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SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 18

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the 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.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code SE1121

2. Project title

Application of RT-PCR for the diagnosis of FMD and other vesicular diseases

3. Contractororganisation(s)

Institute for Animal HealthAsh RoadPirbrightSurreyGU24 0NF     

54. Total Defra project costs £ 585,405(agreed fixed price)

5. Project: start date................ 01 July 2005

end date................. 30 June 2008

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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 Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. 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.

Control of foot-and-mouth (FMD) outbreaks is dependent upon early recognition of infected animals, which requires familiarity with clinical signs of the disease and the ability to accurately and rapidly detect FMD virus (FMDV) in clinical samples using laboratory tests. Of the established diagnostic approaches, virus isolation (VI) in cell culture is considered to be the “gold standard”. This method can be highly sensitive (depending upon the cell culture system used), although it is slow taking between 1-4 days to generate a result. Other assays such as antigen-capture ELISA (Ag-ELISA) are more rapid, but they have lower analytical sensitivity and are inappropriate for use with certain sample types. It is now recognised that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role for the rapid and sensitive detection of FMDV in a wide range of clinical sample types. During a previously funded Defra project (SE1119), automated real-time RT-PCR (rRT-PCR) assays for the detection of FMDV and other viruses (swine vesicular disease virus: SVDV and vesicular exanthema of swine virus: VESV) causing vesicular diseases of livestock were developed. Some of these methods have now been adopted by the OIE (included into the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals). The aim of SE1121 is to build on our previous work, to further define and improve the performance of the automated rRT-PCR assays, and to explore novel and alternative approaches for FMDV detection in the field and the laboratory.

Improvements to laboratory-based FMDV diagnosisDuring this project, we have completed the validation of the rRT-PCR for the detection of VESV. This assay can be performed together with the rRT-PCRs for FMDV, SVDV and VSV (details obtained under an MTA from Plum Island) under identical amplification conditions. Therefore, in the event of suspect cases of vesicular disease in livestock, we are able to simultaneously test for the presence of these 4 viral agents. The sample through-put of this parallel-plex format is lower than a multiplex assay (where all the targets are included the same tube): however, the analytical sensitivity of these individual targets is not compromised by potential competitive interaction between the different assays. SOPs describing assays these for the parallel detection of FMDV (two separate genome targets), SVDV and VESV have been incorporated into the laboratory contingency plans for FMD diagnosis at Pirbright. During the recent FMD outbreaks (in August 2007), these rRT-PCR were used as a front-line diagnostic tool for samples submitted from laboratory investigation. In addition to testing clinical material collected from all the infected premises, the rRT-PCR was also used to support an active surveillance program of high risk herds surrounding FMDV infected farms in northern Surrey. During these outbreaks, the majority of samples received from suspect cases were blood samples (mixture of EDTA and serum tubes). Learning from these recent experiences, in our follow-on project (SE1124), we

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aim to develop optimised and improved protocols using new high through-put robotic equipment to process blood samples that might be received during an outbreak.

As part of our work to improve the robustness of rRT-PCR methods, we have explored a new strategy to prepare internal-controls that are suitable for RT-PCR assays that target an RNA virus such as FMDV. The OIE (World Organization for Animal Health) guidelines for the validation and quality control of diagnostic PCR tests recommend that internal controls are included in the assay to verify negative results. These controls confirm that substances that are inhibitory to the enzymatic RT-PCR steps are absent from the test samples. Based on a plant virus (Cowpea mosaic virus: CPMV), the system developed during this project provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious. In light of the increasing acceptance of rRT-PCR methods for routine diagnosis within Europe, we have coordinated a proficiency testing scheme for National FMD Reference Laboratories. This work followed on from a pilot study undertaken during SE119, where 5 laboratories participated in a ring-trial using “live” FMDV to evaluate the sensitivity and specificity of RT-PCR assays and cell cultures methods in routine use. For the first time, we have also prepared inactivated samples suitable for testing by laboratories that do not have containment facilities (or permission) to handle “live” FMDV. These exercises provide valuable equivalence data for quality assurance accreditation schemes and also give an indication of the relative performance of the different RT-PCR assays in use at these laboratories.

Development and evaluation of hardware platforms and chemistries suitable for field detection of FMDVLaboratory-based rRT-PCR methods can generate a result within a few hours of receipt of a sample. However, the time taken to transport suspect clinical material to a central laboratory (such as Pirbright) can be lengthy, and this delay can preclude laboratory confirmation. During this project, we have evaluated a portable PCR instrument (Bioseeq™; Smiths Detection, Watford) for the detection of FMDV, to assess whether this equipment can potentially be used for field diagnosis of FMD. The results from this preliminary study were encouraging, indicating that it is possible to develop a sensitive rRT-PCR assay for FMDV on the Bioseeq™ platform which can generate a result within 60 minutes. Building upon these results, we have been working in partnership with Smith Detection (and Defra and other stakeholders) to design a bespoke hardware platform suitable for field detection of FMDV. In contrast to the previous versions of the Bioseeq™, this new machine will be able to extract nucleic acid template, in addition to performing PCR amplification, and will therefore make molecular diagnosis of FMD in the field (by a non-specialist) an achievable goal. As an alternative to PCR, we have also explored the use of isothermal (single temperature) amplification methods for the detection of FMDV. During SE1121, we have evaluated a nucleic acid sequence based amplification (NASBA) assay for FMDV. One of the detection formats tested could be performed using a temperature-controlled water bath and a 96-well microplate spectrophotometer. We have also evaluated a loop-mediated amplification (LAMP) assay for SVDV to partner a similar RT-LAMP assay for FMDV that has been developed at IAH-Pirbright. Since the specific amplification step for both of these assay formats occurs at a constant temperature, there is less reliance on expensive equipment. Furthermore, the products of the RT-LAMP assay are generated in abundance so that positive LAMP reactions can be visualised with the naked eye. During the proposed follow-on project (SE1124), we plan to perform further validation of this technology and (funding permitting from an external collaborator) will design, fabricate and test an isothermal device suitable for the field detection of FMDV.

