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Evidence Project Final Report

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 Evidence 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 websiteAn Evidence Project Final Report must be completed for all projects.

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Project identification

1. Defra Project code SE1127

2. Project title

Development and evaluation of improved diagnostic tests for vesicular viral diseases of livestock

3. Contractororganisation(s)

The Pirbright InstituteAsh RoadPirbrightWokingSurreyGU24 0NF

54. Total Defra project costs £ 2,694,702(agreed fixed price)

5. Project: start date 01/07/2012

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 23

end date 29/03/2015


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing Evidence Project Final Reports 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 In these cases, such information should be detailed in a separate annex (not to be published) so that the Evidence Project Final Report can be placed in the public domain.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.

Foot-and-mouth disease (FMD) is an infectious disease of cloven-hoofed livestock (cattle, sheep, pigs and goats) and one of the most widespread epizootic animal diseases. FMD remains endemic in many countries in Africa and Asia. In addition, sporadic outbreaks of disease frequently occur in countries that are normally free from disease such as in the UK 2001 and 2007, and more recently in Bulgaria (during 2011), Japan, South Korea, Russia, and extensively in PR China. These outbreaks have reinforced concerns about how readily the disease can pass across international borders. FMD is difficult and expensive to contain since the causative virus (FMDV) is highly contagious and spreads rapidly through susceptible animals. In the event of an introduction into the UK, successful control and eradication of FMD is dependent upon early recognition of infected animals to reduce the potential for onward transmission of the virus to cause subsequent outbreaks. Accurate tests that can either detect FMD virus in clinical specimens, or FMDV-specific antibodies in sera play central roles in these activities.

The aim of this 33-month project was to maintain and improve laboratory-based diagnostic methods for FMD, as well as evaluate technologies that allow rapid testing to be achieved in the field or in regional laboratories. As part of our work to continuously update the molecular tests that are used for FMD diagnosis, during SE1127 we have optimised a new automated nucleic acid extraction protocol to allow us to rapidly process large numbers of samples that might be received to the FMD National Reference Laboratory during an outbreak. A particular focus of this work has been to evaluate practical approaches that can be used to provide a capability for early FMD diagnosis within a herd prior to the onset of clinical signs, since such tests have the potential to reduce the un-necessary slaughter of uninfected animals. We have shown that milk samples represent a useful non-invasive sample that can be easily pooled for FMD surveillance purposes, and that FMD virus can be detected in milk samples as early as 2-3 days after direct contact with infected cattle (approximately 1.5 days prior to the development of clinical signs). We have evaluated new automated methods that can be used to process milk samples for subsequent testing by real-time RT-PCR, and demonstrated that these molecular methods are superior to conventional virological diagnostic methods.

For FMDV-specific ELISAs, in collaboration with international partners we have undertaken work to validate new FMDV antigen and structural protein antibody assay kits that adopt monoclonal antibodies rather than polyclonal antisera as FMDV-specific reagents. For future use as a universal capture ligand in these tests, we have now established a pipeline to generate recombinant solubilised αvβ6, and also developed a novel assay that can detect FMDV (based on AlphaScreen technology) that may have


application for the basis of a rapid diagnostic test. Work has also continued to validate improved ELISAs that can be used to identify animals that have been previously exposed to FMD infection, particularly those within a vaccinated population according to the EU Directive and recommendations of OIE. In this context, we have we have developed and validated new tests that detect specific antibodies against four FMDV non-structural proteins (3ABC, 3CD, 3D, 2C), two peptides (2B and 3B) and validated these assays using a panel of reference sera and field sera. Encouraging pilot data has also been generated for novel multiplex (Luminex) assay that contains six FMDV non-structural proteins, as well as a new IgA specific ELISAs formats for SAT2 and Asia-1 serotypes and a new IgA assay that utilises recombinant antigen for serotype A that can potentially be used to accurately recognise FMDV carrier animals. Furthermore, during SE1127 we have validated a new 3ABC competitive test from IDVet®, France and shown that both the formats of this test have equivalent specificity and sensitivity with the established Prionics PrioCHECK® FMDV NS 3ABC test (now from Thermo).

During SE1127, we have explored new assay formats that can achieve rapid diagnostics in field settings. Work in this area has evaluated “dry-down” reagents that allow real-time RT-PCR to be performed on mobile equipment, and has explored simple-to-use protocols that can be applied to process clinical samples for down-stream testing by inexpensive isothermal (RT-LAMP) assays for differential diagnosis of FMD for two other vesicular diseases of livestock (swine vesicular disease and vesicular stomatitis). These exciting assay formats have been validated in the field using samples collected from on-going studies in East Africa (sample collection and field work funded from other sources). We have also explored the possibility of developing a new assay format based on the protease activities of two viral enzymes, which for one of these assays (Lpro) shows more promise than the 3Cpro as the cross-reactivity is low with the comparable enzyme for a closely related picornavirus (bovine rhinovirus). During this project we have investigated the potential of using FMDV lateral-flow devices (LFD’s) for dry transportation of clinical material for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by transfection using electroporation.

Together, the outputs from this project will provide Defra with increased confidence in the use of a portfolio of tests for diagnosis and surveillance in the event of future outbreaks of FMD. We continue to recommend methods for adoption by the OIE (to be included into the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals). The value of these tests was highlighted during the FMD outbreak in 2007 where sensitive molecular methods (automated real-time RT-PCR) were applied as front-line diagnostic tools in support of the surveillance and eradication programme, and an antigen strip test was used for triage of samples in the National Reference Laboratory. Furthermore, validated serological tests were available to support a “vaccinate-to-live” policy if this was required by the UK Government.

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 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 Exchange).The project objectives are arranged in 3 themes to address different diagnostic scenarios and priorities:Theme 1: Improved tests, reagents and protocols for FMD virus detectionTheme 2: Improved tests, reagents and protocols for FMDV serologyTheme 3: Field tools for FMD diagnosisWithin these different themes, specific progress made towards the listed objectives is outlined below:


Theme 1: Improved tests, reagents and protocols for virus detection

1.1 Updating real-time RT-PCR assays for the detection of vesicular disease viruses

1.1a - For FMDV and other vesicular disease viruses: during this project, we have initiated work to evaluate new automated platforms that can be used for high-throughput extraction of nucleic acids for molecular analysis. These experiments have used a panel of representative sample types to compare the performance of a 96-well platform (manufactured by MagMax/Kingfisher) with the currently used instruments (MagNA pure and Qiagen BioRobot) that are now at the end of their useful working life. The MagMAXTM-96 Viral RNA Isolation Kit is designed for rapid high throughput purification of viral RNA and DNA from liquid samples in 96 well plates. The system is able to process 96 samples in less than 30 minutes, in comparison with the MagNA Pure LC extraction robot, which can process 32 samples in 90 mins. A new RNA extraction protocol has been the established and compared with the method outlined in the SOP used by the FMD Reference Laboratory. Direct comparison of results generated for 146 samples are shown in Figure 1.

