Source: NATIONAL ANIMAL DISEASE CTR submitted to
TOOLS FOR DIFFERENTIATION OF HIGH CONSEQUENCE PATHOGENS AND ENDEMIC VIRUSES
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0405941
Grant No.
(N/A)
Project No.
3625-32000-070-00X
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 1, 2002
Project End Date
May 31, 2007
Grant Year
(N/A)
Project Director
HORST R L
Recipient Organization
NATIONAL ANIMAL DISEASE CTR
(N/A)
AMES,IA 50010
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113310110120%
3113410110120%
3113510110140%
3113610110110%
3113899110110%
Goals / Objectives
Provide means for differentiation between eradicated/exotic viruses and endemic viruses that produce clinical manifestations similar to eradicated/exotic viruses.
Project Methods
The approach is to develop real time and/or multiplex PCR tests. Two types of tests will be developed. The first type of test will be for specific viruses that may be confused with eradicated or exotic viral pathogens. Viruses that have clinical manifestations that resemble those observed with controlled viruses will be identified. Two panels of viruses for specific diagnostic tests are proposed. One is a panel of viruses that cause hemorrhagic lesions and/or abortion similar to those observed with controlled viruses such as classical swine fever virus (CSFV) or African swine fever virus. This panel would include pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), porcine reproductive and respiratory syndrome virus (PRRSV), parvovirus, infectious bovine rhinotracheitis (IBR) virus, and bovine adenovirus. The other panel would include viruses that cause vesicles or oral lesions and may be confused with foot and mouth disease virus (FMDV). This panel would include blue tongue virus (BTV), ecthyma (orf) virus, bovine papular stomatitis virus, bovine viral diarrhea virus (BVDV), IBR, epizootic hemorrhagic disease virus (EHDV), malignant catarrhal fever virus (MCFV), deer adenovirus, vesicular stomatitis virus, and vesicular exanthema virus of swine. The second type of PCR test will be a differential test for viral families or genera. Viral genomic sequences obtained from public sequence data banks or from researchers at the NADC will be searched for sequence motifs that are both conserved within a family or genus and unique to that family or genera. The purpose would be to identify primers that could be used to determine the family or genus of an unknown virus. The Diagnostic Virology Laboratory/APHIS will play an integral part in test design, development, and validation. Tests will be adapted for use in rapid detection devices such as the Smart Cycler (Cepheid) and RAPID (Idaho Technology). Special appropriation for Homeland Security ($1,500, 000). BSL-Exempt; Recertified 10/17/04.

Progress 06/01/02 to 05/31/07

Outputs
Progress Report Objectives (from AD-416) Provide means for differentiation between eradicated/exotic viruses and endemic viruses that produce clinical manifestations similar to eradicated/exotic viruses. Approach (from AD-416) The approach is to develop real time and/or multiplex PCR tests. Two types of tests will be developed. The first type of test will be for specific viruses that may be confused with eradicated or exotic viral pathogens. Viruses that have clinical manifestations that resemble those observed with controlled viruses will be identified. Two panels of viruses for specific diagnostic tests are proposed. One is a panel of viruses that cause hemorrhagic lesions and/or abortion similar to those observed with controlled viruses such as classical swine fever virus (CSFV) or African swine fever virus. This panel would include pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), porcine reproductive and respiratory syndrome virus (PRRSV), parvovirus, infectious bovine rhinotracheitis (IBR) virus, and bovine adenovirus. The other panel would include viruses that cause vesicles or oral lesions and may be confused with foot and mouth disease virus (FMDV). This panel would include blue tongue virus (BTV), ecthyma (orf) virus, bovine papular stomatitis virus, bovine viral diarrhea virus (BVDV), IBR, epizootic hemorrhagic disease virus (EHDV), malignant catarrhal fever virus (MCFV), deer adenovirus, vesicular stomatitis virus, and vesicular exanthema virus of swine. The second type of PCR test will be a differential test for viral families or genera. Viral genomic sequences obtained from public sequence data banks or from researchers at the NADC will be searched for sequence motifs that are both conserved within a family or genus and unique to that family or genera. The purpose would be to identify primers that could be used to determine the family or genus of an unknown virus. The Diagnostic Virology Laboratory/APHIS will play an integral part in test design, development, and validation. Tests will be adapted for use in rapid detection devices such as the Smart Cycler (Cepheid) and RAPID (Idaho Technology). Special appropriation for Homeland Security ($1,500, 000). Accomplishments Title � Development of real-time PCR assays that differentiate pestiviruses Problem � Differentiation of the recognized pestivirus species, BVDV1, BVDV2, BDV and CSFV, is important because CSFV was eradicated from the U. S. 40 years ago while the other three species are still endemic. What was done � In order to differentiate CSFV from the endemic pestivirus species, real time PCR assays specific for BVDV1, BVDV2, BDV and CSFV were developed and bench validated. Benefit � Better tests for differentiating eradicated viruses from endemic viruses will benefit regulatory agencies and market sectors based on export of animal and animal products. Title � Development of prototype tests using surface-enhanced raman scattering (SERS) as the readout technology Problem � Timely identification of an endemic virus when an eradicated/exotic virus is suspected would allay public fears and allow commerce to proceed more quickly. What was done � Prototype tests using surface enhanced raman scattering (SERS) were developed and refined. Benefits � Use of this novel readout technology has the potential to decrease time and sample preparation required for testing while increasing sensitivity. Faster, simpler, more sensitive tests will benefit producers, diagnostic laboratories and regulatory agencies. The work of this project supports National Program 103, Animal Health, Component 4: Countermeasures to prevent and control respiratory disease and Component 5: Countermeasures to prevent and control reproductive and neonatal disease.