Commercialisation of outputs from SE1121Assay developments have been constantly reviewed to ensure any intellectual property is protected. Several commercial opportunities have arisen from the outputs of this project (such as interest in the novel CPMV particles and the validation data for field-portable detection assays). Advice has been provided by Gencom through regular meetings during the course of the project.

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Project Report to Defra8. 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; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

PROJECT OBJECTIVESThe original project proposal had the following 7 objectives. 1. Generation of standards for RT-PCR methods for the detection of FMDV and Inter-

laboratory comparison of protocols2. Construction and evaluation of a encapsulated control suitable for the validation of

negative RT-PCR results3. Design of multiplex RT-PCR assays for differential diagnosis of vesicular disease viruses4. Design and evaluation of a “macro PCR array” card for the detection and partial

characterisation of FMDV5. Optimisation of template extraction and RT-PCR protocols for use with prototype mobile

PCR equipment.6. Evaluation of alternative viral enrichment strategies such as immunocapture methods 7. Assessment of the suitability of isothermal approaches that can be used to amplify

RNA/DNA for FMDV diagnosis.

The progress towards these objectives was reviewed at a meeting with Defra at month 18 of the project. At this time, modifications were made to some of the specific project milestones to accommodate new opportunities that had arisen since the original proposal was submitted. Where changes were made to the objectives, these are indicated below in the detailed description of the results.

Objective 1: Generation of standards for RT-PCR methods for the detection of FMDV and Inter- laboratory comparison of protocols

During the previous project (SE1119), we coordinated a pilot study to evaluate diagnostic assays in use in 5 European reference laboratories for the detection of FMDV. We concluded that this prototype panel of samples was suitable for external quality assurance of diagnostic tests. However, the inclusion of more negative samples (to test for diagnostic specificity) was recommended. During SE1121, we have extended this exercise to include all of the National FMD Reference laboratories in Europe (and additional laboratories elsewhere in the world). Of the 30 countries agreeing to participate in this study, 25 were from EU member countries and 5 from Non-EU countries. Two panels of coded samples were prepared and sent to participating laboratories for testing by RT-PCR assays. The first (‘live’) panel was sent to 16 laboratories and consisted of 4 FMD viruses of serotype O (2x), A and Asia 1. In addition to RT-PCR, this panel was also used to assess the performance of virus isolation methods employed by the different laboratories. The second panel sent to 28 laboratories comprised 10 non-infectious FMDV samples prepared by binary ethylenimine (BEI) inactivation and included FMD viruses of serotypes O, A, Asia 1, SAT 1 and SAT 2. The results from this exercise were presented to the Meeting of the EC community reference laboratories for FMD (Brussels, November 2007) and at the recent EU Epizone project meeting in Brescia, Italy (see presentations: Ebert et al., 2008).

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In total, 30 laboratories agreed to receive at least one of the panels for evaluation of their RT-PCR methods. Samples in Panel 1 were detected by all laboratories that reported results for RT-PCR: however dilution studies indicated that these assays differed in analytical sensitivity. In addition to the inherent performance properties of the RT-PCR assays, a number of other factors could have underpinned these differences in sensitivity between laboratories. These include: method used to prepare dilution series, criteria used to define a CT value to assign positive/negative cut-off for samples, differences in the nucleic acid extraction protocols and other reagents and also the competence of staff performing the test. These findings highlight the limitations of this type of study that attempt to compare the inherent performance of assays, at the same time as undertaking inter-laboratory proficiency testing.

Real-time RT-PCR methods are being increasingly adopted for routine use: in this study, 16 laboratories reported results for Panel 2 using this format (Table 1). Results for Panel 2 showed that 18/24 of the laboratories successfully detected FMDV in all the positive samples and correctly discriminated the 5 negative samples in the panel. Two laboratories (laboratory codes 4 and 5) failed to detect SAT viruses, possibly due to nucleotide mismatches with primers and/or probes, while 5 laboratories scored a false positive result for at least one of the negative samples (Table 1). The majority of labs used conserved regions of the 5’ UTR and/or 3D as the diagnostic target.

Table 1: Results from the inter-laboratory proficiency testing scheme coordinated during 2007. Results for 24 laboratories (coded) are shown with partners of the EU Epizone project highlighted in red. Samples were scored as positive (■) or negative (□) by the different laboratories. Where real-time RT-PCR was used CT values for the samples are indicated in the boxes. Five laboratories (codes 1, 11, 21, 23 and 38) performed multiple RT-PCR tests upon the submitted material: in these cases a consensus of the results from both tests was used to determine the status of the samples.

Lab O A SAT 1 SAT 2 Asia 1 SVD Target12.66 9.83 16.52 25.06 15.64 Neg Neg Neg Neg Neg 5'UTR12.10 8.21 10.07 10.10 15.51 Neg Neg Neg Neg Neg 3D

3 21.0 19.6 28.5 36.3 24.6 Neg Neg Neg Neg Neg 5'UTR4 23 20 28 Neg 26 Neg Neg Neg Neg Neg 5'UTR5 Pos Pos Neg Pos Pos Neg Neg Neg Neg Neg 3D7 20.5 17.1 24.8 27.3 21.6 Neg Neg Neg Neg Neg 5'UTR8 16 13 17 19 19 Neg Neg Neg Neg Neg 5'UTR

26.9 19.0 21.3 20.6 27.7 Neg Neg Neg Neg Neg 3D23.5 19.9 29.3 Neg 27.7 Neg Neg Neg Neg Neg 5'UTR

12 21 17 19 18 24 Neg Neg Neg Neg Neg 3D13 24 19 24 32 26 Neg Neg Neg 37 Neg 5'UTR14 18 14 16 16 21 Neg Neg Neg Neg Neg 3D15 Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg to confirm16 Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg 3D17 20.86 16.58 28.15 40.89 24.24 Neg Neg 34.3 35.45 Neg 5'UTR 18 Pos Pos Border Border Pos Neg Neg Neg Neg Neg 3D20 15.47 11.77 12.22 13.63 19.23 Neg Neg Neg Neg Neg 3D