Figure 1: A cluster graph showing the mean cycle threshold values (Cts) of diagnostic samples tested by real-time RT-PCR using nucleic acid extracted on the MagMAX and MagNA Pure platforms for samples from Nigeria, Sri Lanka, Ethiopia, Malaysia, Tunisia, Algeria and Zimbabwe. The yellow boxes indicate the current diagnostic cut-off for positive samples (Ct=32).

Overall, the MagMAXTM Express 96 extraction robot with the MagMAXTM-96 viral RNA isolation kit performs to an equivalent standard compared to the current MagNA Pure LC extraction robot, for a variety of sample types and FMDV serotypes that might be received to the UK National Reference Laboratory for FMD. The only exception applies to EDTA whole blood, where an alternative extraction procedure (adopting an alternative MagVETTM lysis buffer in place of the MagMAXTM lysis buffer in conjunction with the rest of the MagMAXTM kit) has been investigated and found to be suitable. The procedure can be performed with no contamination between wells, and in a short time (<30 mins). The lysis buffer has a long shelf life (up to 6 months) both at room temperature, and with sample addition at -80oC, allowing the current storage protocol for diagnostic submissions to be maintained. These evalution results have formed part of a dosier that has been submitted to UKAS in order to extend the scope of our ISO/IEC 17025 testing portfolio, and will facilitate the preparation of new improved SOPs to describe FMD diagnostics methods in the Plowright Building (BBSRC National Virology Centre).

1.1b – For FMDV, development of a non-infectious encapsidated positive control RNA for molecular assays to detect FMDVPositive controls are an important component of the quality-control of molecular tests used for diagnosis of livestock diseases. For high consequence agents such as FMDV, the positive controls required to monitor template extraction, reverse transcription and amplification steps usually consist of material derived from


infectious viruses. Therefore, their production is dependent upon the use of high containment facilities and their deployment carries the risks associated with inactivation of “live” FMDV. During SE1127, we have developed a novel non-infectious positive control that encodes FMDV RNA sequences that are encapsidated within Cowpea mosaic virus (CPMV) particles. This surrogate RNA has been engineered to contain sequences from the 5’UTR and 3D regions of FMDV targeted by many molecular assays (conventional RT-PCR, real-time RT-PCR and RT-LAMP). These sequences were inserted into a movement-deficient version of CPMV RNA-2 which is rescued from cowpea plants (Vigna unguiculota) by inoculation with RNA-1. In order to evaluate the performance of these encapsidated RNAs, nucleic acid prepared from a 10-fold dilution series was tested using a range of molecular assays (see Figure 2C for results for rRT-PCR). Results generated by using the molecular assays confirmed RNA-dependent amplification and the suitability of these particles for use in a range of diagnostic tests. Moreover, these CPMV particles were highly stable for periods of up to 46 days at room temperature and 37°C. Recombinant CPMV can be used to produce high yields of encapsidated RNAs that can be used as positive and negative controls and standards in molecular assays. This approach provides an RNA surrogate that can be potentially used outside of containment laboratories as an alternative to inactivated infectious virus for molecular diagnostic testing.

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Figure 2: A: A cartoon outlining the cloning strategy used to create an engineered FMDV surrogate RNA within a movement deficient (MP) CPMV particle. The identity of the different plasmids are indicated and the location of Cauliflower mosaic virus 35S promoter (35S), as well as regions encoding CPMV MP, partially deleted MP (MP del), large coat protein (L), small coat protein (S), 3’ end of CPMV RNA-2 (3’) and the Agrobacterium nopaline synthase terminator (nos). The location of the artificial construct (FMDV) is shown in pBIN-mim-AvrII-mp del FMDV; B: Recombinant Cowpea Mosaic Virus particle containing surrogate FMDV RNA sequences visualised by electron microscope, C: Detection of FMDV surrogate viral RNA in the recombinant CPMV particle using the RT-qPCR targeting FMDV 3D. Points shown represent mean CT ± SD of triplicate determinations of a 10-fold dilution series of the CPMV particle (starting at 1 µg/µL) in negative bovine epithelium suspension.

1.1c - For swine vesicular disease virus [SVDV]: The established real-time RT-PCRs (rRT-PCRs) for the laboratory diagnosis of SVDV target the 5’ untranslated region of the genome. Although these assays are able to detect a wide range of different field strains, previous validation exercises have shown that these tests fail to detect a small number of SVD viruses in contemporary viral lineages. Therefore, new assays with improved diagnostic sensitivity are required. Using new sequence data for SVDV (generated by University of Copenhagen in collaboration with the Pirbright Institute), we have worked with the Italian SVD Reference Laboratory in Brescia (Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini": IZSLER) to design and evaluate new rRT-PCR assays. The candidate assays target the highly conserved 3D-encoding region of the genome and have been evaluated using a panel of RNA samples generated from representative SVDV strains. Current data indicate that one of these new tests has improved diagnostic sensitivity and in constract to the established SVD rRT-PCR test used for routine diagnosis (named SVD-2B-IR) is able to detect representatives of all of the SVDV lineages. Validation of this new assay is on-going at IZSLER and we anticipate that if these results confirm the high diagnostic senstivity of this assay that it will replace the rRT-PCR test employed for routine testing at The Pirbright Institute (in the National Reference Laboratory for SVD).

1.2 Evaluation of herd-level preclinical diagnostic indicators


There is a need for rapid diagnostic tests to enable early detection of FMDV in cattle. In this context, FMDV detection in milk presents unique opportunities to use non-invasive sampling approaches for surveillance and early detection both prior to and during a FMD outbreak. During SE1127, we have evaluated the effectiveness of preclinical indicators of FMDV infection and to evaluate a high-throughput screening approach using rRT-PCR which could be scaled up for use to screen large numbers of samples from bulk milk tanks. Samples were collected from experimentally infected dairy cattle to evaluate the diagnostic window of detection in milk and to gauge the impact of FMD infection on milk production (cattle study was funded from a separate source). Four Jersey dairy cows were infected via direct contact with two cattle that had been inoculated with FMDV (O/ME-SA/PanAsia strain, isolate O/UKG/2001) within the high-containment facilities at The Pirbright Institute. Diagnostic tests were carried out in real-time on fresh samples collected over the 28 days of the experiment. These tests included virus isolation (in IB-RS2 and BTY cells) and routine pan-serotypic rRT-PCR targeting 3D used in the UK National Reference Laboratory for FMD and which results were compared with a new optimised rRT-PCR protocol for milk. The greatest window for virus detection was by rRT-PCR in the milk up to 21 days post contact, and both rRT-PCR protocols detected virus for a longer period than seen for virus isolation. In addition to the in-vivo study described, samples collected from field cases (including 12/13 milk samples collected from IP2 during the UK 2007 outbreak) were used as a positive cohort to evaluate diagnostic sensitivity. The data implies that rRT-PCR of milk (obtained from a bulk tank) could detect a single infected cow, in a large herd, in the early stages of infection. This suggests that milk could be an excellent sample type for the detection of FMDV and could be adopted to support FMD surveillance in the event of an outbreak. Further validation of the rRT-PCR test using milk samples collected from dairy cattle in FMD endemic countries is planned (Tanzania and Kenya).

