General enquiries on this form should be made to:Defra, Science Directorate, Management Support and Finance Team,Telephone No. 020 7238 1612E-mail: research.competitions@defra.gsi.gov.uk

SID 5 Research Project Final Report

SID 5 (2/05) Page 1 of 17

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.

A SID 5A form must be completed where a project is paid on a monthly basis or against quarterly invoices. No SID 5A is required where payments are made at milestone points. When a SID 5A is required, no SID 5 form will be accepted without the accompanying SID 5A.

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 SE 1119

2. Project title

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

3. Contractororganisation(s)

Dr N P FerrisInstitute for Animal Health, Pirbright LaboratoryAsh RoadPirbrightSurreyGU24 0NF

54. Total Defra project costs £ 541,957

5. Project: start date................ 01 July 2002

end date................. 30 June 2005

SID 5 (2/05) Page 2 of 17

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.

SID 5 (2/05) Page 3 of 17

An automated real-time (r) reverse transcription polymerase chain reaction (RT-PCR) procedure has been evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, blood, milk and oesophageal-pharyngeal samples from the 2001 UK FMD outbreak and from experimentally infected animals. A Roche MagNA Pure LC robot was programmed to automate the nucleic acid extraction from samples and for automation of the liquid pipetting of samples and reagents for the subsequent RT and PCR amplification steps. The diagnostic results were in broad agreement with conventional virus isolation (VI) in cell culture and showed that the RT-PCR was at least equivalent to VI and was more sensitive than antigen ELISA on epithelial suspensions. An approach of using either ELISA plus RT-PCR combined or else RT-PCR alone could be used as the laboratory diagnostic tool(s) and would considerably advance the issue of laboratory diagnostic test result.

The performance of the automated rRT-PCR was further compared to VI and antigen ELISA for FMD diagnosis on a collection of epithelial samples which had been submitted to the WRL for FMD from 18 countries over an 18 month period. The results showed that all VI positive plus VI and ELISA positive samples combined were also positive by RT-PCR. FMD virus genome was detected in a minimum of an additional 18% of samples. Furthermore, the RT-PCR generated definitive diagnostic results within a day in contrast to up to 4 days to define some positive and all negative samples by VI. The study demonstrated that the rRT-PCR provides an extremely sensitive and rapid procedure for improved laboratory diagnosis of FMD.

Further evaluations of the rRT-PCR for FMD diagnosis were undertaken by retrospective analysis of two sample subsets from confirmed cases arising from the 2001 UK outbreak. Firstly, epithelia which were negative by both ELISA and VI and secondly, others which were negative by ELISA on epithelial suspension but positive by VI. There was broad agreement between RT-PCR and VI/ELISA but with the caveat that that the routine RT-PCR procedure did not detect a group of related virus isolates from Wales. These viruses had evidently evolved during the outbreak and had a nucleotide substitution in the RT-PCR probe site that precluded their detection by the routine diagnostic probe but which could be detected by a new probe designed to better fit the nucleotide sequence of the UK causative virus. The results demonstrated the extreme sensitivity and rapidity of the rRT-PCR for FMD diagnosis but illustrated the need for constant monitoring of representative field virus strains by nucleotide sequencing to ensure that A comparison of Pirbright’s rRT-PCR for FMD and another developed at the Plum Island Animal Disease Center, USA, that targets a different conserved region of the FMD virus genome, showed that there was concordance for 90% of clinical samples which were examined from the WRL for FMD sample collection covering a 40 year submission period. The comparison showed a number of samples failing to produce a signal in one assay while giving a positive signal in the other. Nucleotide sequencing of selected isolates highlighted nucleotide substitutions in the primer and/or probe regions to suggest an explanation for false-negative results. The results provided further confirmation of the need for the continuous monitoring of circulating field virus strains to ensure the adequacy of primer/probe design and suggested that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMD virus.

The FMD rRT-PCR procedure has been further validated on sample types of milk and oesophageal-pharyngeal (probang) samples. It correlated closely with VI in detecting FMD virus in all milk components from experimentally infected cattle and detected virus in milk for longer post inoculation than VI. The detection limit was greater by RT-PCR than VI and, in contrast to VI, detected virus genome following heat treatment that simulated pasteurisation. RT-PCR was also able to detect FMD virus in preservative milk. The evaluation showed that automated rRT-PCR is quicker and more sensitive than VI and can be used to detect FMD virus in whole milk as well as milk fractions from infected animals. Optimised rRT-PCR was more sensitive than VI in detecting FMD virus persistence through examination of probang samples collected from cattle in 6 herds during a field study in Zimbabwe. Five of these herds had reported outbreaks of FMD, 1 to 5 months previous to sampling and laboratory results showed that infection had been due to virus of type SAT 2 in 2 herds, while SAT 1 was responsible for infection in 3 others.

A prototype proficiency panel of samples has been prepared by the WRL for FMD and distributed to 4 other FMD laboratories within the EU to participate in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. The specificity and sensitivity of the RT-PCR tests was generally high while the sensitivity of cell cultures was variable from high in one laboratory, moderate in two and low in two others. The prototype panel of samples would appear suitable as a basis for finalising a panel for provision to other FMD laboratories that wish to evaluate their procedures for FMD diagnosis, with the inclusion of more truly negative samples and an extension in the serial dilution range of one or more of the included FMD positive sample titration series.

rRT-PCR procedures have been developed and/or evaluated for the other vesicular viruses which produce clinically identical disease to FMD (and thus allow a differential diagnosis), namely swine vesicular disease

SID 5 (2/05) Page 4 of 17

(SVD), vesicular stomatitis (VS) and those of the Vesivirus group of the Caliciviridae. Two independent sets of primers and probe were designed from the 5’ untranslated region of the SVD virus genome and although both sets failed to detect one virus isolate (different isolate for each set), the assays successfully detected virus preparations from all phylogenetic groups of SVD virus. The assays were specific and provide sensitive and rapid alternatives to ELISA and VI for SVD diagnosis.

An automated rRT-PCR was next evaluated for SVD diagnosis on a range of samples from 4 inoculated and 3 in-contact pigs in comparison with VI and ELISA. The inoculated pigs developed a significant viraemia and clinical disease and transmitted disease to the in-contact animals. The latter, however, developed only a short-lived, low-level viraemia and no clinical disease. RT-PCR and VI were comparable in detecting SVD virus in serum and nasal swabs up to 6 days after infection and, although virus was isolated from the faeces of a few pigs for a longer period, indicated that the rRT-PCR is a useful method for SVD diagnosis in clinically or subclinically affected pigs.

RT-PCR and sequencing analysis has demonstrated high nucleotide identities within the IRES between 33 representative SVD virus isolates and support the choice of this region as a diagnostic target and provide information for the improvement of laboratory-based molecular assays for detection. In contrast to the relative conservation of the IRES element, there was considerable nucleotide variability in the spacer region located between the cryptic AUG at the 3’ end of the IRES and the initiation codon of the polyprotein. Interestingly, 11 SVDV isolates had block deletions of between 6 and 125 nucleotides in this region. Nine of these isolates were of recent European origin and were phylogenetically closely related. In vitro growth studies showed that selected isolates with these deletions had a significantly reduced plaque diameter and grew to a significantly lower titer relative to an isolate with a full-length 5’UTR. Further work is required to define the significance of these deletions and to assess whether they impact on the pathogenesis of SVD.