17.10 13.10 15.09 14.20 24.41 Neg Neg Neg Neg Neg 3D14.39 11.59 12.29 13.21 22.40 Neg Neg Neg Neg Neg 3D19.82 15.4 16.33 18.73 23.28 Neg Neg Neg Neg Neg to confirmPos Pos FMD+SVD Pos Pos Pos SVD Neg Neg Neg Neg to confirm

27 37.25 17.33 28.32 22.36 27.41 Neg Neg Neg Neg Neg 3D29 Pos (5+) Pos (5+) Pos (3+) Pos (5+) Pos (4+) Pos (+) Neg Neg Pos (2+) Pos (+) 5'UTR31 Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg 2B33 Pos Pos Pos (+) Pos Pos Pos Neg Pos (+) Pos (+) Pos to confirm36 36.89 18.34 30.93 21.21 28.83 Neg Neg 31.93 Neg Neg 3D37 Pos Pos Pos Pos Weak Pos Neg Neg Neg Neg Neg 3D

27.38 20.15 19.90 19.33 25.83 Neg Neg Neg Neg Neg 3DPos Pos Neg Neg Pos Neg Neg Neg Neg Neg 3D

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Lab O A SAT 1 SAT 2 Asia 1 SVD Target12.66 9.83 16.52 25.06 15.64 Neg Neg Neg Neg Neg 5'UTR12.10 8.21 10.07 10.10 15.51 Neg Neg Neg Neg Neg 3D

3 21.0 19.6 28.5 36.3 24.6 Neg Neg Neg Neg Neg 5'UTR4 23 20 28 Neg 26 Neg Neg Neg Neg Neg 5'UTR5 Pos Pos Neg Pos Pos Neg Neg Neg Neg Neg 3D7 20.5 17.1 24.8 27.3 21.6 Neg Neg Neg Neg Neg 5'UTR8 16 13 17 19 19 Neg Neg Neg Neg Neg 5'UTR

26.9 19.0 21.3 20.6 27.7 Neg Neg Neg Neg Neg 3D23.5 19.9 29.3 Neg 27.7 Neg Neg Neg Neg Neg 5'UTR

12 21 17 19 18 24 Neg Neg Neg Neg Neg 3D13 24 19 24 32 26 Neg Neg Neg 37 Neg 5'UTR14 18 14 16 16 21 Neg Neg Neg Neg Neg 3D15 Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg to confirm16 Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg 3D17 20.86 16.58 28.15 40.89 24.24 Neg Neg 34.3 35.45 Neg 5'UTR 18 Pos Pos Border Border Pos Neg Neg Neg Neg Neg 3D20 15.47 11.77 12.22 13.63 19.23 Neg Neg Neg Neg Neg 3D

17.10 13.10 15.09 14.20 24.41 Neg Neg Neg Neg Neg 3D14.39 11.59 12.29 13.21 22.40 Neg Neg Neg Neg Neg 3D19.82 15.4 16.33 18.73 23.28 Neg Neg Neg Neg Neg to confirmPos Pos FMD+SVD Pos Pos Pos SVD Neg Neg Neg Neg to confirm

27 37.25 17.33 28.32 22.36 27.41 Neg Neg Neg Neg Neg 3D29 Pos (5+) Pos (5+) Pos (3+) Pos (5+) Pos (4+) Pos (+) Neg Neg Pos (2+) Pos (+) 5'UTR31 Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg 2B33 Pos Pos Pos (+) Pos Pos Pos Neg Pos (+) Pos (+) Pos to confirm36 36.89 18.34 30.93 21.21 28.83 Neg Neg 31.93 Neg Neg 3D37 Pos Pos Pos Pos Weak Pos Neg Neg Neg Neg Neg 3D

27.38 20.15 19.90 19.33 25.83 Neg Neg Neg Neg Neg 3DPos Pos Neg Neg Pos Neg Neg Neg Neg Neg 3D

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Uninfected Cell Supernatant1

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These proficiency testing scheme (PTS) exercises provide valuable equivalence data for quality assurance accreditation schemes and also give an indication of the relative performance of the different RT-PCR assays in use at these laboratories. Future PTS will address important diagnostic activities of National Reference Laboratories and will incorporate the guidelines set out in the proposed ISO 17043/43 standard.

Objective 2: Construction and evaluation of a encapsulated control suitable for the validation of negative RT-PCR results

Guidelines for the validation and quality control of diagnostic PCR tests recommend that internal controls are included in the assay to verify negative results. These controls confirm that substances that are inhibitory to the enzymatic RT-PCR steps are absent from the test samples. During the previous project (SE1119), we developed artificial RNA constructs by in-vitro transcription. Unfortunately these “naked” RNA transcripts are unstable and prone to degradation by cellular RNases. Furthermore, since viral genomes are typically encapsidated or reside in intra-cellular compartments, RNA transcripts that do not require a lysis step to release nucleic acid

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template only provide limited validation of the RNA extraction process of an assay. The development of control RNA molecules which are encapsidated in particles is one strategy that can be deployed to more closely replicate the conditions encountered by the viral RNA template within clinical samples.

Summary (abstract) of findings published in King et al., 2007:During SE1121 we generated novel encapsidated RNA particles and evaluated their performance as in-tube internal controls in diagnostic rRT-PCR assays for the detection of RNA viruses. This project was a collaboration between the IAH and the laboratory of Dr George Lomonossoff at The John Innes Centre (Norwich). A cassette containing sequences of 2 diagnostic primer sets for FMDV and SVDV was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious. In light of the results from this study, Genecom (representing IAH interests) and John Innes Centre (Norwich) have discussed establishing a commercial service to generate similar particles for other diagnostic tests.