1.3 Optimisation of improved methods to recover FMDV from RNA and archived samples

The effective laboratory diagnosis of FMD is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material which emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic material. During this project we have investigated the potential of using FMDV lateral-flow devices (LFD’s) for dry transportation of clinical material for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by transfection using electroporation. Positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were detected by the LFD: after which it was possible to recover viral RNA that could be detected using rRT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes (Figure 3). When eluted RNA was directly inoculated onto susceptible cell cultures there was no infectious virus recovered, however following electroporation into BHK-21 cells and subsequent passage, infectious virus could be recovered. The LFDs could be stored for periods of one month at temperatures as high as at 37°C. Therefore, these preliminary results support the use of the LFD to be used for the dry transportation of FMD positive nucleic acids to FMD reference laboratories. We aim to continue this work using additional clinical samples added to the LFD in the field that have been shipped to the UK National Reference Laboratory for FMD and to compare directly to routine virus isolation methods in order to determine whether there is a difference in the efficiency of infectious virus recovery (as part of other on-going projects). An added benefit of using LFDs is that that these can be used in the field to select suitable specimens that are confirmed to be positive for FMDV, and in the event of negative results, the devices can be used to inform rapid re-sampling of animals within a herd. These results provide evidence to indicate that positive lateral flow devices may pose lower biorisk should they be used for transportation of samples between the field and reference laboratories. In view these data, further work to consider and agree appropriate biosecurity and IATA transport guidelines is required so that these new methods can be transitioned into the field for the safe preservation and recovery of FMDV (discussions regarding the possibility of sending non-infectious materials comprising RNA are now being considered within the network of EU National Reference Laboratories).


Figure 3: Visualization of the 24 overlapping RT-PCR products representing the complete FMDV genome generated from a LFD loaded with a clinical sample (UKG 7B/2007). The figure shows a 1.8% agarose gel stained with ethidium bromide (0.2 µg per ml). Lane 1, S-fragment of the 5’UTR, Lanes 2-22 L fragment, Lanes 23 and 24, poly A fragment.

1.4 Production of soluble integrin for use as a capture ligand in FMDV-specific assays

The integrin αvβ6 is the major receptor for FMDV and is used by all field FMD viruses, regardless of serotype, to initiate infection. FMDV binding to αvβ6 is mediated by a highly conserved RGD motif (amino acids are abbreviated using the single letter code) on the outer surfaces of the viral capsid and is dependent on the presence of divalent cations. We had shown previously that viruses representative of all FMDV serotypes bind immobilized αvβ6 in a solid-phase ELISA in an authentic RGD- and divalent cation-dependent interaction. Furthermore, the potential of αvβ6 as a universal virus-trapping reagent in diagnostic assays was demonstrated by showing that the integrin could be substituted for polyclonal sera in a sandwich ELISA for the detection and serotyping of FMDV (Ferris et al., 2005: Figure 4). Thus, αvβ6 has great potential for application in other diagnostic procedures for viral identification and serotyping FMDV. Integrins are non-covalent heterodimers formed by the association of two chains, α and β. Each chain consists of a larger extracellular domain, a single-span transmembrane region (TM) and a short cytoplasmic tail. The ligand binding site is formed by regions of both α and β subunits.

Figure 4: An example (from the National Reference Laboratory for FMD) of the diagnostic utility of the improved Ag-ELISA using recombinant αVβ6 integrin (in these pilot assays solubilized human integrin as used). The current Ag-ELISA was unable to detect (or type) FMDV in 28 field isolates collected in Africa and the Middle East. In contrast, the modified Ag-ELISA using recombinant αVβ6 integrin and characterised serotype-specific monoclonal antibodies was able to successfully detect and serotype these isolates.

In this project we have expressed soluble bovine αvβ6. This was achieved by generating two mammalian


expression plasmids each directing expression of one chain (αv or β6). To allow for secretion of the integrin into the extracellular media a signal peptide was fused to the N termini of each chain. In addition, the TM region and short intracellular domain of each chain was removed and a stop codon inserted immediately after the coding region for the extracellular region. Alternatively, for the β6 chain, an epitope tag was included immediately before the stop codon; this was either a His tag (His6), a FLAG tag or a short 4 amino acid (EPEA) motif. The purpose of the tag is to allow for detection of integrin expression and as an affinity tag to allow for integrin purification. Integrin expression was carried out using a number of mammalian cell lines including Chinese Hamster Cells (CHO) and HEK293T cells using different transfection reagents. Cells were dual transfected with expression plasmids for each chain combining the plasmid for αv with a plasmid for β6; this was carried out for each tagged version of β6. Western blot analyses and ELISA (to detect integrin expression) showed best results (i.e. maximum integrin expression in transfected cell culture media) was achieved using either the FLAG or EPEA tag with HEK293T cells.

The FLAG and EPEA tagged integrins were further evaluated and shown to bind FMDV (this was carried out using FMDV empty capsids produced using a vaccinia virus expression system), but not to virus particles containing a KGA in place of the integrin-binding RGD (Figure 5B). In addition, we confirmed that virus binding could be inhibited by RGD-containing peptides (but not a control, functionally inactive RGE version) or EDTA (which chelates divalent cations) (Figure 5A). Furthermore, preliminary experiments show that the integrin can be purified using the EPEA motif as an affinity tag.

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Figure 5: RGD and cation dependent FMDV binding to soluble αvβ6. (A) FLAG tagged αvβ6 was used to trap FMDV empty capsid (FMDV A22) and binding detected using an anti-FMDV A22 polyclonal sera and HRP-conjugated secondary antibody. Absorbance (virus binding) was determined at 450 nm after the addition of HRP substrate. Human αvβ6 was included as a positive control. A22 only show virus binding in the absence of competition; RGD virus binding in the presence of an RGD peptide; RGE virus binding in the presence of an RGE peptide; EDTA virus binding in the presence EDTA; BBB & Mock Trans are negative controls. (B) FMDV A22 empty capsids were trapped using an anti-type A polyclonal sera and detected using FLAG or EPEA tagged integrin followed by the appropriate secondary antibody/detection-system. Integrin binding was detected for A22 but not A22 capsids with KGA in place of the RGD.

We are now in a position to scale up transfection and evaluate the integrin for use in FMDV diagnosis. This has the additional benefit that the integrin can be used as a replacement for rabbits and/or guinea-pig polyclonal sera as the virus trapping reagent thereby reducing the need for using animals for FMDV diagnosis.

