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United States Department of Agriculture

Agricultural Research Service


Location: Arthropod-borne Animal Diseases Research

2009 Annual Report

1a. Objectives (from AD-416)
Objective 1: Identify biological determinants of disease susceptibility associated with arboviral infections. 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.

1b. Approach (from AD-416)
Arthropod-borne diseases pose significant concerns to the U.S. livestock industry. This project will investigate several biological relationships among host, vector and virus that will lead to improved disease control and risk assessment of emerging and re-emerging, domestic and exotic arboviruses. Biological determinants of arthropod-borne viral diseases of animals can be associated with the insect vector, the arbovirus, or the animal. One potential insect determinant is insect saliva, which may affect arbovirus transmission and subsequent infection. Culicoides sonorensis saliva will be used to examine interactions between saliva, arboviruses, and the immune response of susceptible animals. This may help to identify ways to interrupt disease transmission. A second important arboviral-animal determinant is virus attachment, mediated by cellular receptor(s) and allowing subsequent infection. Candidate receptor molecules for bluetongue virus (BTV) will be identified from sub-cellular fractions. This will provide a better understanding of BTV pathogenesis and may lead to more targeted vaccine strategies. The final biological determinant to be addressed is the effect of virus infection on host selection by insect vectors. The effect of vesicular stomatitis virus (VSV) infection on insect feeding and host defensive behaviors that could affect virus acquisition and transmission will be examined. This may help in the design and implementation of more efficient and cost-effective bite transmission control strategies. Introduction of exotic arboviruses is an ongoing risk and reality. The susceptibility of North American sheep and white-tailed deer to BTV-8, which is causing devastating disease in Europe, will be determined to provide valuable information for risk assessment.

3. Progress Report
Progress has been made on optimizing the saliva collection method, protein purification, and their analysis by one and two dimensional gel electrophoresis. 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.

4. Accomplishments
1. Infection of grasshoppers following ingestion of rangeland plant species harboring vesicular stomatitis virus. During vesicular stomatitis virus (VSV) outbreaks, infected animals salivate heavily, shedding virus and possibly contaminating pasture plants. ARS scientists had previously shown that grasshoppers could amplify VSV after ingesting it and serve as a source of virus to cattle when they, in turn, ingested the grasshoppers. This transmission cycle is based on the assumptions that virus shed from clinically infected animals would contaminate pasture plants, remain infectious on plant surfaces, and that grasshoppers would become infected by eating the virus-contaminated plants. In a three year study, in collaboration with a rangeland management specialist from USDA ARS High Plains Grasslands Research Station in Wyoming, 14 rangeland plant species were evaluated for their ability to harbor live VSV in order to determine the window of opportunity for grasshoppers to ingest live virus from contaminated plants. Under laboratory conditions, plants were exposed to VSV and infectious virus was measured over time. Virus remained infectious for up to 24 hours on eight of the 14 plant species tested. When grasshoppers were allowed to feed on the plants, they became infected. The scientists next tested a common grasshopper pesticide and found that it killed VSV upon contact. Thus, spraying pastures with a grasshopper pesticide during VSV outbreaks in pastured cattle and horse herds would decrease populations of this VSV-amplifying insect, as well as decontaminate the pasture plants, thereby reducing a viral source for both virus-amplifying plant-eating insects and grazing animals. This is the first report demonstrating the stability of VSV on rangeland plant surfaces. The data support the hypothesis that grasshopper-cattle-grasshopper transmission of VSV is possible. The results of this study could be useful in making disease management decisions during future outbreaks.

Review Publications
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.

Last Modified: 06/27/2017
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