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WORKPACKAGES
4.4. Work package descriptions
Workpackage 3 : Improved FMD virus detection
PARTICIPANTS
Partners 1, 3 and 5
Person-months per participant: 36 10 8
Start date or starting event: Month 1
OBJECTIVES
Improving FMD virus detection has 3 main objectives:
- Providing a tool for validation of NSP-tests without the availability of a gold standard.
- Improving the diagnostic value of FMD virus detection tests in case of mild virus excretion (like in vaccinated/infected animals or in sub-clinical infected sheep, goats or calves).
- Developing mass-screening virus detection methods.
Description of work:
- 3.1 Developing a "rolling-circle amplification" ELISA (RCA-ELISA) - The initial reactions are identical to those performed in the conventional FMD antigen detection ELISA: coating with (polyclonal) rabbit-anti-FMDV strain, addition of suspected sample and controls, addition of guinea pig anti-FMDV strain. Then anti-guinea pig conjugated to a special primer is added. This primer hybridises to an added C-probe. The initialisation of the RCA reaction starts by adding T7 DNA polymerase and incubating the mixture for 30 minutes at 37°C. Detection of the RCA products takes place after adding anti-FITC-peroxidase conjugate and ABTS. The reaction is isothermal (partner 1).
- 3.2 Inadequate growth of the isolate NET/1/2001 on secondary porcine kidney cells by (partner 2):
- Reproducing the observations during virus isolation (VI) of March 2001 in the Netherlands.
- Identification of pathways of the observed effect (blocking infection, blocking of growth or interfering substances).
- Identification of methods to overcome the inadequate growth.
- Methods to be used: VI on different cell types, immunological staining of FMDV on monolayers, RT-PCR for mRNA of cytokines, cytokine ELISAs and blocking studies.
- 3.3 Testing saliva of naïve vaccinated and infected cattle for IgA in parallel with testing of samples (in particular probing samples of cattle) for virus by Plaquetest and PCR (partner 5).
- 3.4 Validation of improved tests compared with NSP-tests and performing Latent class analysis (partners 1, 3 and 5).
Deliverables:
- D.5 - Knowledge about the production of cytokines by FMDV infected porcine cells.
- D.12 - Knowledge to prevent reduction of infection by interferon producing cells and therefore improvement of VI on cell cultures.
- D.17 - Developed and validated RCA-ELISA.
- D.18 - Developed and validated IgA-ELISA and determined correlation of IgA between infection and carrier state in vaccinated cattle.
- D.29 - Comparative tool for validation of NSP-tests without gold standard.
- D.50 - Reports: Scientific publications.
Milestones and expected result:
- M.16 - Validated RCA-ELISA (month 36).
- M.17 - Identified inhibiting factors on porcine kidney cells (month 12).
- M.18 - Validated methods to prevent reduction of infection by interferon producing cells (month 24).
- M.19 - Validated IgA-ELISA (month 36).
- M.20 - Developed comparative tool for the validation of NSP-tests without gold standard (month 48).
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