1.5 Integrin αvβ6: Time resolved fluorescence resonance energy transfer (TR-FRET)

Although some of the current laboratory-based diagnostic methods (such as immunoassays or rRT-PCR) are rapid and can take only a few hours to generate an objective result, the time taken to transport suspect material to the National Reference Laboratory for FMD can be lengthy. In FMD outbreaks where a rapid decision is critical, this delay can hinder laboratory confirmation such that decisions are made based solely on observing clinical signs in the affected animals. These issues have given rise to the idea that the reliability of local clinical diagnosis may be improved using rapid and sensitive tests that can be used closer to the animal with suspect signs of disease. Field-tests that exploit immunoassay and molecular formats have been developed for the detection of FMDV. During SE1127, we have developed a novel


assay (Figure 6) for rapid detection of FMDV, based on AlphaScreen (Perkin Elmer) technology. AlphaScreen is based on detecting energy transfer by diffusion of activated oxygen molecules. For this assay we have used tagged, soluble, bovine integrin αvβ6 (produced in 1.4) and FMDV empty capsids (EC) (FMDV A22), and control empty particles that have a non-functional KGA in place of the integrin-binding RGD. Two populations of integrin (with either a FLAG or EPEA tag) were labelled with an AlphaScreen pair (streptavidin donor to EPEA via a biotinylated anti-EPEA antibody, and an anti-FLAG acceptor) and mixed in the presence of the wild-type FMDV EC or with the KGA version. Only the wild-type EC gave a positive signal thereby demonstrating the use of the assay for detecting FMDV virions and the specificity of the assay for wt capsids.

AlphaScreen

Figure 6: The reaction consisted of wild-type FMDV empty capsids, and FLAG and EPEA tagged αvβ6 labelled with a AlphaScreen acceptor and donor respectively. The αvβ6-FLAG was labelled using a mouse monoclonal anti-FLAG antibody conjugated with the FRET acceptor. The αvβ6-EPEA was labelled using a biotinylated anti-EPEA nanobody and streptavidin conjugated with the donor. The reactions were read after 1h at room temperature. The figure shows the signal (arbitrary units) against different concentrations of the anti-EPEA nanobody. The background signal was determined by omitting either the FLAG (signal: ~900) or the EPEA (signal: ~900) tagged integrin from the reaction, or by substituting empty capsids with KGA mutations at the integrin binding RGD motif (signal: ~2000).

We are now in a position to determine the sensitivity of the assay and evaluate the potential for field diagnostics using clinical samples.

1.6 Further validation of improved antigen detection FMDV ELISAs

During SE1127, a database for monitoring the cross-reactivity of isolates using routinely employed indirect sandwich antigen ELISA was created. These isolates have also been assayed using the current recombinant αvβ6 integrin ELISA (see Figure 4). This will help to investigate cross serotype specificity in future assays. In addition, this process has identified isolates that were positive by virus isolation but unable to be serotyped by indirect sandwich antigen ELISA. These have been grown and organised in preparation for performing the required assays using the αvβ6 integrin and monoclonal antibodies (mAbs). Furthermore, we have assessed and identified the requirements for critical reagents and prioritised those necessary for future production of polyclonal antibodies that will be essential to both outstanding validation and diagnostic work.

In collaboration with IZSLER, a fully evaluated O, A, Asia 1, C and including a pan-FMDV kit is available for FMDV antigen detection and FMDV isolates were selected and prepared for use in evaluating reagents substituting Asia 1 and C with SAT 1 and 2 in this kit format. However, the pan-FMDV is not currently suitable for detecting all SAT 1 and 2 isolates. Therefore, the process of the evaluating suitable reagents for a new kit to include several conjugates/conjugate combinations for SAT1 and SAT2 has been initiated.


A panel of 37 isolates were evaluated against the combinations to establish the best one for use in a prototype kit. However, delays in the evaluation of these conjugates resulted in some technical issues with the kit components and therefore the various conjugate combinations were not fully evaluated. This work will be completed during SE1128.

Recently new mAb combinations for coating and detecting were received from IZSLER for evaluation of antigen detection of SAT 1 and 2. Assaying against panels of SAT 1 and 2 isolates has been initiated and will continue during SE1128. Initial data has identified two suitable combinations that might be suitable for SAT 1 detection. These are coating mAb HD7 and 4E3 and detector mAb 3E11 and in addition capturing mAb HD7and 4E3 and detector mAb HD7. However none of the combinations have 100% sensitivity and this requires careful optimization and validation. The 3E11 mAb was assayed against SAT 2 isolates to establish its suitability for improving sensitivity. Unfortunately, 3E11 does not recognize all the SAT 2 isolates assayed and therefore cannot be used as unique mAb for detecting SATs. The best mAb for SAT 2 has been identified as 2H6 and therefore a mix of mAbs will be required in a kit prototype. In order to increase capacity of producing reagents, the IZSLER laboratory was visited to establish the requirements for producing the mAbs at The Pirbright Institiute. This provided an opportunity to evaluate the requirements of necessary equipment, protocols, and quality assurance specification to have the ability produce these kits at dual sites. Training was provided on all aspects of kit manufacturing.

We need to establish a sustainable source of mAbs using an in vitro system and avoid the use of ascites. It has been agreed that hybridomas of the essential mAbs will be sent to TPI for production in cell culture. The cell supernatant will be evaluated against the existing ascetic fluid mAbs for concordance at both sites. Once available we will evaluate any alternative virus like particles (VLPs). VLPs for serotype A and O are usable but the VLP for Asia 1 is currently unable to be stabilised. Presently there is not a VLP available for serotype C.

1.7 Detection of FMDV 3C protease activity in biological samples

The purpose of this work-package was to explore the potential of using the enzymatic activity of the FMDV 3C protease (3Cpro) in clinical samples as a diagnostic indicator for FMD. During year 1 we decided to include the FMDV L proteases (Lpro) in this WP as it also has the potential for development of a diagnostic assay based on detection of FMDV enzyme activity. The assay uses cleavage of recombinant firefly luciferase (Promega Protease-Glo™ Assay). Target sites for FMDV 3C and Lpro were inserted into firefly luciferase (which renders the luciferase enzyme inactive) and when cleaved by the corresponding viral enzyme luciferase is activated and the resulting light intensity measured after the addition of a luciferase substrate. Using this assay we tested a number of substrates for 3Cpro (Table 1). This included all of the known cleavage sites in both viral and cellular targets.

FMDV 3C: We varied the length (i.e. 7, 6, 5 or 4 residues either side of the point of cleavage) of the VP1/2A target site to determine the influence of sequence length on the assay. This analysis showed that best results were achieved using a P1/2A site of 10 residues (5 either site of the cleavage site). For this assay we used purified FMDV 3Cpro. To reduce the cost of purification we cloned cDNA for FMDV 3Cpro coding region into the Promega pCMVTNT vector and used this in a Promega TNT Quick Coupled Transcription/Translation assay to produce 3C activity. We also created inactive version of 3Cpro (that can be used as negative controls) by site-directed mutagenesis at critical residues in the active site of the enzymes. The TNT produced 3C was shown to perform as well as purified 3C. Importantly, the assay using the 3C VP1/2A targets site gave good results when using infected cell lysates (but not by a control lysate prepared from mock-infected cells) establishing that the assay can be used with infected samples. We then tested if the 3C of bovine rhinitis virus (BRV: a related Aphthovirus) could cut FMDV target


sequences.

For this we produced substrates based on the target sequence of 3C pro of BRV and produced BRV 3Cpro (and an inactive version) using the Quick Coupled TNT system assay as described above. This showed that BRV 3C gave a positive signal with FMDV target sites and a higher than expected cross specificity (Figure 7).