Internal mimics (artificial RNA constructs) have been developed to act as controls to validate negative results for both the FMD and SVD TaqMan assays. They have been optimized for use in the diagnostic assays using two-colour probes and have been shown to perform satisfactory in trial assays.

Sequences of primers and probes for VSV diagnosis were obtained from Plum Island and a duplex rRT-PCR assay was set up to be compatible with the FMD and SVD rRT-PCR procedures. The assay was less sensitive than a hemi-nested (hm) PCR developed at Pirbright some years ago for detection of the VSV New Jersey serotype while the reverse was true for detection of VSV Indiana 1. However, both assays were less sensitive than VI. The rRT-PCR failed to detect viruses of subtypes 2 and 3 of the Indiana serotype while some isolates of the former subtype were detected by the hmRT-PCR but not the latter. Although Indiana viruses of these two subtypes are seldom implicated in vesicular disease outbreaks of VSV, additional primer/probes need to be designed for comprehensive coverage for VS diagnosis.

Sequence data covering the polymerase-capsid region was generated from Vesivirus San Miguel sea-lion virus serotypes 2, 4, 10, 13, vesicular exanthema virus serotypes B51, G55, J56 and an Oklahoma virus isolate. These sequences were aligned with existing Vesivirus sequences to design primers/probe sets from conserved regions. Two sets of fluorogenic primers/probe sets were designed for use in an rRT-PCR. The results of preliminary evaluations show that the assays are sensitive and specific and that viruses of all Vesivirus serotypes are detected by the two primers/probe sets except those of SMSV-8 and SMSV-12. These virus serotypes had previously been undetected by conventional RT-PCR and may suggest that these caliciviruses belong to a different group.

Agreements should soon be in place with two commercial manufacturers to test their mobile PCR equipment designed for on-site FMD diagnosis.

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);

SID 5 (2/05) Page 5 of 17

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 relevance

Vesicular virus diseases are a major threat to the livestock industry of the UK. Among them is foot-and-mouth disease (FMD), a highly contagious disease of cloven-hoofed animals. World-wide it is the most important constraint to trade in livestock and animal products. Other viruses produce vesicular diseases which are indistinguishable from FMD, necessitating laboratory investigation for a definitive diagnosis. Differential diagnosis is required from vesicular stomatitis (VS), swine vesicular disease (SVD) and a group of viruses commonly referred to as marine caliciviruses which include vesicular exanthema of swine and San Miguel sea lion virus within the Vesivirus group. The risk of entry of exotic viruses into the UK has increased in recent years through increased movements of animals and their products from central to western Europe, increased movement between countries within the EU, expanding illegal trade and increased travel which has enhanced the chances of importing virus in products from endemic regions. Livestock in the UK are not vaccinated against these viruses so rapid spread is to be expected should virus gain entry and cases not be quickly identified. Thus, effective control and eradication is dependent upon early reporting of disease and rapid and accurate diagnosis in the laboratory. Definitive diagnosis requires detection of virus or antigen in vesicular epithelium (the preferred specimen). Serology is not favoured for the primary diagnosis of FMD but is essential for the diagnosis of SVD when virus strains are not virulent. Diagnostic procedures must be continuously assessed and refined to ensure their optimal performance in the face of the evolution of pathogens and to incorporate technical developments in related fields of virology.

The Pirbright Laboratory houses the OIE/FAO World Reference Laboratory for FMD (WRL) which maintains a surveillance service for FMD strains worldwide by providing a diagnostic and serotyping referral service for member countries. Annually the WRL typically receives 600-700 samples from 25-30 different countries; however, over 16,500 samples were received by the WRL during the course of the 2001 FMD outbreak in the UK and 300 from Ireland.

The assays used for FMD diagnosis at the time of the UK 2001 outbreak were the best then available. Approximately 90 percent of positive samples during the UK 2001 FMD outbreak were confirmed within 3-4 hours of laboratory receipt. However, the remaining 10 percent of positive samples required amplification of the virus through cell culture passage; a situation which, in extreme cases, can take up to 4 days and which can exacerbate disease control measures. Ideally, laboratory investigations should therefore be made more sensitive to allow for the more rapid issue of conclusive results to speed the investigation of suspected cases of FMD and other vesicular diseases. One approach is through the development of RT-PCR procedures which hold high promise for application to FMD and vesicular virus diagnosis.

Whereas the ELISA is most suited to detecting FMD virus in epithelial samples, virus isolation and RT-PCR can be used on a wide range of sample types including blood, probangs and milk. During the recent FMD outbreak in the UK, there was much interest in the possibility of screening milk for FMD virus, since this can be used for preclinical diagnosis and since tests on bulk tank milk could be used to screen entire herds. From what is known on the amounts of FMD virus shed in milk and of the potential sensitivity of RT-PCR methods, RT-PCR should be suitable for screening bulk milk samples for FMD virus, but optimal procedures need to be developed and the feasibility of the approach needs to be verified.

Immunocapture RT-PCR could enable RNA extraction to be simplified, which is an important constraint to use of the RT-PCR in conjunction with portable PCR equipment, outside of specialist laboratories. Another variant of this approach would be to utilise chromatographic strip tests as virus particle capturing devices to enable a simplified form of RNA extraction from the concentrated virus particles in the result line of the strip test. Before all such methods can replace virus isolation in cell culture they must be fully validated. Internal controls (mimics) would be useful to ensure the reliability of negative RT-PCR test results.

Currently, different regions of the FMD virus genome are targeted by RT-PCR for diagnosis and for molecular epidemiology, since the priority for the former is recognition of all strains wheras for the latter it is inter-isolate discrimination. As more and more virus FMD virus strains are sequenced, an expanding database of FMD virus strain sequences from distinct regions and of diverse molecular characteristics becomes available. This may enable the development of serotype-specific primers for use in both diagnosis and for phylogenetic studies.

Scientific objectives of the project

The project has the following objectives ;

SID 5 (2/05) Page 6 of 17

1. Evaluate TaqMan and SYBR Green RT-PCR methodology in combination with robotic techniques for FMD diagnosis using universal primers

2. Develop ‘mimics’ as controls to validate negative results in the TaqMan RT-PCR for FMD diagnosis3. Evaluate TaqMan RT-PCR methodology for SVD diagnosis4. Validate procedures for the detection of FMD virus in milk and probang samples5. Design and evaluate alternative FMD primers for TaqMan RT-PCR methodology in combination with robotic

techniques to facilitate consequent sequencing of the products6. Design and evaluate type-specific primers for serotype-specific tests for FMD diagnosis7. Analyse test line reactants on chromatographic (pen-side) strip tests by RT-PCR for confirmation of FMD

diagnosis8. Evaluate TaqMan RT-PCR methodology for VSV diagnosis9. Evaluate TaqMan RT-PCR methodology for vesivirus diagnosis10. Evaluate ‘portable’ PCR machines for FMD diagnosis

Objective 1Universal and serotype specific primers have been designed and demonstrated to be specific by conventional RT-PCR for detection of the vesicular viruses and have proved useful supports for diagnosis by the validated procedures of ELISA and isolation of virus in cell culture. They have facilitated rapid molecular analysis of field samples to provide important epidemiological information on the source of outbreaks. Although some samples were found to be positive by RT-PCR but negative by ELISA and virus isolation, early RT-PCR procedures have tended to be slightly less sensitive than virus isolation. Alternative PCR formats have been developed and evaluated in attempts to improve the sensitivity and speed of PCR methodology for FMD diagnosis and serotyping (including PCR-ELISA and immunocapture ELISA. The most promising laboratory based method has been the fluorogenic RT-PCR using amplicon-specific (e.g. TaqMan) or non-specific (e.g. SYBR Green) to provide quantitative results quickly but developments and evaluations of Taqman assays have been pursued in preference to SYBR Green assays as the former (using a specific probe) provides confirmation of the amplified product and this approach is more widely favoured in medical and virological fields elsewhere. Preliminary studies had been carried out during the course of the 2001 UK FMD outbreak on the application of a real-time (r)RT-PCR for FMD. Further exploitation of this methodology combined with the use of automatic procedures for nucleic acid extraction, reverse transcription and presentation for PCR amplification offered the best expectation for increasing the speed and throughput of laboratory investigation of suspected cases of FMD and other vesicular diseases.