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Figure 1, Panel A: The cloning strategy used to prepare the recombinant CPMV particles containing the artificial cassette. The identity of the parent plasmid is indicated in the centre of the vectors. Larger EML inserts contained additional sequence down-stream of the fluorogenic probe site introduced during the first round PCR step of the construction process. Panel B: The agarose-gel shows results of PCR analysis for 4 pGEM-T easy clones (EMS-11 & EMS-12 containing the 64 bp insert and EML-31 & EML-32 containing the 295 bp insert) using the 3 separate diagnostic primer sets (1F/1R, 2F/2R and 3F/3R; corresponding to FMDV [5’UTR]; FMDV [3D] and SVDV respectively). These amplicons were also detected using a fluorogenic TaqMan® probe targeting the middle of the construct (: see panel A).

In common with the internal controls based on bacteriophage MS2, these CPMV particles do not provide information relating to the quality of the RNA template present in the sample material. Therefore, even in the event of a positive signal with the internal control it is still possible that a false negative result may arise if the RNA template of the sample encompassing the virus is severely degraded. For exotic viruses such as FMDV, which is endemic in areas of Asia and Africa often distant from centralised laboratories, the quality of the sample when received for testing can

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be a particular concern. Assays are currently under evaluation utilising control rRT-PCR reactions run in parallel to detect “house keeping” genes that confirm the integrity of host RNA template in the clinical material. Together with internal controls, these approaches can be used to provide confidence in the results generated by diagnostic rRT-PCR assays. These particles provide confidence in the reporting of negative results in a RT-PCR assay and will be particularly useful for inclusion into field-based tests performed by “non-specialists”.

Objective 3: Design of multiplex RT-PCR assays for differential diagnosis of vesicular disease viruses

During this project, we have completed the validation of the rRT-PCR for the detection of VESV. This assay can be performed together with the rRT-PCRs for FMDV, SVDV (developed during SE1119) and VSV (details obtained under an MTA from Plum Island) under identical amplification conditions. Therefore, in the event of suspect cases of vesicular disease in livestock, we are able to simultaneously test for the presence of these 4 viral agents.

Summary (abstract) of findings published in Reid et al., 2007:Marine caliciviruses form a distinct lineage within the genus Vesivirus (family Caliciviridae). This group includes vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) and other related viruses which have been proposed to be marine in origin isolated from a variety of terrestrial and marine animals. Rapid and reliable detection of marine caliciviruses is important as these viruses appear to be widespread and can cause vesicular disease in a wide variety of susceptible hosts including pigs and experimentally infected cattle where clinical signs cannot be easily distinguished from foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). A real-time RT-PCR assay targeting conserved nucleotide sequences in the RNA-dependent RNA polymerase (3D) region of the genome successfully detected cell culture-grown virus preparations of more than thirty marine calicivirus serotypes. Only the atypical SMSV serotypes 8 and 12 failed to be detected, which provided further indication of genetic divergence between these and the other calicivirus serotypes said to be marine in origin. The real-time RT-PCR assay also specifically amplified RNA from samples collected following experimental inoculation of pigs with VESV. No cross-reactivity was demonstrated when the assay was tested with RNA prepared from representative viruses of FMD, SVD and VS. The real-time RT-PCR assay described is a sensitive and specific tool for detection and differential diagnosis of these viruses from other vesicular-disease causing viruses.

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Figure 2: Data from three real-time RT-PCR assays run in parallel for marine caliciviruses (grey bars), SVDV (red bars), FMDV (green bars) and VSV (blue bar) to provide differential diagnosis of 24 selected viruses causing vesicular disease. The CT values obtained for the independent assays of representative serotypes/strains of FMDV, SVDV, VSV and marine caliciviruses with the primers/probe sets are shown. A positive result (CT value of <40.0) was obtained with each primers/probe set on homotypic viruses. None of the primers/probe sets showed a significant cross-reaction against heterotypic viruses. Note: weak SVDV signal generated for FMDV/C sample is below the positive threshold for the assay.

SID 5 (Rev. 3/06) Page 8 of 18

SOPs describing assays for the parallel detection of FMDV (two separate genome targets), SVDV and VESV have been incorporated into the laboratory contingency plans for FMD diagnosis at Pirbright. The sample through-put of this parallel-plex format is lower than a multiplex assay (where all the targets are in the same tube): however, the analytical sensitivity of these individual targets is not compromised by potential competitive interaction between the different assays. This format is appropriate for initial cases of suspect vesicular disease in the UK, where samples numbers are not normally high. A single multiplexed screening test that simultaneously detects and differentiates FMDV from look-alike disease viruses would be desirable. Fluorescent probes for rRT-PCR detection have broad emission spectra which limit multiplexing capacity to the four or five discrete optical channels typically present in most commercial real-time PCR instruments. As described above, we currently have 5 assays for the differential diagnosis of vesicular viral diseases. Together with internal controls, this would certainly challenge most currently available real-time PCR equipment.

During the course of SE1121, an opportunity arose to evaluate a highly multiplexed RT-PCR format that might allow differential detection of a number of viruses that can cause vesicular disease in livestock. We were approached by the Lawrence Livermore National Laboratory (LLNL), USA to undertake an evaluation of a Luminex assay that they had developed in a project funded by US Department of Homeland Security (DHS) Science and Technology Directorate. At the 18 month project meeting with Defra, it was agreed to modify the proposed work to include an evaluation of this assay format. Luminex xMAP technology is a multiplexed high-throughput detection system with many applications for nucleic acid detection. The Luminex array offers up to 100 independent channels using microspheres (5.6 μm) embedded with varying ratios of two fluorescent dyes. User-defined surface modifications can include oligonucleotides, antibodies, peptides or other macromolecules. Typically, a mixed suspension of functionalized microspheres is mixed with the sample to bind analytes which are then labeled with a fluorescent reporter and analyzed using a specialized flow-cytometer. The assay provides end-point detection with qualitative results by comparing fluorescence responses (median fluorescence intensity, MFI) of each microsphere class to cut-off values. Recent nucleic acid applications of the Luminex array include the detection and differentiation of Classical swine fever virus from other pestiviruses, human respiratory viruses, human papillomavirus and human influenza A virus typing.

