Progress 02/01/07 to 12/10/08
Outputs
Progress Report Objectives (from AD-416) Arthropod-borne viruses (arboviruses) are important pathogens of livestock and have a significant impact on the US livestock industry. These viruses have a complex life cycle and often infect not only animals but also humans. The rapid spread of West Nile virus shows the danger from invasive arboviruses. We will characterize viral and host proteins important in the infections cycle. The pathogenesis of epizootic hemorrhagic disease virus (EHDV) infection in cattle is not well understood. We will determine if the difference in EHDV virulence between cattle and deer is related to endothelial cell tropism. We will also examine the effect on the fetus of EHDV infection of pregnant cattle. Effective control of arbovirus infections requires sensitive and specific diagnostics and efficacious vaccines. We will develop tests for the detection of viral antibody, viral particles, viral antigens, and viral nucleic acids. Epizootics caused by West Nile virus and vesicular stomatitis virus result in significant morbidity and mortality in the equine and bovine industries. At present there is no vaccine for these viruses. We will develop and evaluate vaccines for both of these viruses. We will (1) elucidate mechanisms of infections with arboviruses and pathogenesis with epizootic hemorrhagic disease virus; (2) clarify the role of Culicoides saliva in arbovirus infections; and (3) develop additional diagnostic tests for arboviruses and more efficacious preventative measure for arboviruses. 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. Significant Activities that Support Special Target Populations The following work aligns with the Genetic and Biological Determinants of Disease Susceptibility Component of the Animal Health Action Plan. Glycosaminoglycan antagonists significantly reduced productive infection of bovine pulmonary artery endothelial cells by BTV. Preliminary experiments showed that sub terminal sialic acid plays a role in BTV binding to susceptible vertebrate cells. A fluorescent focus overlay assay was developed to directly quantitate reduction of BTV binding by antagonists to susceptible cells. This assay has been adapted to study BTV binding to insect cell receptors. The information from these studies forms the basis to design more effective control measures for arboviruses. To determine the neutralizing epitope of the VP2 protein of EHDV, mouse hybridomas were produced, screened for production of neutralizing monoclonal antibodies, potential secreting hybridomas were identified and frozen down for future scale up and evaluation in neutralization and epitope mapping studies. The incidence of serum antibodies in sheep on ranches in Wyoming affected by the 2007 bluetongue outbreak was determined. ABADRL�s competitive ELISA was used to determine the incidence of infection and antibody titer in animals on the affected premises. Possible reactivation and transmission in response to Culicoides feeding and in response to stress as modeled by dexamethasone injection was tested using 4 naturally infected sheep. Skin biopsies and blood samples pre- and post-treatment were all negative by VI and PCR, and comparison of BTV antibody titer before and after the experiments showed no increase. These assays confirm in sheep what has been shown in cattle that these hosts are not likely to be a long term source of virus for new infections. It was determined that 31% of serum samples from hunter-killed deer in Louisiana submitted during 2005 were positive for BTV. Six samples were positive for BTV-1, demonstrating that the single report of this exotic BTV serotype in 2004 was not an isolated case. The impact of the spread of a new BTV serotype across the nation relative to disease in domestic and wild ruminants is difficult to estimate but could be significant. The effects of ivermectin and ivermectin with clorsulon on bluetongue and EHDV transmission was studied in sheep, ponies, and elk. The drugs did not kill C. sonorensis but double the standard dose of ivermectin in sheep reduced BTV infection rates in biting midges. The addition of clorsulon had no significant effect on EHDV susceptibility in midges. The WNV gene encoding the envelope protein, was cloned but protein expression was unsuccessful. It was determined that the DNA construct containing the intact WNV envelope gene is toxic in both bacterial and mammalian systems. Attempts to clone chimeric constructs consisting of both the envelope gene and combinations of the biological adjuvant, C3D, were also unsuccessful due to toxic effects. This illustrates the constraints and limitations on the cloning and expression of material to be used for DNA and subunit vaccine preparations. Technology Transfer Number of New/Active MTAs(providing only): 9
Impacts
(N/A)
Publications
- Zhou, Y., Nijland, M.J., Miller, M.M., Ford, S.P., Nathanielsz, P.W., Brenna, J.T. 2008. The Influence of Maternal Early to Mid-Gestation Nutrient Restriction of Long Chain Polyunsaturated Fatty Acids in Fetal Sheep. Lipids. 43(6):525-531
- Wilson, W.C., Bernard, K., Isreal, B.A., Mecham, J.O. 2008. Bluetongue virus serotype 17 sequence variation associated with neutralization. Virus Research. DNA Sequence 2008 Jun;19(3):237-240
- Balasuriya, U., Nadler, S.A., Wilson, W.C., Pritchard, L., Smythe, A.B., De Santis, P., Nianzu, Z., Tabachnick, W.J., Maclachlan, N. 2008. The NS3 Proteins of Global Strains of Bluetongue Virus evolve into Regional Topotypes Through Negative (purifying) Selection. Veterinary Microbiology. 2008 Jan 1;126(1-3):91-100