An automated fluorogenic rRT-PCR procedure has been evaluated for FMD diagnosis through its ability to detect FMDVs of all serotypes and a series of papers have either been published, are in press or in preparation – abstracts of which follow :

Reid et al., 2003a - AbstractAutomated fluorogenic (5’ nuclease probe-based) reverse transcription polymerase chain reaction (RT-PCR) procedures were evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, heparinised or cltted blood, milk and oesophageal-pharyngeal fluid (‘probang’) samples from the United Kingdom (UK) 2001 epidemic and on sera from animals experimentally infected with the outbreak O FMD virus strain. A MagNA Pure LC was initially programmed to automate the nucleic acid extraction and RT procedures with the PCR amplification carried out manually by fluorogenic assay in a GeneAmp 5700 Sequence Detection System. This allowed 32 samples to be tested by one person in a typical working day or 64 samples by two people within 10-12h. The PCR amplification was later automated and a protocol developed for one person to complete a single test incorporating 96 RT-PCR results within 2 working days or for two people to do the same thing in around 12 h. The RT-PCR results were directly compared with those obtained by the routine diagnostic tests of ELISA and virus isolation in cell culture. The results on blood, probing and milk samples were in broad agreement between the three procedures but specific RT-PCR protocols for such material have to be fully optimised as perhaps have the positive-negative acceptance criteria. However, the automated RT-PCR achieved definitive diagnostic results (positive or negative) on supernatant fluids from first passage inoculated cell cultures and its sensitivity was greater than ELISA on suspensions of vesicular epithelium (ES) and at least equivalent to that of virus isolation in cell culture). The combined tests of ELISA, virus isolation in cell culture and RT-PCR might. Therefore, only be required for confirmation of a first outbreak of FMD in a previously FMD-free country. Should a prolonged outbreak subsequently occur, then ELISA plus RT-PCT or else RT-PCR alone could be used as the laboratory diagnostic tool(s). Either approach would eliminate the requirement for sample passage in cell culture and considerably advance the issue of laboratory test results.

Shaw et al. 2004 - AbstractThe performance of an automated real-time reverse transcription polymerase chain reaction (RT-PCR) was compared to virus isolation (VI) in cell culture and antigen detection enzyme-linked immunosorbent assay (ELISA) for the laboratory diagnosis of foot and mouth disease (FMD). The World Reference Laboratory for FMD in Woking, the United Kingdom, examined a collection of 334 epithelia received from eighteen countries between August 2002 and January 2004. The results showed that all VI positive (n = 195) and VI and ELISA positive

SID 5 (2/05) Page 7 of 17

samples combined (n = 204) were also positive by RT-PCR. Depending on the cut-off used, FMD virus genome was detected in a minimum of an additional sixty samples (18% of all samples tested). Furthermore, the RT-PCR generated results in less than one day from test commencement in contrast to up to 4 days to define some positive and all negative samples by VI. The study demonstrates that real-time RT-PCR provides an extremely sensitive and rapid procedure for improved laboratory diagnosis of FMD.

Ferris et al. in press - AbstractThere were 2030 designated cases of foot-and-mouth disease (FMD) during the course of the FMD epidemic in the UK in 2001 (including 4 from Northern Ireland). Samples from 1720 of the infected premises (IPs) were received in the laboratory and examined for either the presence of FMD virus (virological samples from 1421 IPs) or both FMD virus and antibody (virological and serological samples from 255 IPs) or antibody alone (from 44 IPs). The time taken to issue final diagnostic results ranged from a few hours (in cases where positive results were obtained by ELISA on epithelia containing sufficient virus to be detected) to several days (for samples either containing low amounts of virus, requiring amplification through cell culture passage or were negative or else were tested for antibody). Retrospective analysis by real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on available material of two sample subsets - firstly, epithelia which were negative by both ELISA and virus isolation (VI) in cell culture and secondly, others which were negative by ELISA on epithelial suspension but positive by VI. There was broad agreement between RT-PCR and VI/ELISA combined but with the caveat that the routine RT-PCR procedure did not detect a group of related virus isolates from Wales. These viruses had evidently evolved during the epidemic and had a nucleotide substitution in the RT-PCR probe site, which prevented their detection by RT-PCR using the routine diagnostic probe. The failure to detect these mutant FMD viruses illustrates the importance of constant monitoring of representative field FMD virus strains by nucleotide sequencing to ensure that the primers/probe set selected for the diagnostic RT-PCR is fit for purpose. The RT-PCR was demonstrated to be an extremely sensitive and rapid procedure that can contribute to improved laboratory diagnosis of FMD and influence the adoption of suitable control measures. No evidence of FMD virus, antibody or nucleic acid was found in approximately 23% (390/1730) of IPs from which samples were received lending credence to the view that the incidence of FMD during the outbreak was over-reported.

King et al. in press – AbstractRapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of two independent real-time RT-PCR (rRT-PCR) assays to detect FMDV in clinical samples obtained from suspect FMD cases. There was concordance between the results generated by the two assays for 88.1% (347/394) of RNA samples extracted from archival suspensions of epithelial tissue. The comparison between the two tests highlighted 19 FMDV isolates (13 for the 5’UTR and 6 for the 3D assay) which failed to produce a signal in one assay while giving a positive signal in the other assay. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer and/or probe regions thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV.

SID 5 (2/05) Page 8 of 17

Ferris et al in preparation – AbstractFive laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five sets of 20 coded samples were prepared from 10 vesicular epithelia and comprised 16 samples from 6 FMD virus positive epithelia representing 4 different serotypes (2 type O, 2 type A and 1 each of types Asia 1 and SAT 2), 2 from samples which had been found to be negative by antigen detection ELISA and virus isolation (VI) in cell culture and 2 from swine vesicular disease (SVD) virus positive epithelia. Some of the FMD virus positive samples were prepared from 10-fold serial dilutions of 3 of the initial suspensions – 5 further serial dilutions of type O BHU 39/2004, 2 of type A IRN 5/2003 and 3 of type Asia 1 PAK 20/2003. Each laboratory tested the samples by one or more of its available RT-PCR procedures and inoculated cell cultures that it routinely uses for FMD diagnosis in attempts to isolate virus, the specificity of which was confirmed by antigen ELISA. The specificity and sensitivity of the RT-PCR tests was generally high while the sensitivity of cell cultures was variable from high in one laboratory, moderate in two and low in two others. The prototype panel of samples would appear suitable as a basis for finalising a panel for provision to other FMD laboratories that wish to evaluate their procedures for FMD diagnosis with the inclusion of more truly negative samples and an extension in the serial dilution range of one or more of the FMD positive sample titration series.