Figure 3: Schematic of the Luminex multiplex RT-PCR assay. In the presence of target nucleic acid, the biotinylated forward primer is extended during the PCR. The PCR product is hybridized to the microsphere array, whereby the extended forward primer binds to the complementary probe-labeled microsphere. The complex is labeled with fluorescent reporter (SAPE) then analyzed using a Bio-Plex flow cytometer. The fluorescence (570 nm) of bound reporter molecules is measured, and the median fluorescence intensity is calculated for each microsphere class, then compared to a cut-off to indicate the presence or absence of a target nucleic acid sequence in a sample

Conveniently, the FMDV targets used for this assay were identical to those already being employed for routine diagnosis by rRT-PCR (namely 5’UTR and 3D).

Summary (abstract) of findings published in Hindson et al., 2008:A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock.

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This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 truenegative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.

Objective 4: Design and evaluation of a “macro PCR array” card for the detection and partial characterisation of FMDV

As an alternative to the Luminex multiplexing format (described in objective 3), during SE1121 we undertook an evaluation of a multiplex “Microfluidic” card format for the serotyping and characterisation of FMDV. This assay format is essentially a parallel-plex assay format (see http://marketing.appliedbiosystems.com/mk/get/TAQMAN_LDA_1006_LANDING), which has been expanded to encompass up to 96 different oligo-primer sets. This work was undertaken in collaboration with FairIsaac (Minneapolis, USA) and the University of California (Davis Campus). FMDV samples (n=32) from all seven serotypes were tested during a visit of Dr Garry Kelley to Pirbright during February 2006. The results of this evaluation showed that the assay was broadly able to discriminate between different FMDV serotypes. However, in its current format, the assay has a lower analytical sensitivity than real-time RT-PCR which would significantly limit its use for routine diagnosis. This issue is being addressed by the developers of the test. However, it has not been possible to evaluate a modified version of this test during the lifetime of SE1121.

Objective 5: Optimisation of template extraction and RT-PCR protocols for use with prototype mobile PCR equipment.Rapid and accurate diagnosis is necessary for effective control of FMD. Some laboratory-based methods are rapid (generating a result within a few hours), but the time taken to transport suspect clinical material to a central laboratory can be lengthy, and this delay can preclude laboratory confirmation in the event of an FMD outbreak. Notably, the implementation of the control strategy adopted by the UK government in the 2001 epizootic: to slaughter the animals on infected premises within 24 hours, meant that in the majority of cases, diagnosis based on clinical signs was not substantiated by laboratory investigation. Retrospective analyses have indicated that reliance upon clinical diagnosis alone resulted in over-reporting of FMD, since the presence of FMD virus (FMDV) on 23% of farms designated as infected premises could not be corroborated by laboratory methods. These factors have given rise to the idea that rapid and sensitive technologies that can be used in situ (without transferring the samples to a central laboratory) have the potential to improve the reliability of FMD diagnosis. The use of rapid diagnostic assays was recommended in two major reports concerning the 2001 outbreak (Royal Society Report on Infectious Diseases of Livestock, 2002 & Foot and Mouth Disease 2001: Lessons to be Learned Enquiry, 2002). Previous studies (at the IAH and elsewhere) have developed rapid reverse-transcription polymerase chain reaction (RT-PCR) assays for FMDV detection: however limitations in the hardware and some aspects of the protocols used have restricted the adoption of these assays for the field detection of FMDV. The aim of this objective is to develop and test an FMDV-specific RT-PCR suitable for field use.

During SE1119, a two-step rRT-PCR was developed for the detection of FMDV. We have now transferred this assay to a one-step rRT-PCR format making the test simpler and more rapid to

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perform in the laboratory. In addition to improved laboratory diagnosis of FMD, this format is more suitable for platforms that might be deployed for field detection.

Summary (abstract) of findings from Shaw et al., 2007An automated one-step rRT-PCR protocol was optimised and evaluated for the routine diagnosis of FMD. Parallel testing of RNA samples (n = 257) indicated that this assay has a diagnostic sensitivity at least equivalent to the automated two-step rRT-PCR protocol previously used for the laboratory detection of FMDV. This more rapid and economical one-step protocol will play a key role in contingency planning for any future outbreaks of FMD in the United Kingdom.

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Figure 4: Comparison between one-step and two-step rRT-PCR assays on RNA samples (n = 257) of the vesicular disease viruses FMD, SVD, VS and marine caliciviruses and an isolate of PPRV. * represents identical “No CT” results for 61 nucleic acid samples comprising SVDV, VSV, marine calicivirus and PPRV and negative epithelium using both assays.

We subsequently transferred this one-step rRT-PCR assay to a portable real-time PCR machine (Bioseeq™; Smiths Detection, Watford: see Figure 5A). This machine was developed for the “first responder” market and our work with FMDV represented the first RNA-specific assay that had been developed on this platform.

Summary of findings from King et al., 2008A one-step real-time RT-PCR targeting the 5’UTR of the FMDV genome was adapted for use on the Bioseeq™. Initial experiments to assess the performance of the Bioseeq™ were performed using RNA samples prepared using an automated extraction robot (MagNA PURE LC, Roche) to optimize components of the RT-PCR such as Mg2+ ion and enzyme concentration. Twenty five µl reactions were prepared containing 6 mM MgSO4, 0.2 mM of each dNTP, 20 pmol of each primer, 7.5 pmol FAM-TAMRA dual labeled fluorogenic probe and 1 µl of the SuperScript™ III RT/Platinum® Taq mix (Invitrogen, Paisley, UK). One drop of mineral oil overlayed each reaction. RNA template was amplified using a single thermocycling programme consisting of an RT step (60°C for 600 seconds) followed by 50 cycles of PCR amplification (95°C for 20 seconds and 60°C for 30 seconds). RT-PCR products of the expected size (97 bp) could be visualized (in a 2% agarose-gel, data not shown) indicating that the correct region of the FMDV genome had been successfully amplified. For subsequent assays, the Bioseeq™ Support Software (ver 1.24, Smiths Detection) was used to assign cycle threshold (CT) values for the RT-PCR. The limit of detection of the real-time RT-PCR on the Bioseeq™ was comparable to the laboratory-based diagnostic assay, although the CT values produced on the Bioseeq™ were higher for the corresponding RNA samples. The variability of the Bioseeq™ modules was assessed by testing an identical RNA