Table 1: Relative signal for different 3C and Lpro target sites: For the results shown, the target sequence was 12 residues, 6 either side of the cleavage site. Scrambled versions of the targets sites were used as negative controls and to determine the assay background signal. The number of + symbols indicates the relative signal strength; -/+ or -indicate low or no cleavage detected respectively

FMDV 3CRelative Luc

activityFMDV Lpro

Relative Lucactivity

Viral targets VP2/VP3 - L/P1 ++++VP3/VP1 -VP1/2A +++++2B/2C +2C/3A +++3A/3B1 -3B1/3B2 -3B2/3B3 -3B3/3C -3C/3D -

Cellular targets EIF4G1 ++ EIF4G1 -/+EIF4G2 -/+

FMDV Lpro: We used the same approach to investigate the potential of the assay to detect the enzyme activity of the FMDV Lpro (table 1). The best results were obtained using a target site for the FMDV L/P1 cleavage site with a sequence length of 14 residues. This assay shows more promise than the 3C assay as the cross-over signal with the FMDV Lpro target sites gave a relatively low background with the BRV Lpro (Figure 7).


FMD VP1/2A + FMD 3C

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Figure 7: Cross reactivity of BRV 3C and Lpro for FMDV targets: Shown is the mean luminescence for FMDV P1/2A with FMDV 3C (lane 1) and BRV 3C (lane 2); and for FMDV L/P1 with FMDV Lpro (FMDV Lb) (lane 3) and BRV Lpro (BRV Lb) (lane 4).

Overall conclusion: We are now in a position to determine the sensitivity of the assay and evaluate the potential for field diagnostics using clinical samples. The assay for detecting activity of the FMDV Lpro shows more promise than the 3C assay as the cross-reactivity is low for the Lpro of BRV. These assays will also be useful to investigate pharmacological inhibitors of 3C and Lpro for the development of antiviral reagents

1.8 Preparation and analysis of PTS panels and maintenance of QA standards

During SE1127, we have continued to coordinate annual intra-laboratory proficiency testing schemes (PTS) to cover laboratory tests used to diagnose vesicular diseases. These exercises provide vital evidence to support accreditation to IEC/ISO 17025 and also provide insights into the different diagnostic systems that are employed in the different National Reference Laboratories in EU member states and elsewhere on the world (where they participate in the PTS). During 2013 and 2014 PTS exercises were organised that employed two separate panels of FMDV samples comprising (i) infectious material and (ii) BEI inactivated non-infectious material in order to evaluate the performance of cell culture, antigen-immunoassay and molecular diagnostic tests. The main coordinating work of the PTS is funded from the EU (via the annual project that supports the European Union – Reference Laboratory for FMD [EU-RL for FMD], or via the EuFMD (to support our work as the World Reference Laboratory for FMD [WRLFMD] by the Food and Agriculture Organisation [FAO]). However, work within SE1127 has provided expertise to help identify suitable material for the PT panels and test these materials prior to shipment to the laboratories. Performance of The Pirbright Institute diagnostic systems is reviewed by UKAS during annual external ISO 17025 assessment.

Theme 2: Improved tests, reagents and protocols for FMDV serology

2.1 Validation of new 2B and 3B NSP confirmatory tests

At an international workshop held under the EU Improcon project at Brescia, Italy, four commercial tests (PrioCHECK FMDV NS, CHEKIT FMD 3ABC, UBI FMDV NS ELISA, SVANOVIR FMDV 3ABC-Ab ELISA), one in-house test (3ABC trapping-ELISA from IZS Brescia) and the OIE index test NCPanaftosa were validated. The specificity of the tests ranged from 97.2% to 98.5% at the first round of screening and the specificity was further increased to 98.3% to 99.7% after retesting the nonspecific samples. Sensitivity to detect vaccinated cattle that became carriers ranged from 68% to 94% depending on the test used. A suggestion was made to increase the sensitivity and specificity of the tests by using more than one NSP antibody assay to differentiate infection in vaccinated animals (DIVA) (Brocchi et al., 2006, Paton et al.,


2006). Moreover it has been suggested that the confidence in detection of infection may be increased by using multiple FMDV antigens in a multiplex ELISA (Perkins et al., 2006, Perkins et al., 2007). The use of multiple antigens to gain high sensitivity and specificity to detect FMD infection have been demonstrated in South American FMD control by combining 3ABC NSP ELISA with enzyme-linked immunoblot assay (Bergmann et al., 2000). Therefore, a multiplex approach has been further extended in this project by using multiple antigens in a liquid array system to detect antibody responses against multiple NSPs in single well so that the confidence to detect infection could be increased to obtain more conclusive results in a single testing. During SE1127, we have developed and validated 2B and 3B peptide tests and 4 other recombinant non-structural protein tests (3ABC, 3CD, 3D, 2C). Receiver Operator Characteristics (ROC) and Bayesian analysis revealed a good sensitivity, specificity and area under curve (AUC) for these tests except for the 2C NSP test. Three tests (2B, 3B & 3ABC) were found with comparable sensitivity & specificity to PrioCHECK® FMDV NS (relative performance is summarised in Table 2, below).

Table 2: Individual test sensitivity for the NSP panel samples and field outbreak samples in comparison with Prionics test sensitivity and specificity.

Test N AUC [95% CI] p Se (%) Sp (%) LR+ LR-

PanelPrionics 1027 0.995 [0.990 - 1.000] Ref 91.67 99.39 151.40 0.08

2B 1027 0.996 [0.992 - 0.999] 1.00 91.67 99.19 113.552 0.083B 1027 0.962 [0.917 - 1.000] 0.807 88.89 98.39 55.05 0.11

3ABC 1027 0.964 [0.925 - 1.000] 0.673 75.00 99.19 92.90 0.253D 1027 0.913 [0.846 - 0.980] 0.09 72.20 97.20 61.17 0.45

3CD 1027 0.940 [0.897 - 0.982] 0.051 72.20 98.58 66.85 0.532C 1027 0.780 [0.704 - 0.856] 0.000 63.80 76.69 2.97 0.40

Field outbreak sera

Prionics 224 0.994 [0.986 - 0.000] Ref 96.86 96.92 31.47 0.0322B 224 0.988 [0.976 - 1.000] 1 96.23 98.46 62.5472 0.03833B 224 0.987 [0.974 - 0.999] 1 97.48 93.85 15.8412 0.0268

3ABC 224 0.985 [0.971 - 0.999] 0.735 96.23 95.38 20.849 0.03963D 224 0.875 [0.830 - 0.920] 0 75.47 95.38 18.1918 0.4542

3CD 224 0.848 [0.797 - 0.898] 0 71.70 98.46 57.14 0.6032C 224 0.764 [0.692 - 0.835] 0 82.39 60.00 2.0597 0.2935

Further studies showed that the sensitivity & specificity of the detection of antibodies against FMD virus are increased by combining these 3 tests (2B, 3B & 3ABC) in parallel and serial testing with the PrioCHECK® 3ABC NSP test. By combining these individual tests with the PrioCHECK® 3ABC NSP in parallel, an increase of sensitivity up to 99% was achieved, whereas in serial testing the specificity was estimated to be 99.99% (Figure 8). Therefore the 2B, 3ABC and 3B NSP tests could be used as confirmatory tests in conjunction with Prionics 3ABC test to detect FMDV infection in vaccinated populations with more confidence.