Objective 2

The OIE guidelines for the validation and quality control of PCR assays recommends that internal controls are included into the assay to test for substances inhibitory to the enzymatic reaction and to validate negative results. The aim of this objective was to generate artificial templates for SVDV and FMDV real-time RT-PCR assays. When added to the diagnostic real-time RT-PCRs, successful amplification of these RNA transcripts confirms that there are no substances that can inhibit the RT-PCR present, thereby helping to validate negative results generated in the assays.

PCR was used to engineer plasmids (pGEM-T easy, Promega) containing the diagnostic primers used for the FMDV (Reid et al., 2002) or SVDV (Reid et al., 2004) real-time RT-PCRs. Intervening sequence present on the diagnostic target containing the recognition site for the TaqMan® probe was removed and replaced by a fragment of the human -actin gene containing the sequence 5’-CAT GCC ACC CTG CGC CTA GAC CT-3’ recognised by an alternative TaqMan® probe. For each assay, two sets of plasmids which generated amplicons of approximately 100 bp (similar to that of the diagnostic target – see figure 2) or 350 bp were produced. We speculate that RT-PCR amplification of the transcript generated from the larger plasmid (Figure 2) is thermodynamically less favourable, therefore RNA transcripts produced from these plasmids will have less potential to compete with the diagnostic target which could lead to a loss in analytical sensitivity.

pGEM-T easyT7

promoter Sal IpGEM-T easy

T7promoter Sal I

Figure 1: Schematic outline of the plasmid used to generate RNA mimics for FMDV and SVDV real-time RT-PCRs. Black boxes represent diagnostic primer sites and grey box shows region containing the alternative TaqMan® probe site (obtained from a fragment of the human -actin gene). Plasmids were prepared by restriction enzyme digestion (Sal I) prior to in-vitro transcription.

Correct sequence of the individual plasmids was confirmed by DNA sequencing (Beckman CEQ 8000). RNA transcripts were synthesised from the plasmids using in-vitro transcription (Megascript, Ambion) using T7 RNA polymerase (Figures 1 and 2). DNase I treatment was subsequently used to remove contaminating plasmid DNA from the preparation after which the quantity and quality of RNA present was assessed (Agilent bioanalyser).

SID 5 (2/05) Page 9 of 17

TaqMan® probeFOR REV

WT amplicon (>100 bp)

(FAM)

Mimic TaqMan® probe(VIC™)

SHORT MIMIC RNA TRANSCRIPT (~100 bp)

FORT7 REVSal I

Mimic TaqMan® probe(VIC™)

LONG MIMIC RNATRANSCRIPT (~350 bp)

FORT7 REVSal I

A

B C

2% agarose gel showing difference in sizes of mimic amplicons vs WT virus[example shows data for SVDV]

CO

LOU

R 1

FAM

CO

LOU

R 2

HEX

/VIC

A

TaqMan® probeFOR REV

WT amplicon (>100 bp)

(FAM)

Mimic TaqMan® probe(VIC™)

SHORT MIMIC RNA TRANSCRIPT (~100 bp)

FORT7 REVSal I

Mimic TaqMan® probe(VIC™)

LONG MIMIC RNATRANSCRIPT (~350 bp)

FORT7 REVSal I

A

B C

2% agarose gel showing difference in sizes of mimic amplicons vs WT virus[example shows data for SVDV]

CO

LOU

R 1

FAM

CO

LOU

R 2

HEX

/VIC

A

Figure 2: A schematic overview of the artificial TaqMan® mimic RNA constructs for FMDV and SVDV. The TaqMan® probe site ( ) was disrupted in the mimic by the insertion of a fragment of human -actin and was detected by a second probe ( ). An agarose gel gel showing the different size products generated after RT-PCR are shown. The location of PCR primers (FOR and REV) are shown.

The alternative probe (see sequence above) used to recognise the mimic was labelled with either VICTM (Applied-Biosystems) or HEX (Sigma-Genosys) dyes and was detected in a separate channel of the real-time PCR machine (MX4000, Stratagene) from that used to measure fluorescence derived from the diagnostic target (FMDVV or SVDV). Initial experiments using serial dilutions of the RNA transcripts were performed to select an appropriate dilution for use in the real-time RT-PCR (see Figure 3). At the same time, parallel experiments (with and without reverse-transcriptase) were also performed to demonstrate that (at the dilution selected) the product generated is RNA dependent, and not a consequence of carry-over from the plasmid template used for in-vitro transcription (data not shown).

Dilution of RNA

20

2530

3540

4550

No Ct

1.E-10 1.E-08 1.E-06 1.E-04

Ct v

alue

clone 153 -large

Dilution of RNA

20

2530

3540

4550

No Ct

1.E-10 1.E-08 1.E-06 1.E-04

Ct v

alue

clone 153 -large

Figure 3: Dilution series of RNA transcript for FMDV detected by real-time RT-PCR. Results shown are for RNA generated from plasmid clone 261 (FMDV long transcript).

SID 5 (2/05) Page 10 of 17

FAM Channel (MX4000) HEX Channel (MX4000)FAM Channel (MX4000) HEX Channel (MX4000)

Figure 4: Amplification plots showing the use of the artificial mimic in the diagnostic real-time RT-PCR. FMDV is detected in the FAM channel and the mimic target is detected in the HEX channel. Plots represent the same epithelial sample containing mimic (brown line) or without mimic (blue line). At this dilution of mimic (clone 261) there is no inhibition of the FMDV-specific signal in the assay.

These RNA mimics have been optimised for use in the diagnostic assays using two-colour probes without impacting upon the analytical sensitivity of the assay (Figure 4). Occasionally, we have found that the amplification of the mimic target is unsuccessful when the FMDV viral load is high (probably due to less favourable amplification kinetics compared with the diagnostic target). However, since the rationale for adding the mimic is to validate negative results, these failures do not retract from the use of the mimic in the diagnostic assays.

Future work:In summary: the mimics have been generated and optimized for use in the diagnostic assays using two-colour probes and have been shown to perform satisfactory in trial assays. Unfortunately these “naked” RNA transcripts are relatively unstable since they are prone to degradation by RNAses. Furthermore, since a lysis step is not required to release the mimic RNA, these mimics only provide limited validation of the RNA extraction process where viral genome is typically encapsulated by the viral capsid or resides in intra-cellular compartments. During the next follow-on defra project (SE1121) we will use a novel approach to develop an encapsulated RNA control and validate its use in the real-time PCRs for FMDV and other vesicular disease viruses. Similar reagents have recently been developed (using patented technology developed by Ambion based on RNA phages) for inclusion in assays to detect other RNA viruses. This work will be a collaborative project between IAH and the John Innes Centre (Norwich).