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sample prepared from the reference FMDV isolate O1 Manisa. These results showed that mean inter-module variability (CV) was 6.5% (all 6 modules) in comparison with mean intra-module variability of 4.7% (n = 5 separate runs). These preliminary results were encouraging, indicating that it is possible to develop a sensitive RT-PCR assay for FMDV on the mobile Bioseeq™ platform which can generate a result within 60 minutes. Clearly, effective preparation of template RNA is an important consideration for the deployment of this technology in the field since the presence of tissue-derived factors may inhibit the RT-PCR. This aspect was previously recognised as a limitation to the implementation of molecular methods for field detection of FMDV. In pilot experiments we were able to show that FMDV could be directly detected in “raw” unprocessed material. Approximately 25 mg of biopsy lesion sample from a cow infected with the O1-Manisa isolate of FMD virus) was placed into a disposable Nalgene® bottle containing 25 ml of PBS and 6 ball-bearings. The bottle was shaken for 3 min after which 1 µl of the resulting suspension was tested directly by real-time RT-PCR using the Bio-Seeq™. However, this method was not robust to changes in tissue concentration since there was only a narrow dilution range over which the assay would detect FMDV in the sample. Building upon these results, since 2004 we have been working in partnership with Smith Detection (and Defra and other stakeholders) to design a bespoke hardware platform suitable for field detection of FMDV. In contrast to the previous versions of the Bioseeq™, this new machine (Bioseeq™ Vet: see figure 5B) will be able to extract nucleic acid template, in addition to performing PCR amplification, and will therefore make molecular diagnosis of FMD in the field (by a non-specialist) an achievable goal.

Figure 5: Portable PCR equipment for field use: [A] Bioseeq™: a portable, battery operated real-time PCR machine weighing approximately 3 Kg with 6 independently programmable modules. The instrument can be directly connected to a PC to allow programming of protocols and recording of data, or can be operated in the field employing preset protocols and the in-built LED screen to present assay results. NOTE: this version of the Bioseeq™ does not perform nucleic acid extraction [B] Bioseeq™ Vet: bespoke mobile PCR system for field use. This case contains 5 independently programmable modules which incorporate nucleic acid extraction protocols. The case can be immersed in disinfectant (for decontamination). Further details can be found at http://www.smithsdetection.com/eng/veterinary_diagnostics.php.

The FMDV-specific assay that will be transferred to the Bioseeq™ Vet is currently being developed and validated at the IAH (in collaboration with Smiths Detection). This evaluation will involve an assessment of the diagnostic sensitivity and specificity of the test in comparison with the rRT-PCR in routine use in the laboratory. Once the laboratory-based work at IAH has been completed, we aim to field-trial the assay in Turkey (in collaboration with FAO and EC) later in 2008.

Objective 6: Evaluation of alternative viral enrichment strategies such as immuno-capture methods

At the 18 month review meeting, it was agreed that the work undertaken in this objective would be modified to evaluate a proximity ligation assay (PLA) for FMDV. In contrast to rRT-PCR assays that detect FMDV genome, this approach uses specific antibodies to detects intact FMDV antigen in a sample (Figure 6). The basis of the PLA is that FMDV specific-antibodies binding target proteins are coupled to oligonucleotide strands. These oligonucleotides can be joined by ligation

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when two or more such reagents are brought into proximity by binding to the same target molecule or target molecule complex. The DNA ligation products are subsequently detected by PCR amplification using fluorogenic probes to detect the amplified product. The performance of the PLA was compared to Ag-ELISA and rRT-PCR used for the routine laboratory diagnosis of FMD.

Figure 6: An outline of the amplification strategy used for proximity ligation assay (PLA: left-hand side of cartoon) compared with reverse transcriptase PCR on FMDV-RNA (right-hand side of cartoon). For the proximity ligation analysis, 1 ml of sample was incubated with proximity probes (antibodies extended with single-stranded oligonucleotides) directed against FMDV surface proteins. In the next step, free ends of the oligonucleotide extensions on the probes bound to the same viral particle were ligated. The newly formed DNA sequence was amplified and detected by real-time PCR.

Summary (abstract) of findings published in Nordengrahn et al., 2008:A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. This work was a collaboration between the IAH, Svanova (Uppsala, Sweden) and Instituto Zooprofilattico Sperimentale della Lombardia e dell´Emilia Romagna (Brescia, Italy). The monoclonal antibody chosen for this study (designated 1F10) has been previously shown to be capable of recognising all seven FMDV serotypes in both a trapping ELISA and a sandwich ELISA. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMDV antigen detection.

Objective 7: Assessment of the suitability of isothermal approaches that can be used to amplify RNA/DNA for FMDV diagnosis

As an alternative to using RT-PCR amplification in field-based assays, we have also explored the use of isothermal (single temperature) amplification methods for the detection of FMDV. Since

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amplification occurs at a single temperature, these assay formats may be ideal for incorporation into simple-to-use and inexpensive tests that can be deployed in the field.

Nucleic acid sequence-based amplification: summary of findings published in Lau et al., 2008:A study was conducted to evaluate the performance of a nucleic acid sequence-based amplification (NASBA) assay for the detection of FMDV. NASBA is a continuous, isothermal and enzyme-based method to amplify single-stranded RNA and is therefore particularly suited to RNA virus detection. The technology employs three enzymes: reverse-transcriptase, ribonuclease-H and T7 RNA polymerase, a set of target-specific forward and reverse oligonucleotide primers and two types of detection probes. The forward primer has a 5’ extension containing the promoter sequence for T7 bacteriophage DNA-dependent RNA polymerase, while the reverse primer has a 5’ extension containing a complementary binding sequence for a DNA oligonucleotide detection probe labelled with a ruthenium (Ru)-based electrochemiluminescent (ECL) tag for electromagnetic detection (Figure 6A). NASBA amplification can occur at a constant temperature (41 °C): therefore the process can be performed in a heat-block or even in a temperature-controlled water bath which circumvents the need for expensive thermocycling equipment. Two detection methods: NASBA-electrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzyme-linked oligonucleotide capture (NASBA-EOC) were evaluated (Figure 7).

