insects. Insects inoculated with VSV will be used for RNA extraction. Various RNA extraction methods will be tested and quality of RNA evaluated. RNA samples will be tested utilizing current real-time PCR.
In FY 2008 we continued refining some of the real-time PCR methodologies previously developed under this project. We finished the validation of the Capripoxvirus and Rinderpest virus tests and transferred all the information to APHIS. In addition we carried out the full-length genomic characterization of exotic Vesicular Stomatitis strains from Brazil and Argentina that had never been characterized. We also carried out partial sequencing of a number of exotic viruses from outbreaks reported in Brazil and Argentina over the last 20 years. This information is necessary to design real-time RT-PCR capable of detecting all strains of VSV causing disease in South America. Additionally FMDV strains from Afghanistan were sequenced and determined to be detectable by the real-time RT-PCR test previously developed under this project. We also tested our current real-time RT-PCR for Vesicular Stomatitis in infected and non-infected insects and showed that the test is cable of detecting infected insects. This project will terminate in FY 2009.
The progress of this project relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.
Balinsky, C.A., Delhon, G., Smoliga, G.R., Prarat, M., French, R.A., Geary, S.J., Rock, D.L., Rodriguez, L.L. 2008. Rapid Preclinical Detection of Sheeppox Virus by a Real-Time PCR Assay. Journal of Clinical Microbiology. 46(2):438-442.