Objective 3

The evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus has now been completed and two papers describing the results have been published and another one recently accepted for publication – abstracts of these papers appear below :

Reid et al., 2004 - AbstractDifferential detection of swine vesicular disease virus (SVDV) from the other vesicular disease viruses of foot-and-mouth disease (FMD), vesicular stomatitis (VS) and vesivirus is important as the vesicular lesions produced by these viruses are indistinguishable in pigs. Two independent sets of primers and probe, designed from nucleotide sequences within the 5' untranslated region (UTR) of the SVDV genome, were evaluated in a real time (5' nuclease probe-based or fluorogenic) PCR format. Although both primers/probe sets failed to detect one isolate, the assays successfully amplified RNA extracted from epithelial suspensions (ES) and cell culture grown virus preparations from clinical samples representing all currently designated phylogenetic groups of SVDV. Furthermore, no cross-reactivity was demonstrated when these primer/probe sets were tested with RNA prepared from all seven serotypes of FMD virus (FMDV) and from selected isolates of VS virus (VSV), vesivirus and teschoviruses. These assays provide sensitive and rapid alternatives to supplement the routine procedures of ELISA and virus isolation for SVDV diagnosis. The two independent sets of primers/probe can be used routinely while only one of the primers/probe sets would typically be used in SVDV diagnosis during an outbreak.

Reid et al., 2004 - Abstract

SID 5 (2/05) Page 11 of 17

Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus isolation, and ELISA for antigen and antibody) were used in parallel. Each inoculated pig developed a significant viraemia and clinical disease, and excreted virus, which was transmitted to the in-contact animals. The latter, however, developed only a short-lived, low-level viraemia and no clinical disease. The RT-PCR and virus isolation were generally compatible in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs.

Shaw et al. in press – AbstractSwine vesicular disease virus (SVDV) is a picornavirus closely related to the human pathogen Coxsackievirus B-5. In common with other picornaviruses, the 5’ untranslated region (5’ UTR) of SVDV contains an internal ribosomal entry site (IRES) that plays an important role in cap-independent translation. The aim of this study was to use RT-PCR and sequencing to characterise a fragment of the 5’ UTR encompassing the entire IRES. Sequence analysis demonstrated high nucleotide identities within the IRES between 33 representative SVDV isolates. These data support the choice of this region as a diagnostic target and provide information for the improvement of laboratory-based molecular assays to detect SVDV. In contrast to the relative conservation of the IRES element, there was considerable nucleotide variability in the spacer region located between the cryptic AUG at the 3’ end of the IRES and the initiation codon of the polyprotein. Interestingly, 11 SVDV isolates had block deletions of between 6 and 125 nucleotides in this region. Nine of these isolates were of recent European origin and were phylogenetically closely related. In vitro growth studies showed that selected isolates with these deletions had a significantly reduced plaque diameter and grew to a significantly lower titer relative to an isolate with a full-length 5’UTR. Further work is required to define the significance of these deletions and to assess whether they impact on the pathogenesis of SVD.

Objective 4

Two papers have been accepted for publication which relate to the use of rRT-PCR for the detection of FMD virus in milk and probang fluids. The abstracts of the two papers are reproduced below :

Reid et al. in press - AbstractFoot-and-mouth disease virus (FMDV) can be excreted in milk and thereby spread infection to susceptible animals in other holdings. The feasibility of using real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic tool for detection of FMDV in milk was assessed by studying the excretion of virus from experimentally-infected cattle. Fore- and machine milk samples were collected over a 4-week period from 2 dairy cows infected with FMDV and from 2 in-contact cows held in the same pen. The whole, skim, cream and cellular debris components of the milks were tested by automated rRT-PCR and results compared to virus isolation (VI) in cell culture. The onset of clinical signs of FMD in all 4 cows correlated with viraemia, and the presence of FMDV in other clinical samples. rRT-PCR results matched closely with VI in detecting FMDV in all milk components and generally coincided with, but did not consistently precede, the onset of clinical signs. rRT-PCR detected FMDV in milk up to 23 days post inoculation which was longer than VI. Furthermore, the detection limit of FMDV in milk was greater by rRT-PCR than VI and, in contrast to VI, rRT-PCR detected virus genome following heat treatment that simulated pasteurisation. rRT-PCR was also able to detect FMDV in preservative-treated milk. In conclusion, this study showed that automated rRT-PCR is quicker and more sensitive than VI and can be used to detect FMDV in whole milk as well as milk fractions from infected animals.

Sammin et al. in press – AbstractDuring a field study in Zimbabwe, clinical specimens were collected from 403 cattle in six herds, in which the history of foot-and-mouth disease (FMD) vaccination and infection appeared to be known with some certainty. Five herds had reported outbreaks of disease one to five months previously but clinical FMD had not been observed in the sixth herd. A trivalent vaccine (SAT 1, SAT 2 and SAT 3) had been used in some of the herds at various times either before and/or after the recent outbreaks of FMD. The primary aim of this study was to evaluate the performance of serological tests for the detection of SAT-type FMD virus infection, particularly ELISAs for antibodies to non-structural proteins of FMDV (NSPEs) and solid phase competition ELISAs (SPCEs) for each of serotypes SAT 1 and SAT 2. Secondary aims were to examine NSP seroconversion rates in cattle that had been exposed to infection and to compare virus detection rates by virus isolation and real-time, reverse transcriptase polymerase chain reaction (rtRT-PCR) tests on both oesophago-pharyngeal (OP) fluids and nasopharyngeal (NP) brush swabbings. In addition, the hooves of sampled animals were examined for the presence/absence of growth arrest lines as clinical evidence of FMD convalescence. Laboratory tests provided evidence of FMD virus infection in all six herds. SAT 2 viruses were isolated from OP fluids collected at two outbreak locations in Northern Zimbabwe, whereas SAT 1 viruses were isolated from three FMD-affected herds in

SID 5 (2/05) Page 12 of 17

Southern Zimbabwe. Optimised rtRT-PCR was more sensitive than virus isolation at detecting FMDV persistence and when the results of both methods were combined for OP fluids, between 12 and 35% of the cattle sampled in convalescent herds were deemed to be “carriers”. In contrast, NP swabs yielded only two virus positive specimens. The overall seroprevalence varied with different NSPEs from 48% to 67%, compared to 74% and 82% by homologous SPCE and virus neutralization test respectively. However if serological test results were only considered for those cattle in which persistent infection with FMD virus was demonstrated, 73 to 91% scored seropositive in different NSPEs.

Objective 5

No progress has been made against this objective.

Objective 6

Whilst the real-time RT-PCR method developed for the detection of FMDV has proven to be highly sensitive, it retains the draw-back of not providing any indication as to the serotype of any virus present; a potentially important consideration for the initial diagnosis of an outbreak and for its control. There are now an increased number of nucleotide sequences of FMDVs in Genbank and together with our own sequence results generated at Pirbright should facilitate primer/probe set design for evaluation and will allow this topic to be addressed in the follow-up project SE1121.

Objective 7

We have demonstrated that the rRT-PCR can confirm the FMD diagnosis achieved through the use of a lateral flow device (LFD) through its examination of the reactant excised from the positive test line of the LFD. Objective 8

VSV sequences of primer and minor groove binding probes have been obtained from Plum Island and primer/probes obtained from Sigma for rRT-PCR evaluation. The VSV-New Jersey (NJ) probe was labelled with VIC and the VSV-Indiana 1 probe with 6-FAM and the assay followed a similar protocol to that used for FMD and SVD. Pilot studies showed that a duplex assay generated similar signals as using the probes individually, being specific for the serotype and that there was no spectral overlap between channels. Other pilot studies were undertaken to show that both altering the annealing/extension step temperature to 55oC from 60oC and 3 different probe concentrations had no effect on assay performance.