Figure 8. Bayesian analysis of in-house 3ABC, 2B and 3B confirmatory tests in parallel and serial testing with Prionics test

Furthermore, during SE1127 we have validated a new 3ABC competitive test from IDVet®, France and compared the performance of this test with Prionics 3ABC test. The new test has two formats. One is with overnight incubation and the other one is two hours incubation after loading of serum samples. Both the formats have equivalent specificity and sensitivity with the Prionics test. Further we used our in-house test (described above) as confirmatory tests to this new IDVet test and found equal or slightly more sensitivity and specificity than the combination of Prionics tests and our in-house test (Table 3).

Table 3: - NSP tests diagnostic parameters estimated for the detection of infection in bovine serum panel. N = number of positive and negative samples tested; AUC: Area under curve; p value denotes the significance level for difference of AUC between the ID Vet tests (reference) and in-house tests; κ = kappa statistic; Agreement (%) = percentage of agreement between in-house tests and ID Vet tests.

2.2 Development of next-generation SP ELISAs

Prototype solid phase ELISAs kits have been evaluated for O, A Asia 1 and SAT 2 and are ready for commercialisation upon a commercial agreement with IZSLER. Cross validation was performed against the gold standard VNT. Prototype kits for O, A and Asia 1 were evaluated for sensitivity and specificity which was well correlated with the gold standard VNT. There are a number of outstanding validation actions to be performed upon receipt of kits from IZSLER. This includes evaluation of cross serotype reactivity. The serotype O kit suffers from relatively low sensitivity probably due to an inherently lower specificity necessitating a higher cut-off. Furthermore, the SAT 1 prototype kit suffers from coating stability problems and IZSLER have been establishing the cause, which is thought to be antigen stability issue. Once the prototype kits are available these will be provided to TPI and evaluated.

The SAT 2 prototype kit has been evaluated using 630 sera from field samples from Tanzania and other third parties. Feedback received from IZSLER has indicated the kit is performing well and can now be produced commercially. In the small numbers tested against the gold standard VNT, a sensitivity of a


Test N AUC [95% CI] p Se (%) Sp (%) κ (L/S) Agreement (%)

ID Vet L/S 1027 0.99/0.99 Ref 91.67/91.67 99.29/99.50 Ref Ref2B 1027 0.99 [0.99-0.99] 1.00/1.00 91.67 99.10 0.76/0.76 98.25/98.783B 1027 0.96 [0.91-1.00] 0.88/0.89 88.89 98.39 0.71/0.71 97.76/97.983ABC 1027 0.96 [0.92-1.00] 0.68/0.78 75.00 99.09 0.72/0.70 98.15/97.993D 1027 0.91 [0.84-0.98] 0.09/0.08 72.20 97.17 0.53/0.52 96.10/95.913CD 1027 0.94 [0.89-0.98] 0.06/0.08 72.20 98.59 0.60/0.61 97.17/97.192C 1027 0.78 [0.70-0.85] 0.00/0.00 63.80 76.69 0.08/0.09 75.73/75.78

100% and specificity of 86% was observed.

We are now in the position to evaluate these kits in relation to post-vaccination monitoring. Furthermore, once VLPs are readily available they will be evaluated when the prototype kits are received from IZSLER.

The existing in-house SP ELISAs are based on semi-purified whole virus antigen and not highly sensitive to detect antibodies against African viruses. Further SP ELISAs cross react between the serotypes and as seen in IgA tests using capsid antigen the assay may be specific for specific antigen. Therefore during SE1127, new FMDV solid phase (SP) ELISAs have been developed for O, A, SAT1 and SAT2 using sucrose gradient purified natural viral capsids of African origin. These ELISAs have comparable performance to the Brescia or Pirbright SP ELISAs. Furthermore, O, A and SAT2 SP ELISAs from Prionics and O serotype SP ELISA from ID Vet have been validated using samples from field outbreaks and negative sera obtained Italy. All the ELISAs have good sensitivity and specificity. However a cross reactivity has been seen between these SP ELISAs where as a reduced cross reactivity has been seen in in-house SP ELISAs where capsid had been used as antigen.

2.3 Development of a multiplex antibody assay using different recombinant NSPs and peptides

A multiplex assay using 6 NSPs (2B, 3B, 3AB, 3ABC, 3D & 3CD) has been developed in SE1127. Out of these 6 proteins/peptides 2B, 3D and 3CD were recorded as outstanding and by joining 2B and 3CD protein tests in multiplex assay the best sensitivity and specificity was achieved and were found higher than or similar to the sensitivity and specificity of the PrioCHECK® FMDV NS test. A small validation has been carried out with NSP bovine serum panel and some outbreak field samples (Table 4). These test further needs to be validated in larger scale in cattle, sheep and goats and pig sera including the serum sample from vaccinated animals. The 2B, 3D and 3CD tests performed well to detect infection in the bovine serum panel. The 2B test showed 80.56% sensitivity whereas both the 3D and 3CD tests showed 86.11% sensitivity (Table 4).

Table 4. Detection of infection in bovine serum panel and field samples by Multiplex assay

2.4 Development and evaluation of FMDV-specific IgA antibody ELISAs

We have shown that whereas DIVA serology using non-structural protein ELISAs is a sensitive measure of previous infection, the IgA test is particularly relevant for detection of carrier animals. This is a significant breakthrough, since high sensitivity/specificity detection of residual carrier cattle is still perceived as a major stumbling block to the use of vaccination-to-live policies for FMD control. The option for detection of carrier animal by PCR of probang samples is not easy or economical to perform in extremely large sets of that might be received from animals during post-outbreak surveillance activities. Till date we have developed and validated IgA assays for serotypes O, A, Asia and SAT2 that could detect the FMD carrier cattle in an equivalent manner to animals detected by virus isolation and RT-PCR.Earlier work had focused on serotype O, but we have now carried out extensive validation for serotype A using saliva and/or nasal samples from vaccinated and subsequently infected cattle that had been


Test N AUC Se (%) Sp (%) LR+ LR-Bovine serum panel

2B 336 0.99 80.56 99.33 120.8 0.23B 336 0.98 69.44 98.00 34.7 0.3

3ABC-GST 336 0.88 44.44 97.00 14.8 0.63D 336 0.99 86.11 98.00 51.7 0.1

3CD 336 1.00 86.11 99.33 129.2 0.13AB-His 336 0.97 66.67 97.33 28.6 0.3

Field2B 459 0.99 86.79 99.33 260.4 0.13B 459 0.98 78.62 98.00 39.3 0.2

3ABC-GST 459 0.94 65.41 97.00 21.8 0.43D 459 1.00 98.11 98.00 49.1 0.0

3CD 459 1.00 97.48 99.33 146.2 0.03AB-His 459 0.98 79.25 97.33 34.0 0.2

collected together from various archives. These samples originated from 7 homologous and 5 heterologous vaccine potency tests of A serotype viruses carried out at the laboratories of CVI, Lelystad, The Netherlands and FLI, Germany. Saliva/nasal fluids from 581 naïve cattle were tested in the IgA test to check the assay’s specificity. 250 saliva samples from the field originated from cattle known to have been vaccinated with serotype A. As with the O serotype test, the A serotype IgA test detected FMD virus carriers amongst FMDV infected cattle. ROC analysis revealed that the IgA assay has good area under curve (AUC), specificity (Sp) and sensitivity (Se) (Figure 9).More recently, two further IgA tests have been developed for serotype SAT2 and Asia1 of FMDV and evaluated using saliva/nasal samples. An inactivated sucrose gradient antigen for these tests has been prepared from a field virus that has caused recent FMD outbreaks in Tanzania. Several hundred saliva/nasal samples have been tested in this assay and the results have been compared to the NSP antibody assay: the results showed similar performance as previously shown for the detection of O and A serotypes. Similarly the saliva and nasal samples have been tested from a potency test of Asia1 and an IgA ELISA has been developed which needs further validation in large scale