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Figure 7: Schematic illustration of the principle of NASBA-ECL (A) and NASBA-EOC (B) detection methods. [A]: The biotin-labelled capture probe is attached to the magnetic bead through a biotin-streptavidin interaction. Its 3’ end is complementary to a sequence conferred to the product by the forward primer. The ruthenium-labelled detection probe is complementary to a sequence conferred to the product by the reverse primer. Detection is at 620 nm in an ECL reader. [B]: The biotin labelled capture probe is attached to the well of a streptavidin-coated microtitre plate (grey). As in NASBA-ECL, its 3’ end is complementary to a sequence on the product. The DIG-labelled detection probe is complementary to a sequence conferred to the product by the reverse primer. After substrate addition and incubation (see method), the reaction was stopped and absorbance at 405nm measured in a spectrophotometer.

The diagnostic sensitivity of these assays was compared with other laboratory-based methods using 200 clinical samples collected from different regions of the world. Assay specificity was also assessed using samples (n = 43) of other viruses that cause vesicular disease in livestock and genetic relatives of FMDV. Concordant results were generated for 174/200 (87.0%) of suspect FMD samples between NASBA-ECL and real-time RT-PCR (Figure 8). In comparison with the virus isolation (VI) data, the sensitivity of the NASBA-ECL assay was 92.9%, which was almost identical to that of the real-time RT-PCR (92.4%) for the same set of samples. There was broad agreement between the results of the NASBA-ECL and the simpler NASBA-EOC detection method for 97.1% of samples. In conclusion, this study provides further data to support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance of FMDV

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Figure 8: Plot of real-time RT-PCR results compared with NASBA-ECL resultsThe four quadrants indicate the regions of within and outside the cut-off values. Compatibility is seen in the top right (positive) and bottom left (negative).

Loop-mediated isothermal amplification: summary of findings published in Blomström et al., 2008:Loop mediated amplification (LAMP) also amplifies specific nucleotide sequences. However, unlike PCR, a denatured template is not required and DNA is generated in abundance so that positive LAMP reactions can be visualised with the naked eye. LAMP has been reported to have equivalent (or improved) analytical sensitivity when compared to PCR methods for virus detection. During SE1121, we worked (in collaboration with SVA, Uppsala) to develop and evaluate a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SVDV. This assay partners a similar RT-LAMP assay for FMDV that has been developed at IAH-Pirbright as part of a MoD/Cabinet Office funded project (see: Dukes et al., 2006: Arch Virol. 151: 1093). The SVDV RT-LAMP assay detects the virus rapidly, within 30–60 min and the result is visualised either by gel-electrophoresis or by the naked eye through the addition of SybrGreen. A collection of 28 SVDV isolates were tested positive, while heterologous viruses such as foot-and-mouth disease virus and vesicular stomatitis virus remained negative. The performance of the RT-LAMP was compared directly with real-time PCR using RNA from clinical samples including nasal swabs, serum and faeces. For nasal swabs and serum the sensitivity of the RT-LAMP was shown to be at least equivalent to real-time PCR. Interestingly, for faecal samples the RT-LAMP assay was even more sensitive than real-time PCR, possibly because it is less sensitive to inhibitory substances. This RT-LAMP assay provides a number of benefits for the diagnosis of SVD, since it is sensitive and rapid, and as the isothermal amplification strategy used is not reliant upon expensive equipment it is particularly suited for “front line” diagnosis of SVD in modestly equipped laboratories, in field situations or in mobile diagnostic units.

Future work

The follow-on project (SE1124) will have objectives to (i) further improve high-throughput laboratory-based RT-PCR assays foe the detection of FMDV and (ii) evaluate mobile assays for the rapid detection of FMDV in the field.

The specific objectives for this follow-on project are:

A: Improvements to the laboratory-based real-time RT-PCR assay1: Develop and validate improved automated protocols to process large numbers of blood

samples for real-time RT-PCR.2: Evaluate automated sample homogenisation methods for epithelial samples.3: Optimise improved protocols for the detection of FMDV in “probang” samples4: Planning, implementation and analysis of inter-laboratory comparative exercises for RT-

PCR methods to detect FMDV.B: Improvements to FMDV detection in the field using molecular approaches

5: Evaluation of mobile PCR equipment and assays for field detection of FMD.

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6: Development of a "disposable" isothermal assay for FMDV.7: Evaluation of laboratory and/or field-based RT-PCR assays to confirm weak lateral-flow

device reactors.C: Application of new molecular technologies for vesicular disease diagnosis

8: Identification of unique signatures for the different FMDV serotypes and development of assays to detect and discriminate the 7 serotypes of FMDV.

9: Evaluation of novel molecular detection strategies for FMDV diagnosis.

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References to published material9. 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.

Peer-reviewed publications

1. Ferris N. P., King D. P., Reid S. M., Hutchings G. H., Shaw A. E., Paton D. J., Goris N., Haas B., Hoffmann B., Brocchi E., Bugnetti M., Dekker A. and De Clercq K. (2006) Foot-and-mouth disease virus: a first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR detection methods. Veterinary Microbiology 117: 130-140.

2. Reid S. M., King D. P., Shaw A. E., Knowles N. J. Hutchings G. H., Cooper E. J., Smith A. W. and Ferris N. P. (2007) Development of a real-time reverse transcription polymerase chain reaction assay for detection of marine caliciviruses (genus Vesivirus). Journal of Virological Methods 140 (1-2): 166-173.

3. Shaw A. E., Reid S. M., Ebert K., Hutchings G. H., Ferris N. P. and King D. P. (2007) Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-and-mouth disease. Journal of Virological Methods 143 (1): 81-85.