A sensitivity of the rRT-PCR was next compared to VI and a hemi-nested (hn) RT-PCR, developed at Pirbright some years ago, with the following results :

NJ probe (HEX) Indiana probe (FAM) nested PCR VI (RS1) VI (BHK)NJ 10-2 36.07 50 + 5/5+ 5/5+NJ 10-3 39.43 50 + 5/5+ 5/5+NJ 10-4 43.56 50 + 5/5+ 5/5+NJ 10-5 50 50 + 5/5+ 5/5+NJ 10-6 50 50 - 2/5+ 3/5+NJ 10-7 50 50 - - -NJ 10-8 50 50 - - -NJ 10-9 50 50 - - -

NJ 10-10 50 50 - - -In 10-2 50 28.05 + 5/5+ 5/5+In 10-3 50 31.84 + 5/5+ 5/5+In 10-4 50 34.25 + 5/5+ 3/5+In 10-5 50 37.74 - 5/5+ 1/5+In 10-6 50 50 - 2/5+ -In 10-7 50 50 - - -In 10-8 50 50 - - -In 10-9 50 50 - - -In 10-10 50 50 - - -

The hnRT-PCR was able to detect both VSV serotypes and was more sensitive than the rRT-PCR assay for detection of VSV-NJ but was less sensitive at detecting VSV-Indiana 1. However, both RT-PCR assays appeared less sensitive than VI.

SID 5 (2/05) Page 13 of 17

A panel of representative VSV samples from the WRL for FMD sample collection were next evaluated by the duplex rRT-PCR and hnRT-PCR procedures and the specificity of the assays challenged by examining other non-VSV rhabdoviruses and virus isolates of FMD and SVD, with results as follows :

SEROTYPE VI/ELISA NJ probe (HEX) Indiana probe (FAM) hnRT-PCRIND1 VSV1 RS1 27/8/87 NO CT 20.50 -IND1 VSV5 RS2 1/9/87 NO CT 23.32 +IND1 VSV In INDC EX FA 50 BHK2 18/1/93 NO CT 20.44 -IND1 VSV1 OS 26/8/87 NO CT 22.16 +IND1 VSV5 OS 26/8/87 NO CT 27.30 +IND1 VSV IND 16677 BHK3 COLOMBIA 17/6/92 NO CT/NO CT 26.56/31.06 -IND1 VSV IND 15156 BHK3 COLOMBIA 17/6/92 NO CT/NO CT 26.42/NO CT -IND1 VSV-IND COL 4/93 RS1 13/8/93 NO CT 28.57/28.38 -IND1 VSV IND1 COSTA RICA 72 BHK3 26/4/98 NO CT/NO CT NO CT/NO CT -IND1 VSV IND1 EL SALVADOR 71 BHK3 25/4/98 NO CT/NO CT 28.51/29.38 -IND1 VSV IND1 COSTA RICA 79 BHK2 11/12/97 NO CT/NO CT 20.58/18.48 -IND1 VSV IND1 EL SALVADOR78 BHK3 24/4/98 NO CT/NO CT 30.35/29.47 -IND2 VSV NEW COCAL RS1 3/10/96 NO CT NO CT -IND2 VSV ARGENTINA EX CPECA RS1 28/10/96 NO CT NO CT +IND2 VSV In2 RIBEIRAO 2A/79 BHK3 NO CT NO CT +IND2 VSV 7 COCAL OS 1987 NO CT NO CT +/-IND2 VSV COCAL OS 7/10/87 NO CT NO CT +IND2 VSV IND2 MARABA BRASIL83 BHK2 2/4/98 NO CT/NO CT NO CT/NO CT -IND2 VSV IND2 MAIPU ARGENTINE BHK7 24/4/98 NO CT/NO CT NO CT/NO CT -IND2 VSV IND2 RANCHARIA BRASIL66 BHK4 26/4/98 NO CT/NO CT NO CT/NO CT -IND3 VSV ALAEAS EX CP FA RS1 3/10/96 NO CT NO CT -IND3 VSV IND3 AGULHAS NEGRAS BRASIL/86 BHK4 16/1/92 NO CT/NO CT NO CT/NO CT -IND3 VSV IND3 ESPINOSA BRASIL77 BHK7 27/4/98 NO CT/NO CT NO CT/NO CT -NJ VSV3 RS1 19/11/87 23.65 NO CT -NJ VSV FS 4 BHK1 7/11/87 30.37 NO CT +NJ VSV NJ COLORADO 10 BHK2 18/1/93 24.69 NO CT +NJ VSV2 RS1 28/8/87 28.28 NO CT +NJ VSV NJ COLORADO 10 RS2 18/1/93 23.35 NO CT +NJ VSV3 OS 26/8/87 24.58 NO CT +NJ VSV NJ-COLORADO OS 22/3/2005 24.67 NO CT +NJ VSV4 OS 26/8/87 27.77 NO CT +NJ RV31 VSV-NJ 15/88 CP21634/91 OS 29/7/91 31.05/29.22 NO CT/NO CT +NJ VSV-NJ COL 1/93 RS1 13/8/93 43.11/39.27 NO CT/NO CT -NJ VSV-NJ COL 3/93 RS1 13/8/93 38.03/36.12 NO CT/NO CT -NJ VSV-NJ EL SALVADOR BHK4 25/4/98 32.98/29.08 NO CT/NO CT -NJ VSV-NJ NICARAGUA 71 BHK3 26/4/98 30.39/28.55 NO CT/NO CT -NJ VSV NJ ECUADOR 85 BHK3 26/4/98 34.82/30.05 NO CT/NO CT -NJ VSV-NJ COSTA RICA 66 BHK2 26/4/98 36.08/31.55 NO CT/NO CT -

non-VSV rhabdovirus ISFAHAN 10-1 6/7/84 RS2 29/1/91 NO CT/NO CT NO CT/NO CT -FMDV O1 MANISA NO CT NO CT -FMDV MAY 4/2003 NO CT NO CT -SVDV HKN 1/80 NO CT NO CT -SVDV HKN 1/82 NO CT NO CT -

Every VSV-NJ isolate was detected using the duplex rRT-PCR assay, although VSV-NJ COL 1/93 RS1 was only weakly positive, while the hnRT-PCR was only able to detect 8/15 VSV-NJ isolates. The rRT-PCR assay detected 11/12 VSV-IND-1 isolates, although VSVIND 15156 BHK3 COLOMBIA was detected on one out of two occasions, while the hnRT-PCR detected 3/12 VSV-IND-1 isolates. The rRT-PCR assay is specific for VSV-NJ and VSV-IND-1 serotypes and did not amplify isolates of VSV-Indiana-2 or -3 serotypes. In contrast, the hnRT-PCR did detect 4/8 VSV-IND-2 isolates but not those of VSV0-IND-3. Neither PCR assay amplified another Rhabdovirus, Isfahan or isolates of SVDV and FMDV.

