2009 Annual Report
Subobjective 1A. Assess the role of insect salivary proteins on the pathogenesis of bluetongue virus in relevant target vertebrate hosts.
Subobjective 1B. Assess the role of insect salivary proteins on the pathogenesis of vesicular stomatitis virus in relevant target vertebrate hosts.
Subobjective 1C. Identify and characterize the vertebrate host receptors for bluetongue virus.
Subobjective 1D. Assess vesicular stomatitis virus-induced physiological variations and determine their affect on vector-host selection.
Objective 2: Determine the host-range specificity of exotic bluetongue viruses.
Subobjective 2A: Determine the susceptibility of U.S livestock to exotic bluetongue virus.
Subobjective 2B: Determine the susceptibility of U.S wildlife to exotic bluetongue virus.
A Specific Cooperative Agreement has been developed between ABADRL and CSU so that white-tailed deer and sheep studies can be conducted in their facilities.
An Interagency Reimbursable Agreement has been developed between ABADRL and APHIS, NWRC. An amendment to the agreement was completed to increase funding for CSU in order to fund the rearing and weaning of 16 white-tailed deer.
Sixteen white-tailed deer fawns have been purchased and are being reared by NWRC and CSU staff.
A bluetongue virus (BTV) transovarial transmission study in Culicoides sonorensis is nearly completed.
Genetic diagnostic assays for emerging and re-emerging insect transmitted viruses affecting livestock and wildlife were developed. This includes: BTV, epizootic hemorrhagic disease virus (EHDV), and vesicular stomatitis virus (VSV).
Testing was done on samples from lambs born in spring 2008, throughout the 2007 Wyoming outbreak area. This cohort of lambs was a sentinel population since they could not have been exposed to the virus during the outbreak. All of the tested lambs were antibody negative, showing no evidence of the virus circulating during the summer of 2008.
Membrane proteins from cattle pulmonary artery endothelial cells and Vero Maru (VM) cells were prepared and analyzed by SDS-PAGE. A binding ELISA was used to examine virus attachment to cells.
Subgenomic cloning was performed with the gene encoding VP7. These clones will be used to express control proteins for use in the receptor experiments. The techniques developed will also be applied to the cloning and expression of truncated VP2 to use in defining and characterizing mammalian receptors for orbiviruses.
An immunofluorescent detection assay was developed for in situ detection of BTV in insect cells. This assay is specific for BTV and does not detect the related EHDV. The technique is being applied to the direct detection of BTV and EHDV in Culicoides insects. This is a novel detection assay that contributes to the development of novel control strategies.
Additional BTV and EHDV baculovirus expressed VP7 was produced and titrated for use in diagnostics developed at the ABADRL.
Mice were immunized with EHDV for preparation of hybridomas to this virus. The objective is to isolate neutralizing monoclonal antibodies to EHDV that will be used to define neutralizing epitopes on this virus.
Deer, elk, cattle, bison, and goats in Colorado were tested for EHDV exposure after the 2008 outbreak.
Three species of Lutzomyia from Colorado and Wyoming were tested for VSV. No virus was detected.
Ticks were collected from a chronic wasting disease (CWD) positive moose and tested for CWD protein by ELISA. None was detected.
The effects of melezitose and stachyose on adult longevity and virus susceptibility in Culicoides sonorensis were examined.
The effects of ivermectin on the susceptibility of Culicoides sonorensis (Diptera: Ceratopogonidae) to BTV and EHDV were examined.
Mecham, J.O., Brown, P.L., Mcholland, L.E. 2009. In Situ Immune Infrared Fluorescent Staining for Detection and Quantification of Bluetongue Virus in Cullicoides Insect Cell Culture. Journal of Virological Methods. 158, 110-113.
Wilson, W.C., O Hearn, E.S., Tellgren-Roth, C., Stallknecht, D., Mead, D., Mecham, J.O. 2009. Detection of all eight serotypes of epizootic hemorrhagic disease virus (EHDV) by real-time RT-PCR. Journal of Veterinary Diagnostic Investigation. Vol 21:220-225 Drolet, B.S., Stuart, M.A., Derner, J.D. 2009. Infection of Melanoplus Sanguinipes Grasshoppers Following Ingestion of Rangeland Plant Species Harboring Vesicular Stomatitis Virus. Applied and Environmental Microbiology. 75(10):3029-3033.