Location: Arthropod-borne Animal Diseases Research2011 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
The Culicoides saliva collection method was optimized. Protocols were developed for analysis of proteins by one and two dimensional gel electrophoresis. 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). 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. An In situ fluorescent binding assay was developed for directly measuring binding of virus to mammalian cells. The utility of the assay was demonstrated by competition experiments with putative co-receptors for bluetongue virus. 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. This is a novel detection assay that contributes to the development of novel control strategies. An Interagency Reimbursable Agreement was completed between ABADRU and APHIS, NWRC for the rearing and weaning of 16 white-tailed deer. Importation permit was received for an EU-BTV-8 isolate from the Netherlands. Virus was received at CSU for deer and sheep studies. Two sheep were inoculated with EU-BTV-8 to produce washed blood cell inocula for subsequent studies. Sixteen white-tailed deer fawns were reared and weaned and transferred to CSU large animal containment laboratory. White-tailed deer susceptibility study completed. See accomplishment below. Comparative sequence analysis between the original BTV-8 inoculum and virus isolated from infected deer is ongoing. A Specific Cooperative Agreement has been extended between ABADRU and CSU for BTV - sheep studies at their facilities. Tested sera from sheep infected with EU-BTV-8 for the presence of antibody by the competitive ELISA. Demonstrated a specific antibody response following infection. The blood from sheep infected with EU-BTV-8 was analyzed using real time PCR demonstrated viremia by 3 days post inoculation.
1. Susceptibility of U.S. deer to a European bluetongue virus. Bluetongue (BT) is an insect transmitted disease of cattle, sheep, goats, and deer. World-wide there are at least 26 types of bluetongue virus (BTV), of which 5 types are considered domestic to the U.S. In 2006, bluetongue virus type 8 invaded north-western Europe, resulting in the largest outbreak ever recorded with unprecedented disease levels. In the U.S., white-tailed deer are the sentinel wildlife species for emerging and re-emerging bluetongue outbreaks. To determine their susceptibility to this exotic bluetongue virus type 8 should it come into the U.S., ARS researchers in Manhattan, Kansas infected deer under high containment conditions with type 8 virus. Clinical signs were monitored and blood and tissues tested for signs of infection. They demonstrated that if type 8 bluetongue virus is introduced, North American deer would be very susceptible and would serve as significant virus sources for insects to transmit the virus to sheep and cattle herds. Determining whether there are susceptible animals in the U.S. to various exotic BTV serotypes is critical for risk assessment and predicting future outbreak threats to our food security.
Mecham, J.O., Mcholland, L.E. 2010. Measurement of Bluetongue Virus Binding to a Mammalian Cell Surface Receptor by an In Situ Immune Fluorescent Staining Technique. Journal of Virological Methods.