The duplex rRT-PCR assay was capable of detecting VSV-IND-1 and VSV-NJ, with just one VSV-IND-1 isolate being missed. The assay has been set up to be compatible with the FMDV and SVDV RT-PCR assays currently in use at the WRL. Although both assays were sensitive and highly efficient, neither was as sensitive as VI. When compared with the hnRT-PCR method the real-time RT-PCR assay detected a much higher proportion of the VSV-NJ and VSV-Indiana 1 virus isolates examined. However, the assay did not detect isolates of VSV-Indiana subtypes 2 and 3, in contrast to the hnRT-PCR which detected viruses of the former Indiana subtype. Although viruses of Indiana subtypes 2 and 3 are seldom implicated in outbreaks of clinical disease, additional primer/probe sets need to be designed to accommodate detection of these viruses.

Objective 9

Sequence data covering the polymerase-capsid region has been generated from several viruses representative of different vesivirus serotypes: namely, SMS virus types 2, 4, 10 and 13, VES virus types B51, G55 and J56 and a virus isolate Oklahoma. These sequences were aligned with existing, published Vesivirus sequences to facilitate the design of two primers/probe sets from conserved regions for use in an rRT-PCR for Vesivirus detection.

The results of preliminary evaluations of the rRT-PCR are shown in the following table :

Sample Primers/probe setVirus Serotype 602F/700R/648P 602F/700R/648P

SID 5 (2/05) Page 14 of 17

SMS virus 1 30.69, 29.36*2 29.88, 29.16 503 23.65, 23.064 14.49, 13.63 15.786 11.75, 11.927 12.84, 12.388 47.15, 49.06 509 22.08, 22.3410 28.69, 28.34 18.4311 25.52, 24.4612 50, 50 5013 19.89, 19.6 31.4914 37.92, 38.88

VES virus B1-34 23.19, 24.07 19.96A48 17.76, 19.29B51 34.34, 34.69 34.37C52 17.25, 18.08D53 28.91, 29.14 50E54 17.47, 17.2F55 13.84, 13.74G55 12.07, 12.03 23.97H54 17.99, 17.29I55 19.35, 18.22 17.56J56 16.36, 17.56 20.36K54 32.85, 33.13 16.35, 18.4

Cetacean CV 12.32, 12.93Bovine CV 14.28, 15.04Oklahamoma CV 19.08, 17.35 15.72, 23.37FMD virus Each of all 7 50 50SVD virus 50 50VS virus Both 50 50

* Ct value of ≤40 used to assign sample as positive

These results show that the assays are sensitive and specific and Vesiviruses of all serotypes tested are detected by the two primers/probe sets combined, with the exception of SMSV-8 and SMSV-12. These virus serotypes had previously been undetected by conventional RT-PCR and may suggest that these caliciviruses belong to a different group. Attempts are currently being undertaken to sequence SMSV-12 and thence to design an addition primers/probe set for rRT-PCR evaluation.

Objective 10

Work on this objective is dependent upon establishing links with commercial suppliers of mobile, field-based PCR equipment. A collaborative project involving a commercial company Enigma (formerly part of DSTL), VLA and Pirbright has been setup to trial such a mobile PCR machine (‘PCR-Light’) but the collaboration has been seriously delayed through non-delivery of the machine caused by technical problems in its construction. This machine is designed to extract nucleic acid from the test sample prior to PCR amplification. Discussions have also taken place with another company – Smith’s Detection, Watford - who has expressed interest in establishing assays for FMDV using their equipment (BioSeeq). The current BioSeeq is designed to test up to 6 samples at one time in contrast to the single sample by the PCR-Light. A preliminary trial of this equipment (using unifected pig tissues) was perfomed in August 2004 to confirm that simple sample preparation could yield template that did not contain substances inhibitory to PCR on the Bioseeq. It is anticipated that agreement for the full evaluation phase of the collaboration will be formalised in early September and a BioSeeq will be loaned to Pirbright to allow preliminary trials to take place. These will initially involve transferring the Pirbright FMD RT-PCR assay to the equipment, using it to test epithelia stored in the WRL for FMD sample collection and on material collected from FMDV experimentally infected animals in the Institute isolation compounds.

The problem of sample extraction is of importance to on-site FMD testing and some studies have already been done which might have relevance. Several protocols have been evaluated for the capture of FMD virus antigen on a microtitre plate to enable RNA extraction and reverse transcription to be carried out in the plate prior to PCR amplification of the cDNA product. Initial protocols enabled FMD viral antigen to be captured from suspensions of clinical samples in the well by coating with a cocktail of either type-specific guinea pig or polyclonal rabbit anti-FMD virus immune sera. The viral RNA was extracted by heating the plate for five minutes at 90oC and was

SID 5 (2/05) Page 15 of 17

subsequently detected by conventional PCR amplification with universal primer sets and PCR products analysed by gel electrophoresis. In an alternative protocol, specific primer sets were able to serotype FMD viruses of types O, A, C and Asia 1 after individual wells were coated with homologous type-specific guinea pig or rabbit anti-FMD virus immune serum. Both protocols were specific for FMD virus and their sensitivity was essentially the same as that of conventional RT-PCR. More recently, soluble integrin αvβ6 recombinant protein has been evaluated as a capture ligand combined with the use of real-time PCR involving a universal primers/probe set for amplification of the cDNA product rather than conventional PCR. Preliminary results indicate that αvβ6 captures FMD virus from a clinical sample to facilitate extraction of RNA and eventual detection of genome by the rRT-PCR. This work will be continued in SE1121.

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.

SID 5 (2/05) Page 16 of 17

Peer-reviewed publications:Reid, S.M., Grierson, S.S., Ferris, N.P., Hutchings, G.H. and Alexandersen, S. (2003). Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus. Journal of Virological Methods 107, 129-139.Reid S. M., Ferris N. P., Hutchings G. H., King D. P. and Alexandersen S. (2004). Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus. Journal of Virological Methods 116, 169-176.Reid, S.M., Paton, D.J., Wilsden, G., Hutchings, G.H., King, D.P., Ferris, N.P. and Alexandersen, S. (2004). Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infections in pigs. Journal of Comparative Pathology 131 (4), 308-317.Shaw, A.E., Reid, S.M., King, D.P., Hutchings, G.H. and Ferris, N.P. (2004). Enhanced laboratory diagnosis of foot and mouth disease by real-time polymerase chain reaction. Revue Scientific et Technique de l'Office International des Epizooties 23 (3), 1003-1009.Ferris, N.P., King, D.P., Reid, S.M., Shaw, A.E. and Hutchings, G.H. (2005). Analysis of original laboratory results and retrospective analysis by real-time PCR of virological samples collected from confirmed cases suggest over-reporting of foot-and-mouth disease in the UK in 2001. The Veterinary Record (in press)Sammin, D.J., Paton, D.J., Parida, S., Ferris, N.P., Hutchings, G.H., Reid, S.M., Shaw, A.E., Holmes, C., Gibson, D., Corteyn, C., Knowles, N.J., Valarcher, J.F., Hamblin, P.A., Fleming, L., Gwaze, G. and Sumption, K.J. (2005). Evaluation of laboratory tests for SAT serotypes of foot-and-mouth disease virus with specimens collected from convalescent cattle in Zimbabwe. The Veterinary Record (in press).Shaw, A.E., Reid, S.M., Knowles, N.J., Hutchings, G.H., Wilsden, G., Brocchi, E., Paton, D.J. and King, D.P. (2005). Sequence analysis the 5’ untranslated region of Swine Vesicular Disease Virus reveals block deletions between the end of the internal ribosomal entry site and the initiation codon. Journal of General Virology (in press).King, D.P., Ferris, N.P., Shaw, A.E., Reid, S.M., Hutchings, G.H., Giuffe, A.C., Robida, J.M., Callahan, J.D., Nelson, W.M. and Beckham, T.R. (2005). Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time RT-PCR assays. Journal of Veterinary Diagnostic Investigation (in press)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., Dekker, A. and De Clercq, K. (2005). Foot-and-mouth disease virus: a first ring test to evaluate virus isolation and RT-PCR detection methods. Veterinary Microbiology

