2010 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)
Receptors for arboviruses will be characterized. Mechanisms of virus neutralization in vertebrate and invertebrate cells will be determined. The role of the BTV hemagglutinin in transmission of virus from infected mammalian hosts to insect vectors will be studied. The effect of EHDV infection on the bovine fetus will be investigated. EHDV replication in cattle and deer cells will be studied. The effects of insect saliva proteins on VSV replication in mammalian cells will be examined. Insect genes coding for proteins that affect virus replication in the insect will be identified. Specific diagnostic tests for BTV, EHDV, VSV and WNV will be developed and tested. The potential efficacy of a mucosal vaccine for protection against VSV infections will be tested. Research will be carried out to show that intracellular antibodies can protect against BTV infection.
The following work aligns with the Genetic and Biological Determinants of Disease Susceptibility Component of the Animal Health Action Plan.
The Culicoides saliva collection method was optimized. Protocols were developed for analysis of proteins by one and two dimensional gel electrophoresis.
A Specific Cooperative Agreement has been extended between ABADRU and CSU for BTV - sheep studies at their facilities.
An Interagency Reimbursable Agreement has been completed between ABADRU and APHIS, NWRC for the rearing and weaning of 16 white-tailed deer.
Sixteen white-tailed deer fawns were reared and weaned and transferred to CSU large animal containment laboratory.
Tested sera from white-tailed deer from sheep infected with BTV-8 for the presence of antibody by the competitive ELISA. Demonstrated a specific antibody response following infection.
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. A manuscript describing the assay and its potential use in receptor studies was published in the Journal of Virological Methods.
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.
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.
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.
Introduction of exotic strains of bluetongue virus (BTV) to the US is a constant threat to US livestock. Many exotic strains have been identified in the past few years. Of particular concern for introduction is the European BTV-8 strain (EU-BTV-8). This virus strain has caused devastating disease all across Northern Europe where the disease had not been seen previously. To determine the susceptibility of North American white-tailed deer to EU-BTV-8 infection, an experimental infection study was conducted by ABADRU scientists in collaboration with scientists from Colorado State University and APHIS, National Wildlife Research Center in Fort Collins, CO. Tissue and blood samples were tested for the presence of virus and antibody. Infection resulted in significant clinical disease with peak viremia at 7-10 days. Virus-positive tissues included kidney and liver. This study demonstrates that our North American deer would be highly susceptible to an outbreak of this European BTV-8 were it to be introduced into the US.
Wilson, W.C., Hindson, B., O Hearn, E.S., Hall, S., Tellgren-Roth, C., Torres, C., Naraghi-Arani, P., Mecham, J.O., Lenhoff, R. 2009. A Multiplex Real-time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups. Journal of Veterinary Diagnostic Investigation, Vol 21:6, 760-770.
Allison, A.B., Goekjian, G.H., Potgieter, C., Wilson, W.C., Johnson, D., Mertens, P.P., Stallknecht, D.E. 2010. Detection of a Novel Reassortant Epizootic Hemorrhagic Disease Virus in the United States Containing RNA Segments Derived from Both Exotic and Endemic Serotypes.
Basile, F., Zhang, S., Shin, Y., Drolet, B.S. 2010. Atmospheric Pressure-Thermal Desorption (AP-TD)/Electrospray Ionization-Mass Spectrometry for the Rapid Analysis of Bacillus Spores. Analyst.