Download Research Project Final Report

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SID 5
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Research Project Final Report
Note
In line with the Freedom of Information
Act 2000, Defra aims to place the results
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public domain wherever possible. The
SID 5 (Research Project Final Report) is
designed to capture the information on
the results and outputs of Defra-funded
research in a format that is easily
publishable through the Defra website. A
SID 5 must be completed for all projects.
1.
Defra Project code
2.
Project title
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appropriate.
3.
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SID 5 (Rev. 3/06)
Project identification
SE2003
Ex vivo cell culture methods for the detection and
characterisation of ovine TSEs
Contractor
organisation(s)
Molecular Pathogenesis and Genetics
(formally TSE Molecular Biology)
Department
Veterinary Laboratories Agency
New Haw, Addlestone
Surrey
Postcode KT15 3NB
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 4
£
402,368.00
start date ................
01 January 2006
end date .................
31 December 2008
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They
should be written in a clear and concise manner and represent a full account of the research project
which someone not closely associated with the project can follow.
Defra recognises that in a small minority of cases there may be information, such as intellectual property
or commercially confidential data, used in or generated by the research project, which should not be
disclosed. In these cases, such information should be detailed in a separate annex (not to be published)
so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report
without including references to any sensitive or confidential data, the information should be included and
section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No"
answer.
In all cases, reasons for withholding information must be fully in line with exemptions under the
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(b) If you have answered NO, please explain why the Final report should not be released into public domain
Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
Animal bioassays remain the gold standard for the proof of TSE infectivity in ruminant TSE research, and
strain typing and titre estimation usually require challenge of multiple groups of a panel of mice. However,
these experiments are generally slow and require many mice. Ex vivo cell-based assays offer an
alternative approach and recently Klöhn et al have developed a cell based assay for measuring de novo
infection and titre of mouse-passaged scrapie using cell lines permissive to mouse passaged scrapie
strains (Klohn et al. 2003).
The main limitation of employing a cell based approach in veterinary research and diagnostics is the
scarcity of cell lines permissive to infection with natural TSE strains. The majority of permissive cell lines
can only be infected with rodent passaged strains of scrapie and BSE. One of the few sheep scrapie
permissive cell lines, Rov9, (Vilette et al., 2001) is based on a rabbit kidney epithelial cell line (RK13) that
does not express detectable endogenous PrP and has been engineered to express ovine PrP (VRQ allele)
upon induction with doxycycline.
The primary aim of this project was to identify cell lines directly permissive to ovine scrapie by further
investigation of existing cell lines with proven ability to propagate sheep scrapie, through the development
and characterisation of further Rov lines expressing natural ovine PrP alleles and novel cell lines
permissive to scrapie. The effects of scrapie strain and PrP genotype was examined and the titre of an
established experimental strain of scrapie was assessed using a cell culture approach.
This study has shown the standard scrapie cell assay (SSCA) can detect de novo infection in a subset of
natural scrapie cases, including a 10-6 dilution of high titre brain tissue (estimated to contain 106.84 ID50 /
gram tissue) and low titre non-neural tissue such as adrenal glands. Subcloning of cell lines was
successfully applied to the Rov9 cells and subclones more sensitive to natural scrapie infection were
isolated; this increased sensitivity may be due to the increased proportion of cells expressing membrane
bound PrPC seen in the subclones. However, subcloning of the Rov9 cell line did not increase the range
of natural scrapie cases able to infect the cell lines. Rov cells generated to express other naturally
occurring PrP alleles were not found to be permissive to sheep scrapie. Preliminary data on the strains of
natural scrapie cases that infected Rov9 cells showed them to be ME7-, 87A- or 221C-like (using the
Fraser-Dickinson classification system) and all isolates that were propagated in these cells carried at least
1 VRQ PrP allele. Our data suggests that the strain and homology between the PrP allotype of the cell and
the genotype of the donor animal can contribute to the susceptibility of cell lines to natural scrapie
infection.
Future work would focus on studies aimed at broadening the range of natural scrapie and BSE cases and
tissues that can be propagated ex vivo are outlined and include.
 Ex vivo strain differentiation of VRQ/VRQ sheep scrapie.
SID 5 (Rev. 3/06)
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Isolation of rare de novo infections not detectable by SSCA through post-infection sub-cloning.
Novel delivery. – improve efficiency of using substrate bound prion as inocula.
This was a collaborative project between the Veterinary Laboratories Agency (VLA), UK and Institut
National de la Recherche Agronomique (INRA), France.
Klohn, P. C., Stoltze, L., Flechsig, E., Enari, M. & Weissmann, C. (2003). A quantitative, highly sensitive cell-based
infectivity assay for mouse scrapie prions. Proc Natl Acad Sci U S A 100, 11666-11671
Vilette, D., Madelaine, M. F. & Laude, H. (2000). Establishment of astrocyte cell lines from sheep genetically
susceptible to scrapie. In Vitro Cell Dev Biol Anim 36, 45-49.
1. Establish a standardised de novo infection screening method in-house - The scrapie assay
allows the extensive screening of a cell line, which may be highly heterogeneous in nature, to identify
permissive sub clones, thereby greatly enhancing the possibility of isolating a clone that supports the
propagation of a given TSE agent.
2. Screening of cell lines for scrapie permissive sub-clones - Using the scrapie assay described
above, undertake sub cloning of cell lines (Rov and MovS) to assay for permissiveness to infection from
highly characterised scrapie isolates such as SSBP1 and CH1641. In addition, the study will define
whether different strains can be biochemically discriminated following infection.
3. Transmission of sheep scrapie field cases to susceptible sub-clones - Once susceptible clonal
lines have been isolated, using essentially the same assay format, infectious samples of scrapie field
cases will be screened for infectivity.
4. Investigate novel delivery systems - Improving the TSE infectibility of a cell culture through
increasing individual de novo infection events in a culture will be investigated using TSE infectivity bound
to metal surfaces as proven in mouse and cell studies to be highly infectious i.
5. Determine prion titre using the scrapie cell assay on a well characterised scrapie isolate and
compare with mouse bioassay titre - Estimate the titre of SSBP1 inoculum in clonal Rov cells using the
scrapie cell assay and establish whether prion infectivity can be accurately determined ex vivo.
Importantly, the mouse bioassay will also serve to establish that prion infectivity is transmitted to the cell
culture and provide a correlation between PrPres detection and the infectious agent ex vivo.
6. Investigation of new scrapie permissive cell lines - Our collaborator (INRA, France) will apply their
vast expertise to the development of new permissive cell lines, which could include lines expressing
additional natural PrP alleles.
7. Investigation of new Scrapie permissive cell lines- Our collaborator (INRA, France) will develop a
novel cell line using neuroglial cells established from Prnp 0/0 mice and transfected with a vector coding for
ovine PrP and assess their permissiveness to scrapie.
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
SID 5 (Rev. 3/06)
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possible future work; and
any action resulting from the research (e.g. IP, Knowledge Transfer).
References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
M.H. Neale, S.J. Mountjoy, J.C. Edwards, D. Vilette, H. Laude, O. Windl, G.C. Saunders (2009). Infection of cell
lines with experimental and natural ovine scrapie (manuscrpit currently in internal review process)
i
Flechsig E, Hegyi I, Enari M, Schwarz P, Collinge J, Weissmann C. 2001 Transmission of scrapie by steelsurface-bound prions. Mol Med 7(10):679-84.
SID 5 (Rev. 3/06)
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