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Arthropod-borne viruses (arboviruses) are important pathogens of livestock and have a significant impact on the US livestock industry. These viruses have a complex life cycle and often infect not only animals but also humans. The rapid spread of West Nile virus shows the danger from invasive arboviruses. We will characterize viral and host proteins important in the infections cycle. The pathogenesis of epizootic hemorrhagic disease virus (EHDV) infection in cattle is not well understood. We will determine if the difference in EHDV virulence between cattle and deer is related to endothelial cell tropism. We will also examine the effect on the fetus of EHDV infection of pregnant cattle. Effective control of arbovirus infections requires sensitive and specific diagnostics and efficacious vaccines. We will develop tests for the detection of viral antibody, viral particles, viral antigens, and viral nucleic acids. Epizootics caused by West Nile virus and vesicular stomatitis virus result in significant morbidity and mortality in the equine and bovine industries. At present there is no vaccine for these viruses. We will develop and evaluate vaccines for both of these viruses. We will (1) elucidate mechanisms of infections with arboviruses and pathogenesis with epizootic hemorrhagic disease virus; (2) clarify the role of Culicoides saliva in arbovirus infections; and (3) develop additional diagnostic tests for arboviruses and more efficacious preventative measure for arboviruses. 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. Accomplishments Research progress continues to be made in the development of improved diagnostics for arboviruses. A method for direct detection and titration of bluetongue virus in Culicoides cell culture was optimized. The ability to directly detect and titrate virus replication in these cell lines may facilitate our ability to monitor bluetongue activity and rapidly detect the introduction of exotic serotypes of BTV into the U.S. This accomplishment aligns with the pathogen detection and diagnosis component of the Animal Health Action Plan. Parameters of a method using infrared PCR to detect EHDV in Culicoides are being optimized. Buffer and hybridization conditions are being optimized for using light harvesting water soluble conjugated polymers for detection of BTV RNA on a solid surface. A real-time dealkylation assay for detection of cytochrome P450 enzymes is being developed to measure the effects of viral infections on hepatic function. Additional sequencing of BTV and EHDV genes are providing data to better understand the evolution of these viruses and to develop improved diagnostics. Research on development of DNA and recombinant protein vaccines for WNV was continued. Recombinant proteins were tested in mice for induction of neutralizing antibody. This accomplishment aligns with the pathogen detection and diagnosis component of the Animal Health Action Plan. DNA vaccine for WNV. Procedures for expression and purification of recombinant WNV proteins in mammalian COS cells were improved and pure recombinant proteins conjugated with a biological adjuvant were prepared. The recombinant proteins and DNA encoding the recombinant proteins were compared in vaccination trials for induction of neutralizing antibody in mice. The goals of this research were to reduce the protective dose of recombinant WNV antigen, thus reducing the cost of the antigen in a WNV vaccine, increasing the number of animals that are vaccinated, thus reducing the impact of this disease. This accomplishment aligns with the strategies to control infectious diseases component of the Animal Health Action Plan. Characterization of BTV Receptors on Vertebrate Cells. Virus entry into a host cell is a multi-step process that starts with binding and internalization. Investigations into these mechanism(s) have led to the identification of a carbohydrate binding receptor for BTV. This information forms the basis to design more effective control measures. This accomplishment aligns with the mechanisms of disease component of the Animal Health Action Plan. Development of additional diagnostic tests for arboviruses. Additional tests demonstrating improved specificity, sensitivity and/or speed were developed for arbovirus diagnosis. These include a real time PCR that detects all 24 serotypes of EHDV, and an IR-RT-PCR assay for BTV. A detection method based on light harvesting water soluble conjugated polymers (CPs) for rapidly identifying nucleic acid targets with extremely high sensitivity recently developed by Sirigen, Inc. (Santa Barbara, CA) was applied to bluetongue diagnosis. To more easily determine the effects of viral infections on hepatic function an N- dealkylation real-time assay was developed for cytochrome P450 enzymes and shown to be specific for CYP1A1 and 2D6. These accomplishments align with the pathogen detection and diagnosis component of the Animal Health Action Plan. Significant Activities that Support Special Target Populations ABADRL scientists are working with deer farmers in Oklahoma to identify the vectors and serotype of bluetongue virus that is currently causing disease and death in farmed deer. This research being conducted through collaboration with Oklahoma State University, Department of Entomology and Plant Sciences. Technology Transfer Number of New CRADAS and MTAS: 2 Number of Active CRADAS and MTAS: 3 Number of Web Sites managed: 1
Impacts
(N/A)
Publications
- Kato, C.Y., Mayer, R.T. 2007. An improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared- dye-labeled primers for reverse transcriptase PCR. J. of Virological Methods, V. 140, Issues 1-2, March 2007, p. 140-147.
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