Presentations:Reid, S.M., Grierson, S.S., Ferris, N.P., Hutchings, G.H. and Alexandersen, S. (2002). Evaluation of a automated RT-PCR systems to accelerate FMD diagnosis. Association of Veterinary Teachers and Research Workers, Scarborough 25-27, March 2002. Research in Veterinary Science 72 (Suppt. A) 11 31.Reid, S.M., Ferris, N.P. and Alexandersen, S. (2002). Detection of swine vesicular disease virus genome by TaqMan RT-PCR assay. Association of Veterinary Teachers and Research Workers, Scarborough 25-27, March 2002. Research in Veterinary Science 72 (Suppt. A) .Reid, S.M., Hutchings, G.H. and Ferris, N.P. (2002). Detection and serotyping of foot-and-mouth disease virus by immunocapture RT-PCR. Association of Veterinary Teachers and Research Workers, Scarborough 25-27, March 2002. Research in Veterinary Science 72 (Suppt. A) 12 33.Ferris, N.P., Hutchings, G.H., Reid, S.M. and Newman, B . (2002). Laboratory virological investigations relating to the 2001 foot-and-mouth disease outbreak in the United Kingdom. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Izmir, Turkey, 17-20 September 2002, Appendix 4, 58.Reid, S.M., Ferris, N.P., Hutchings, G.H. and Alexandersen, S. (2002). Diagnosis of foot-and-mouth disease virus by automated RT-PCR. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Izmir, Turkey, 17-20 September 2002, Appendix 25, 203-209.Reid, S.M., Ferris, N.P. and Hutchings, G.H. (2002). Comparison of RT-PCR procedures for diagnosis of clinical samples of foot-and-mouth disease virus (serotypes O, A, C and Asia 1) under the European Union Concerted Action group project PL 98-4032. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Izmir, Turkey, 17-20 September 2002, Appendix 26, 210-219. Reid, S.M., Zhang, Z., Murphy, C., Quan, M., Ferris, N.P. and Alexandersen, S. (2002). Research and diagnosis of foot-and-mouth disease: Application of real-time RT-PCR. UK SDS User Meeting, Applied Biosystems, London, November 2002.Reid, S.M., Paton, D.J., Wilsden, G, Hutchings, G.H., King, D.P. and Alexandersen, S. (2003). Detection and quantitation of swine vesicular disease virus by real-time RT-PCR assay. European Society for Veterinary Virology. 6th International Congress of Veterinary Virology. Virus Persistence and Evolution. San-Malo, France, 24-27 August 2003, 99. 42.King, D.P., Reid, S.M., Shaw, A.E., Bashiruddin, J.B., Hutchings, G.H., Knowles, N.J., Alexandersen, S., Ferris, N.P. and Paton, D.J. (2003). An integrated laboratory Approach for the detection and classification of foot-and-mouth disease virus. European Society for Veterinary Virology. 6th International Congress of Veterinary Virology. Virus Persistence and Evolution. San-Malo, France, 24-27 August 2003, 102Reid S.M., Paton D.J., Wilsden G., Hutchings G.H., King D.P., Ferris N.P. and Alexandersen S. (2004). Use of automated real-time RT-PCR to monitor experimental swine vesicular disease virus infection in pigs. Abstracts: Current topics in veterinary science: 58th annual conference of the Association of Veterinary Teachers and Research Workers; Scarborough, 5-7 April 2004. Research in Veterinary Science 2004 76 (Suppt. A) 34 90.Shaw A.E., Reid S.M., King D.P., Hutchings G.H. and Ferris N.P. (2004). Sensitivity and specificity of foot and mouth disease virus RT-PCR: defining a cut-off for diagnosis. Abstracts: Current topics in veterinary science: 58th annual conference of the Association of Veterinary Teachers and Research Workers; Scarborough, 5-7 April 2004 Research in Veterinary Science 2004 76 (Suppt. A) 35 91.Ferris, N.P. (2004). Laboratory virological investigations for the 2001 FMD outbreak in the UK. EUFMD meeting : Workshop on Contingency Planning for FMD laboratory diagnostic activities; Cordoba, Spain 28-30 April 2004.Shaw, A.E., King, D.P., Reid, S.M., Hutchings, G.H., Ferris, N.P. and Paton, D.P. (2004). Sequence analysis of the 5’ untranslated region of swine vesicular disease virus. 7th International Symposium on Positive-Strand RNA viruses, San Francisco, USA, 27 May-1 June 2004.Donal Sammin, David Paton, Geoff Hutchings, Nigel Ferris, Andrew Shaw, Nick Knowles, Satya Parida, Catherine Holmes, Debi Gibson, Mandy Corteyn, Rosa Fernandez and Pip Hamblin (2004. Serological responses in relation to vaccination and infection in Zimbabwe cattle following outbreaks of FMD. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Chania (Crete), Greece, 12-15 October 2004, Appendix 17, 108-122.Nigel Ferris, Scott Reid, Donald King, Geoff Hutchings and Andrew Shaw (2004). Prospects for improved laboratory diagnosis of FMD using real-time RT-PCR. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Chania (Crete), Greece, 12-15 October 2004, Appendix 42, 261-267.Scott Reid, Satya Parida, Donald King, Geoffrey Hutchings, Andrew Shaw, Nigel Ferris, Eric Hillerton and David Paton (2004). Use of automated real-time RT-PCR to detect foot-and-mouth disease virus in milk. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Chania (Crete), Greece, 12-15 October 2004, Appendix 43, 268-276.King, D.P., Reid, S.M., Hutchings, G.H., Shaw, A.E. Ferris, N.P. and Paton, D.J. (2004). A ring-test for the laboratory detection of FMDV by RT-PCR and virus isolation. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Chania (Crete), Greece, 12-15 October 2004, Appendix 79, 479.Paton D. J., Ferris N., Knowles N., Valarcher J., Newman B., King D. P., Reid S. M., Dukes J. and Parida S. International surveillance of and laboratory preparedness for dealing with FMD Fifth McLaughlin Symposia in Infection & Immunity, Galveston, Texas, February 2005.Paton D. J., Ferris N., Knowles N., Valarcher J., Newman B., King D. P., Reid S. M., Dukes J. and Parida S. Laboratory contingency plans for dealing with exotic animal viral diseases such as foot-and-mouth disease. 156th Meeting of the Society for General Microbiology, Heriot-Watt University, Edinburgh, April 2005.Shaw A. E., Reid S. M., Knowles N. J., Hutchings G. H., Wilsden G., Brocchi E., Paton D. J. and King D. P. Sequence analysis of the 5’ untranslated region of swine vesicular disease virus reveals block deletions between the end of the internal ribosomal entry site and the initiation codon. EUROpic, Lunteren, The Netherlands, May 2005.

SID 5 (2/05) Page 17 of 17