Impacts
(N/A)

Publications


    Progress 10/01/05 to 09/30/06

    Outputs
    Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The work of this project supports National Program 103, Animal Health, and more specifically those program components that deal with Pathogen Detection and Disease Control Strategies. Presumptive diagnosis of viral infections in the field are often made based on "classic" clinical signs. Clinical manifestation such as the formation of vesicles in and around the mouth observed with foot and mouth disease virus (FMDV) or hemorrhages and abortions observed with classical swine fever virus (CSFV) are not unique to these eradicated viruses and may be observed after infection with viruses that are presently endemic to the U.S. Further the development of "classical" clinical signs is dependent on viral strain, the animals health status prior to infection and management systems. Implementation of control of recent outbreaks of CSFV in Europe were delayed because the mild clinical signs presented were not immediately associated with CSFV infections. While tests are available that will identify eradicated/exotic viruses such as FMDV and CSFV, tests may not be readily available for viruses that may be confused with them based on clinical signs. In addition, diagnosticians must also be prepared to quickly characterize unknown or rare viruses. Timely identification of an endemic virus when an eradicated/exotic virus is suspected would allay public fears and allow commerce to proceed more quickly. Tests that provide preliminary identification of viral families or genera would speed characterization and help direct subsequent study. 2. List by year the currently approved milestones (indicators of research progress) This CRIS does not have a project plan. It was initiated as the result of "no year" funds set aside to support the development and testing of diagnostics that differentiated high consequence pathogens from endemic viruses. 4a List the single most significant research accomplishment during FY 2006. This accomplishment aligns with the National Program Components: Pathogen Detection and Disease Control Strategies. Two primers were developed using deer adenovirus DNA sequences generated at NADC. Evaluation by Lawrence Livermore National Laboratory determined that these primers appeared to be good candidates for inclusion in a multiplex PCR test for viral diseases that have clinical presentations resembling foot and mouth disease. The specificity of the primers for the deer adenovirus was confirmed, by bench validation, using 150 (100 positive cases confirmed by immunohistochemistry and 50 cases with diagnoses other than adenovirus). Analysis of data is in progress. Additionally, virus isolation and deer adenovirus specific PCR tests were performed on tissues collected at necropsy to identify potential tissues to be utilized as positive controls in test development. Use of the multiplex will allow an animal that has lesions that look like foot and mouth disease (endemic diseases include bluetongue, epizootic hemorrhagic disease and adenovirus) to be tested rapidly and one time resulting in a quick response time to eradicate the disease should the animal have FMD. 5. Describe the major accomplishments to date and their predicted or actual impact. These accomplishments align with the National Program Components: Pathogen Detection and Disease Control Strategies. Real-time PCR assays were developed for bovine viral diarrhea virus (BVDV), swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 PCV (PCV2). Evaluation of the SIV test was completed and reported in the Journal of Veterinary Diagnostic Investigation. The BVDV test is being transferred to Center for Veterinary Biologics (CVB)/APHIS. A prototype surface- enhanced raman scattering immunoassay has been developed. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Diagnostic tests have been transferred to diagnostic laboratories cooperating in the Specific Cooperative Agreements (SCAs) listed. A bovine viral diarrhea virus (BVDV) test based on PCR is in the process of being transferred to the Center for Veterinary Biologics (CVB), APHIS, USDA. Current Specific Cooperative Agreements: 3625-32000-070-01S. University of California-Davis. Developing tools for rapid nucleic acid based tests for differentiation of deer adenovirus hemorrhagic disease from high consequence viruses. 3625-32000-070-02S. University of California-Davis. Developing tools for the differentiation of endemic viruses from high consequence pathogens: rapid nucleic acid based tests for pestivirus. 3625-32000-070-04S. Iowa State University. Developing rapid nucleic acid based tests for detection of endemic swine viruses. 3625-32000-070-06S. Iowa State University. Development of assay platforms for viral pathogens by real-time monitoring of antigen-antibody binding. 3625-32000-070-08S. Iowa State University. Characterization of swine immune response to selected viruses.