Data No AUC[95%CI] SEOverall 1149 0.934[95%CI] 0.008Carrier 981 0.971[0.958-0.980] 0.006Non carrier 749 0.848[0.820-0.873] 0.020

Figure 9. ROC analysis for the new IgA ELISA for serotype A evaluated for carrier and non-carrier animals

2.5 FMDV-specific IgA ELISAs: replacement of antigens with recombinant capsids

We have substituted inactivated FMDV antigen in the IgA test with a recombinant O serotype FMDV capsid. In the serotype O IgA test, replacing the homologous inactivated antigen with purified empty capsid increased the sensitivity of the test. Out of 32 known carrier animals, this capsid-based IgA ELISA could detect 31 carrier animals whereas using unpurified inactivated antigen we could only detect 29 carrier cattle. Similarly, we have also replaced the inactivated antigen with recombinant A22 FMD empty capsid in the serotype A IgA assay and using saliva and nasal samples from experimental animals all the known carrier animals (detected by virus isolation and RT-PCR) were detected by this new test (Figure 10). Therefore, as a proof of principle we have shown that replacing inactivated antigen with non-infectious viral empty capsid, we could generate kit reagents outside of high containment laboratory. Further analysis showed that all the 14 carriers were detected by both inactivated antigen and capsid antigen in IgA ELISA for serotype A. However it has been seen that A22 capsid is very specific to detect carrier only in A22 infected animals where as it failed to detect many carriers for other A subtypes although all the carriers were detected by using A22 inactivated antigen as shown in the below figure. This suggests that using the specific capsid antigen, SP ELISA could be developed with limited cross-reaction between serotypes. Furthermore, we have recently expressed Asia1 empty capsid in Sendai virus and using this empty capsid we were successful to develop the non-infectious ELISA for Asia1 serotype.


Figure 10: Substitution of capsid antigen with inactivated whole virus antigen for developing A22 IgA ELISA

However, it was also shown that the A22 capsid is very specific to detect carrier only in A22 infected animals where as it failed to detect many carriers for other A subtypes although all the carriers were detected by using A22 inactivated antigen (Figure 11). This suggests that using the specific capsid antigen, SP ELISA could be developed with limited cross reaction between serotypes.

_x0015_A Malyasia 97

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Figure 11: Substitution of capsid antigen with inactivated whole virus antigen for developing serotype A IgA ELISAs

We have also expressed Asia1 empty capsid in Sendai virus and using this empty capsid we were successful to develop the non-infectious ELISA for Asia1 serotype. Currently this work has been extended to determine the specificity and sensitivity of the assay.

Theme 3: Field tools for FMD diagnosis

3.1 Mobile PCR: Evaluation of “dry-down” reagents for the Enigma FL

Currently, clinical samples from suspect cases are normally tested at centralised reference laboratories (such as the Pirbright Institute): a lengthy process that can delay critical decision-making. Mobile RT-PCR and Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) provide realistic option for


rapid, sensitive, in situ detection of FMDV as simple sample preparation, amplification and detection methods can be utilised. A FMDV-specific rRT-PCR has been previously evaluated using mobile (field ready) equipment. However, the development of robust ‘ready-to-use kits’ that utilise reagents compatible with environmental conditions is still required. During SE1127, we have continued to work with Enigma Diagnostics (Porton-Down, UK) to develop a lyophilised FMDV rRT-PCR that can be deployed for field-testing. Initial laboratory validation of these stabilized reagents was performed on a decimal dilution series (10-1 to 10-10) of O1Manisa FMDV spiked in negative bovine epithelium. This dilution series was then run in parallel using the gold standard RNA extraction (MagNA pure) and rRT-PCR and compared to the complete assay run on the Enigma FL (Enigma). In comparison to the routine test using in the FMD Reference Laboratory, the analytical sensitivity was one log10 less when the complete protocol was run on the Enigma FL and attempts to increase the analytical sensitivity by increasing the enzyme concentration and reducing the elution volume did not improve the performance of the test. A small panel of recent epithelial field samples from the WRLFMD archive were analysed on the Enigma FL and compared to the gold standard methods of extraction and rRT-PCR. Complete concordance between the Enigma FL and the gold standard method was observed within this sample set (Figure 12).

Isolate Serotype Sample Enigma FL Report Enigma FL CT Gold standard

TAN 19/2012 SAT2 EPITHELIUM NEGATIVE No CT No CT

TAN 25/2012 SAT1 EPITHELIUM POSITIVE 24 14.45

TAN 22/2012 SAT1 EPITHELIUM POSITIVE 22 14.30

TAN 50/2012 SAT1 FOOT EPITHELIUM NEGATIVE No CT No CT

TAN 39/2012 O EPITHELIUM NEGATIVE No CT No CT

TAN 54/2012 FMDGD FOOT EPITHELIUM POSITIVE 30 26.27

TAN 58/2012 FMDGD FOOT EPITHELIUM NEGATIVE No CT No CT

TAN 60/2012 A MOUTH EPITHELIUM POSITIVE 19 38.83

Figure 12. Comparative performance of the Enigma FL compared to the gold standard rRT-PCR methods: Grey indicates positive samples and the serotype is shown [FMDGD represents specimens that were originally positive by rRT-PCR but negative using other diagnostic methods: for one of these sample repeated testing in this study generated negative results].

Field-testing was then performed in Kenya and Tanzania (Figure 13), to confirm whether the assay and equipment were robust and suitable for routine use in harsh environmental conditions (travel funded by FAO and Wellcome Trust projects). This study accessed clinical samples (blood, epithelium and probang [OP-fluids]) collected from 13 cattle in FMD endemic settings.

Figure 13. Performance of the FMDV-specific mobile rRT-PCR (Enigma FL) in East Africa (Kenya and Tanzania).

These results showed that the dry-down (lyophilised) RT-PCR assay on the Enigma-FL was able to detect FMDV in a range of different sample types from animals at different stages of clinical disease including in probang (OP-fluids) in apparently clinically normal animals.