4. King D. P., Montague N., Ebert K., Reid S. M., Dukes J. P., Schädlich L., Belsham G. J., and Lomonossoff G. P. (2007) Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR. Journal of Virological Methods 146: 218-225.

5. Lau L.-T., Reid S. M., King D. P., Lau A. M. –F., Shaw A. E., Ferris N. P., and Yu A. C. –H. (2008) Detection of foot-and-mouth disease virus by nucleic acid sequence-based amplification (NASBA). Veterinary Microbiology 126 (1-3): 101-110.

6. Blomström A. –L., Hakhverdyan M., Reid S. M., Dukes J. P., King D. P., Belák S., and Berg M. A (2008) one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus. Journal of Virological Methods 147 (1): 188-193.

7. Nordengrahn A., Gustafsdottir S. M., Ebert K., Reid S., M., King D. P., Ferris N. P., Brocchi E., Grazioli S., Landegren U., and Merza M. (2008) Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus. Veterinary Microbiology 127: 227-236.

8. King D. P., Dukes J. P., Reid S. M., Ebert K., Shaw A. E., Mills C. E., Boswell L. and Ferris N. P. (2008) Prospects for rapid diagnosis of foot-and-mouth disease in the field using RT-PCR. Veterinary Record 162 (10): 315-6.

9. Hindson H. J., Reid S. M., Baker B. R., Ebert K., Ferris N. P., Bentley Tammero L. F., Lenhoff R. J., Naraghi-Arani P., Vitalis E. A., Slezak T. R., Hullinger P. J. and King D. P. (2008) Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses. Journal of Clinical Microbiology 46 (3): 1081-1089.

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Presentations

1. Reid S. M., King D. P., Shaw A. E., Knowles N. J., Hutchings G. H. and Ferris N. P. Detection of marine caliciviruses (Genus Vesivirus) by real-time RT-PCR. 7 th International Congress of the European Society for Veterinary Virology, Lisbon, Portugal, September 2006.

2. King D. P., Dukes J. P., Reid S. M., Ebert K., Shaw A. E., Boswell L., Mills C. E. and Ferris N. P. Detection of foot-and-mouth disease virus using field portable equipment. 7 th International Congress of the European Society for Veterinary Virology, Lisbon, Portugal, September 2006.

3. King D. P., Montague N., Ebert K., Reid S. M., Dukes J. P., Ferris N. P., Belsham G. J. and Lomonossoff G. P. Development of a novel encapsidated internal control for real-time RT-PCR. 7th

International Congress of the European Society for Veterinary Virology, Lisbon, Portugal, September 2006.

4. Ebert K., King D. P., Reid S. M. and Ferris N. P., Comparison of RNA extraction methods for RT-PCR diagnosis of FMD. 7th International Congress of the European Society for Veterinary Virology, Lisbon, Portugal, September 2006.

5. Reid S. M., Dukes J. D., Ebert K., Ferris N. P. and King D. P. Diagnosis of FMD by RT-PCR: prospects for mobile and portable assays. Open Session of the Research Group of the Standing Technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Paphos, Cyprus, October 2006.

6. Ferris N. P., King D. P., Hutchings G. H., Li Y., Goris N. and Paton D. J. FOA collaborative studies for FMD standardisation: Phase XIX – virological assays. Open Session of the Research Group of the Standing Technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Paphos, Cyprus, October 2006.

7. Pierce K. E., Dukes J. P., Reid S. M., King D. P., Wangh L. J. LATE-PCR detection of foot and mouth disease virus. 107th General Meeting of the American Society for Microbiology, Toronto, Canada, May 2007.

8. Reid S. M., Mistry R., Dukes J. P., Ebert K., Mills C. E., Boswell L., Ferris N. P. and King D. P. Evaluation of a portable thermocycler for the detection of foot-and-mouth disease virus. 1 st Annual Meeting of the EPIZONE project, Lublin, Poland, May 2007.

9. King D. P., Montague N., Ebert K., Reid S. M., Dukes J. P., Schädlich L., Belsham G. J. and Lomonossoff G. P. Use of a novel recombinant cowpea mosiac virus particle as an internal control for diagnostic RT-PCR. 1st Annual Meeting of the EPIZONE project, Lublin, Poland, May 2007.

10. Mistry R., Pierce K. E., Reid S. M., Dukes J. P., Ebert K., King D. P. and Wangh L. J. LATE-PCR detection of foot-and-mouth disease virus. 13th International Symposium for the World Association of Veterinary Laboratory Diagnosticians, Melbourne, Australia, November 2007.

11. King D. P. Laboratory tools for the detection of foot-and-mouth disease virus. 3 rd Regional Workshop of the SEAFMD Laboratory Network, Hanoi, Vietnam, March 2008.

12. King D. P., Reid S. M., Ebert K., Brocchi E. and Ferris N. P. Field tools for the rapid detection of foot-and-mouth disease virus. 14th Meeting of the OIE sub-commission for Southeast Asia, Hanoi, Vietnam, March 2008.

13. King D. P., Reid S. M., Ebert K., Batten C., Swain A., Bankowska K., Wright C., Cottam E. M., Wadsworth J., Ferris N. P., Haydon D. T., Knowles N. J. and Paton D. J. Molecular assays for FMDV detection: their role during last summer’s outbreaks in the UK. 2nd Annual Meeting of the EPIZONE project, Brescia, Italy, June 2008.

14. Ebert K., Reid S. M., Li Y., Hutchings G. H., Paton D. J., Ferris N. P. and King D.P. Results from the 2007 proficiency testing scheme for RT-PCR methods used to detect foot-and-mouth disease virus. 2nd Annual Meeting of the EPIZONE project, Brescia, Italy, June 2008.

15. Pierce K., Mistry R., Dukes J. P., Reid S. M., Ebert K., King D. P. and Wangh L. LATE-PCR detection of foot-and-mouth virus (FMDV) using the Bioseeq Vet. 2nd Annual Meeting of the EPIZONE project, Brescia, Italy, June 2008.

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