    Impacts
    (N/A)

    Publications


      Progress 10/01/04 to 09/30/05

      Outputs
      1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Presumptive diagnosis of viral infections in the field are often made based on "classic" clinical signs. Clinical manifestation such as the formation of vesicles in and around the mouth observed with foot and mouth disease virus (FMDV) or hemorrhages and abortions observed with classical swine fever virus (CSFV) are not unique to these eradicated viruses and may be observed after infection with viruses that are presently endemic to the U.S. Further the development of "classical" clinical signs is dependent on viral strain, the animals health status prior to infection and management systems. Implementation of control of recent outbreaks of CSFV in Europe were delayed because the mild clinical signs presented were not immediately associated with CSFV infections. While tests are available that will identify eradicated/exotic viruses such as FMDV and CSFV, tests may not be readily available for viruses that may be confused with them based on clinical signs. In addition, diagnosticians must also be prepared to quickly characterize unknown or rare viruses. Timely identification of an endemic virus when an eradicated/exotic virus is suspected would allay public fears and allow commerce to proceed more quickly. Tests that provide preliminary identification of viral families or genera would speed characterization and help direct subsequent study. The work of this project supports National Program 103, Animal Health, and more specifically those components that deal with pathogen detection and diagnosis and strategies to control disease outbreaks. 2. List the milestones (indicators of progress) from your Project Plan. This CRIS does not have a project plan. It was initiated as the result of no year funds set aside to support the development and testing of diagnostics that differentiated high consequence pathogens from endemic viruses. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Complete design and validation of differential diagnostics with collaborators and generate standard operating protocols for implementation in diagnostic laboratories. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The research goals for the remainder of this CRIS are to make test technology developed available to all U.S. diagnostic laboratories and work with these laboratories in developing standard operating procedures based on these tests. 4a What was the single most significant accomplishment this past year? Six prototype tests have been developed and are in various stages of bench validation. 4d Progress report. See individual progress reports. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Real-time PCR assays were developed for bovine viral diarrhea virus (BVDV) , swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 PCV (PCV2). Evaluation of the SIV test was completed and reported in the Journal of Veterinary Diagnostic Investigation. The BVDV test is being transferred to Center for Veterinary Biologics (CVB)/APHIS. A prototype surface- enhanced raman scattering immunoassay has been developed. This test has been described in a manuscript accepted for publication in Analytical Chemistry. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Diagnostic tests have been transferred to diagnostic laboratories cooperating in the SCAs listed. A BVDV tested based on PCR is in the process of being transferred to the Center for Veterinary Biologics (CVB) /APHIS.

      Impacts
      (N/A)