3.2 Development of a multiplex RT-LAMP assay for the detection of viruses causing vesicular diseases in livestock


Because of the clinical similarity of FMD to other vesicular diseases such as SVD and Vesicular Stomatitis (VS) there is a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. Singleplex reverse transcription loop mediated isothermal amplification assays (RT-LAMP) combined with lateral-flow visualisation (RT-LAMP-LFD) have been previously developed for vesicular diseases such as FMD and SVD but there are currently no available multiplex RT-LAMP-LFD assays which would permit rapid discrimination between these ‘look-a-like’ diseases in situ. The objectives of this study were i) to develop two multiplex RT-LAMP assays combined with molecular lateral-flow detection RT-LAMP-LFD); one to detect FMDV and/or SVDV, ii) to evaluate these assays against the equivalent rRT-PCR assays and iii) to develop simple sample preparation methods compatible with field use. The limit of detection of both multiplex assays was demonstrated to be equivalent to that of the equivalent laboratory based rRT-PCR assays when visualised using fluorescence (measured using a real-time PCR machine, Stratagene) or a molecular LFD (Forsite Diagnostics, York) (Figure 14).

Figure 14. Performance of novel multiplex RT-LAMP assays for FMDV, VSV and SVD when analysed against the equivalent gold standard rRT-PCR assay. Data for the VSV RT-LAMP was provided from a project funded by Institute for Infecious Animal Diseases (USA).

Importantly, this study demonstrated that FMDV, SVDV and VSV RNA could be reliably distinguished from a range of epithelial suspensions without the need for prior RNA extraction (Figure 15). Our results outline an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMDV from other vesicular disease viruses in the field.


Figure 15. Performance of multiplex RT-LAMP when assayed directly on clinical samples (no RNA extraction). The upper panels of this figure outline the simple steps (identification of lesions - tissue collection – homogenisation of tissues using the Svanova preparation kit – dilution) that can be used to prepare of epithelial tissue samples for analysis by RT-LAMP. Results shown in the lower panels represent those generated by RT-LAMP (using fluorescence and LFD – described above) and laboratory-based rRT-PCR. Serotype/or isolate designation of individual samples is shown.

3.3 Development of a LFD based on the pan-serotypic detection of NSPs

FMDV 3D protein has been expressed in E-coli under a previous Defra-funded project (SE1125) and sent to a collaborator in India to produce specific monoclonal antibodies (Mabs). Six new Mabs against FMDV 3D protein were obtained from Indian Immunologicals and have been used in an ELISA system to test their binding capacity. However, none of these Mabs have proven suitable in an LFD format and we have not yet been successful in the objective of this project to find a specific Mab that is suitable for the purpose of using in a LFD for the detection of FMDV NSPs.

3.4 Evaluation of type-specific LFDs for the detection of FMDV

No work has been undertaken during SE1127 to address this objective.


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.Publications and articles arising from this work:

Peer-reviewed papers Yamazaki W., Mioulet V., Murray L., Madi M., Haga T., Misawa N., Horii Y. and King D. P. (2013)

Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus. Journal of Virological Methods 192: 18-24

Reid S. M., Mioulet V., Knowles N. J., Shirazi N. Belsham G. J. and King D. P. (2014) Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A, Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East. Journal of Virological Methods 207: 146-153

Waters R. A., Fowler V. L., Armson B., Nelson N., Gloster J., Paton D. J. and King D. P. (2014) Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection. PLoS ONE 9 (8): e105630.

Fowler V. L., Bankowski B. M., Armson B., Di Nardo A., Valdazo-González B., Reid S. M., Barnett P. V., Wadsworth J., Ferris N. P., Mioulet V. and King D. P. (2014) Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices. PLoS ONE 9 (10): e109322.

Madi M., Mioulet V., King D. P., Montague N., and Lomonossoff G. Development of a non-infectious encapsidated positive control RNA for molecular assays to detect foot-and-mouth disease virus. Journal of Virological Methods (IN PRESS)

Review article Paton D. J. and King D. P. (2013) Diagnosis of Foot-and-mouth disease. Developments in

Biologicals (Basel) 135: 117-123.

Invited talks and keynote presentations King D. P. Progress towards the deployment of simple and rapid diagnostic tests away from

centralised laboratories. OIE-Meeting at the 16th International Symposium of the World Association of Veterinary Laboratory Diagnosticians, Berlin, Germany, June 2013.

Invited talk: New tools to detect and monitor the spread of viral diseases of livestock. 1 st European Animal and Plant Symposium, Amsterdam, The Netherlands, February 2014

Invited Talk: King D. P., Armson B., Mioulet V., Madi M. and Fowler V. Simple field tools for the diagnosis of livestock diseases: is this an achievable goal? 9th Conference of Rapid Methods Europe, Noordwijkerhout, The Netherlands, April 2014.

Invited Talk: King D. P. Performing diagnostic assay field trials in an international setting. Workshop presentation: Defining requirements for international trials for conventional and next-generation foot-and-mouth disease virus vaccines and diagnostics. Washington DC, June 2014.

Invited Talk: King D. P. New diagnostic methods. The Pirbright Institute Centenary Day, Guildford, September 2014.

Other presentations at other national and international conferences (oral and poster) Madi M., Montague N., Mioulet V., Lomonossoff G. P. and King D. P. Development of a non-

infectious encapsidated positive control RNA for molecular diagnosis of foot-and-mouth disease.16th International Symposium of the World Association of Veterinary Laboratory Diagnosticians, Berlin, Germany, June 2013.

Waters R., Nelson N., Gloster J., Yamazaki W., Murray L., Paton D. J., Fowler V., Cauisi C. and King D. P. Evaluation of a simple assay format for the detection of foot-and-mouth disease virus using reverse transcription loop mediated isothermal amplification. 7th Annual Meeting of the EPIZONE project, Brussels, October 2013

Burman A., Mioulet V., Shimmon G., Tuthill T., King D. P. and Jackson T. Development of an improved antigen-detection ELISA for the diagnosis of foot-and-mouth disease using recombinant integrin (alpha-v/beta-6) 18th International Picornavirus meeting (EUROPIC), Blankenberge, Belgium, March 2014.

Fowler V. L., Caiusi C., Howson E. L. A., Mioulet V., Madi M. and King D. P. Development and evaluation of multiplex reverse transcription loop mediated isothermal amplification assays combined with lateral-flow visualisation for the discrimination of foot-and-mouth disease from other vesicular diseases. Open Session of the Research Group of the Standing Technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Cavtat, Croatia, October


2014. Howson E., Cleaveland S., Armson B., Mioulet V., King D. P., Kasanga C. J., Sallu R., Clark D.,

Millington S. and Fowler V. L. Realising the potential of simple isothermal molecular tools for field diagnosis of foot-and-mouth disease. Open Session of the Research Group of the Standing Technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Cavtat, Croatia, October 2014.

Armson B., Mioulet V., Doel C., Madi M., Bounpheng M., Lemire K., Das A., Holder D., McIntosh M., Parida S. and King D. P. Real-time RT-PCR for the rapid detection of FMDV in milk. Open Session of the Research Group of the Standing Technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Cavtat, Croatia, October 2014.

Ludi A. B., Li Y., Wilsden G., Mioulet V., Armson B., Adams K., Ryder T., Belgrave S., Hammond J. and King D. P. Results of the 2013 proficiency testing scheme. Open Session of the Research Group of the Standing Technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Cavtat, Croatia, October 2014.


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