      Publications


        Progress 10/01/03 to 09/30/04

        Outputs
        1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Presumptive diagnosis of viral infections in the field are often made based on "classic" clinical signs. Clinical manifestation such as the formation of vesicles in and around the mouth observed with foot and mouth disease virus (FMDV) or hemorrhages and abortions observed with classical swine fever virus (CSFV) are not unique to these eradicated viruses and may be observed after infection with viruses that are presently endemic to the U.S. Further the development of "classical" clinical signs is dependent on viral strain, the animals health status prior to infection and management systems. Implementation of control of recent outbreaks of CSFV in Europe were delayed because the mild clinical signs presented were not immediately associated with CSFV infections. While tests are available that will identify eradicated/exotic viruses such as FMDV and CSFV, tests may not be readily available for viruses that may be confused with them based on clinical signs. In addition, diagnosticians must also be prepared to quickly characterize unknown or rare viruses. Timely identification of an endemic virus when an eradicated/exotic virus is suspected would allay public fears and allow commerce to proceed more quickly. Tests that provide preliminary identification of viral families or genera would speed characterization and help direct subsequent study. The work of this project supports National Program 103, Animal Health, and more specifically those components that deal with pathogen detection and diagnosis and strategies to control disease outbreaks. 2. List the milestones (indicators of progress) from your Project Plan. The project was initiated with special appropriation funds for Homeland Security to support the development and testing of diagnostics that differentiate high consequence pathogens from endemic viruses. The project does not have a certified project plan. 3. Milestones: A. List milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. FY04 - Design and beta testing and/or validation of differential diagnostics. We continued design and beta testing and/or validation of differential diagnostics with collaborators Dr. Leslie Woods, University of California at Davis; Dr. Sharon Hietala, University of California at Davis; Dr. Gene Erickson, North Carolina Diagnostic Laboratory; and Dr. Kyoung-Jin Yoon, Iowa State University. Three new agreements were executed with collaborators Dr. Marc Porter, Iowa State University, for development of Scanning Force Microscopy (SFM) and Surface Enhanced Raman Spectroscopy (SERS) as tools for the detection of viral pathogens through antigen/antibody binding; and Dr. James Roth, Iowa State University, for two conferences to present researchers the latest diagnostic technologies and to gather further information related to diseases of livestock and wildlife. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? While this project does not have a certified project plan that lists milestones, there are research goals for FY 05 and FY 06. They are as follows: In FY 2005 - Complete design and validation of differential diagnostics with collaborators and generate standard operating protocols for implementation in diagnostic laboratories. In FY 2006 - Make test technology available to all U.S. diagnostic laboratories. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004. The goal of this research project is to generate sensitive differential tests that are compatible with standard operations in diagnostic laboratories. Four specific cooperative agreements (SCA) with veterinary diagnostic laboratories located at the University of California at Davis, Iowa State University and the North Carolina Animal Disease Diagnostic Laboratory System (North Carolina Department of Agriculture) are in their second year (SCA's # 1, 2, 3, 4, listed below). All four support cooperative research geared to design and test new diagnostics. In addition a fifth SCA was initiated this year to explore the use of atomic force microscopy and raman spectroscopy as methods of detecting and differentiating antibody/antigen reactions (SCA #5). Two more agreements were initiated in support of two meetings that offered scientists an opportunity to compare data and discuss pathogens that may be passed between domestic and wildlife species and to compare novel diagnostic technologies (# 6 and 7). The titles and participants of all seven agreements are listed below. The accomplishments of each are detailed in separate Report of Progress (AD-421)s. Developing Tools For Rapid Nucleic Acid Based Tests For Differentiation Of Deer Adenovirus Hemorrhagic Disease From High Consequence Viruses Project No: 3625-32000-070-01S Accession: 405345 SY(s): Lehmkuhl, Howard D Woods, Leslie Agreement: 58-3625-2-0143 Organization: Univ Of California Developing Tools For The Differentiation Of Endemic Viruses From High Consequence Pathogens: Rapid Nucleic Acid Based Tests For Pestivirus Project No: 3625-32000-070-02S Accession: 405242 SY(s): Ridpath, Julia Hietala, Sharon Agreement: 58-3625-2-0144 Organization: Univ Of California Developing Rapid Nucleic Acid Based Tests For Detection Of Endemic Swine Viruses Project No: 3625-32000-070-03S Accession: 405221 SY(s): Lager, Kelly Erickson, Gene Agreement: 58-3625-2-0145 Organization: North Carolina Dept Of Agri Project Title: Developing Rapid Nucleic Aid Based Tests For Detection Of Endemic Swine Viruses Project No: 3625-32000-070-04S Accession: 405241 SY(s): Lager, Kelly Yoon, Kyoung-Jin Agreement: 58-3625-2-0146 Organization: Iowa State University Development Of Assay Platforms For Viral Pathogens By Real-Time Monitoring Of Antigen-Antibody Binding Project No: 3625-32000-070-06S Accession: 407556 SY(s): Ridpath, Julia Porter, Marc Murray, Peter Agreement: 58-3625-3-0145 Organization: Iowa State University Project Title: Diseases At The Diseases At The Interface Between Domestic Livestock And Wildlife Species Project No: 3625-32000-070-05G Accession: 407520 SY(s): Ridpath, Julia Roth, James A Palmer, Mitchell Murray, Peter Vacant Agreement: 59-3625-3-0320 Organization: Iowa State University Novel Diagnostic Technologies Project No: 3625-32000-070-07G Accession: 407964 SY(s): Kehrli Jr, Marcus Murray, Peter Roth, James A Agreement: 59-3625-4-0325 Organization: Iowa State University B. Other significant activities None. C. Significant Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Real-time PCR assays were developed for bovine viral diarrhea virus (BVDV) , swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 PCV(PCV2). Evaluation of the SIV test was completed and reported in the Journal of Veterinary Diagnostic Investigation. Currently, evaluation of the PRRSV and PCV2 assays are being completed. BVDV test will be transferred to APHIS in the next two months. These tests will improve on the time required and/or specificity of tests currently in use. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Diagnostic tests have been transferred to diagnostic laboratories cooperating in the SCA's listed above.

        Impacts
        (N/A)

        Publications