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SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 22

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.

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Project identification

1. Defra Project code SE1120

2. Project title

Application of newer technologies for the diagnosis of FMD and other vesicular diseases

3. Contractororganisation(s)

Institute for Animal HealthComptonNewburyBerksRG20 7NN     

54. Total Defra project costs £ £396,290.00(agreed fixed price)

5. Project: start date................ 01 July 2003

end date................. 30 June 2006

SID 5 (Rev. 3/06) Page 2 of 22

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 In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain.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.

The collection of foot-and-mouth virus (FMDV) monoclonal antibodies (mabs) has been catalogued and on-going studies are being undertaken to characterise their activity for potential use in antigen detection ELISA’s and for use in lateral flow devices (LFD) for pen-side diagnosis

A prototype antigen detection ELISA has been developed using a recombinant protein to αvβ6 (the principal integrin cell receptor for FMDV) as the capture ligand. The recombinant integrin bound FMD virus of all seven serotypes but not that of another vesicular disease, swine vesicular disease. Considerable heterotypic cross-reactions were evident when using the integrin capture ligand in combination with guinea pig detecting antibodies but totally type-specific reactions resulted when serotype-specific mabs were used instead of the guinea pig reagents. The specificity of reaction of the integrin capture/ mab detector combination was superior to that of our routinely employed rabbit/guinea pig polyclonal antibody ELISA and offers an improvement for test interpretation. As a universal trapping reagent for all FMD virus serotypes the αvβ6 recombinant protein also has the potential for application in other test procedures for viral identification and in antibody detection assays employed for the diagnosis of FMD.

An International Patent Application following the UK Patent Application of June 2004 -Improved FMDV Test – and based on the use of integrins as a ligand for FMD diagnosis was filed in June 2005.

Concerted efforts to revive the FMDV C1 hybridoma which yielded the mab used in the previously successful LFD for pen-side diagnosis have proved fruitless. Prolonged attempts to identify an alternative candidate have been undertaken and many mabs identified for potential use based on their FMDV pan-reactivity in the conventional antigen ELISA. Prototype LFDs produced by our commercial collaborator (Svanova) based on the use of the selected mabs have been evaluated. However, several of these mabs have failed to work in the LFD or either the specificity of reaction of many of these has been shown to be deficient or else the sensitivity and spectrum of reaction shown to be poor. More recently, three mabs have been shown to be more promising as they work specifically in prototype LFDs and function efficiently for the detection of FMDV serotypes O, A, C and Asia 1, although less well for the three SAT virus serotypes. Another FMDV antigen detection LFD produced by Princeton BioMeditech Corporation, USA The solid phase competition ELISA for the measurement of antibodies to FMDV has been evaluated for serotypes other than the already validated type O assay. The extension of validation for types A, C and Asia 1 has largely been accomplished but for the three SAT serotypes, test development continues.

A variety of cell lines have been obtained to investigate their sensitivity to FMD (plus SVD and VS, where appropriate) virus replication and namely, MAX (SV40 immortalised swine kidney), bovine turbinate, bovine kidney, new-borne swine kidney, new-borne pig trachea, pig ileum, LLC-pig kidney cell lines, a human carcinoma

SID 5 (Rev. 3/06) Page 3 of 22

cell line (SW) and Athos, a procine thyroid cell line. All of these cell lines have inferior sensitivity to our current cell cultures, which are routinely used for FMD/SVD diagnosis (i.e. primary calf thyroid and IB-RS-2 cells). However, a further ‘cell line’ of bovine kidney (BK) cells has only marginally inferior sensitivity for FMDV than primary calf thyroid cells.

Attempts have been made to engineer cell lines with characteristics suitable for the routine propagation of field isolates of FMDV. The study used real-time RT-PCR and flow-cytometry to measure the expression of integrin molecules by a panel of cell cultures in order to understand the surface determinants that are required for FMDV sensitivity. The most sensitive cell cultures were primary bovine thyroid cells and IB-RS-2 cell line which had highest levels of αvβ6and αvβ8 respectively, confirming the importance of these heterodimers as receptors for the virus. In contrast, expression of αvβ3 did not appear to correlate with sensitivity to FMDV in the cell cultures selected. A stable MDBK cell line generated after transfection with a plasmid containing bovine α6 demonstrated increased expression of cell surface αvβ6. Unfortunately, this line showed no increased sensitivity to a field isolate of FMDV, although binding of FMDV was enhanced compared with parental MDBK cells.

A first-generation microarray consisting of probes covering 150 viral ‘species’ has been developed by a consortium of the Veterinary Laboratories Agency and the Institute for Animal Health laboratories at both Compton and Pirbright. The WRL at Pirbright is validating the microarray’s ability to serotype and subtype veterinary viruses using FMDV as a model. Tests have shown that it is capable of discriminating different serotypes of FMDV and that differently related strains within a serotype also appear to give different ‘signatures’. A second-generation array has now been printed that includes oligos for all vesicular diseases and known picornaviruses in order to facilitate differential diagnose of FMD. This second-generation array has the capacity to distinguish 250 viral species from 29 virus families. Current work at Pirbright is focused on validating this array with further FMDVs and related picornaviruses.

Fever and the production of blisters on the feet and tongues are prominent clinical features of acute infection of ruminants and pigs with FMDV. A study has been conducted to evaluate the ability of thermal imaging to monitor the surface temperatures of experimental animals. Its practical value for the diagnosis of FMD seems limited in ruminants because of the variable temperatures detected prior to infection. In pigs there appeared to be a better correlation between progression of disease and hot extremities, although more measurements are needed, especially from uninfected animals. In the field, the camera might be used to help single out animals for examination and sampling.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Project relevanceVesicular 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 (MC) which include vesicular exanthema of swine and San Miguel sea lion virus. 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. The 2001 FMD outbreak in the UK was a dramatic illustration of these points and has highlighted the requirement for effective diagnostic support, both within the laboratory and in the field, for disease control and eradication. The episode also highlighted the urgent need for marked improvements to be made in the sensitivity and speed of high throughput diagnostic tests and for a shift in emphasis to rapid

SID 5 (Rev. 3/06) Page 4 of 22

local diagnosis during an outbreak as an aid to support clinical suspicion, to help reduce the scale of any future outbreak and to target scarce resources (slaughter and vaccination teams).

Definitive diagnosis requires detection of virus or antigen in vesicular epithelium (the preferred specimen). Serology is not favoured for the primary diagnosis of FMD in cattle and pigs but may be valuable in the diagnosis of subclinical FMD in sheep. It is essential for the diagnosis of subclinical SVD when virus strains are not virulent. Serology is needed also for epidemiological studies, import/export testing to demonstrate freedom from infection and in vaccine evaluation. In countries where FMD vaccination is carried out, a swift assessment of the serotype involved and on the suitability of available vaccines is essential for disease containment and control. 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 virus strains worldwide by providing a diagnostic and serotyping referral service for member countries. Annually the WRL typically receives up to 600-700 samples from 25-30 different countries; however, during the course of the recent FMD outbreak in the UK over 16,000 samples were received and a further 300 from Ireland. In the course of performing diagnostic procedures, non-vesicular viruses, principally bovine and porcine enteroviruses (BEV and PEV), have been isolated. Thus sensitive tests are required for those viruses also.

The laboratory assays used for FMD diagnosis are highly sensitive and are the best currently available. The majority of positive samples can be reported as such within 3-4 hours of sample receipt. However, poor quality positive samples which contain low concentrations of virus, below the detection limit of the ELISA, and negative samples cannot be defined until samples have been inoculated onto cell cultures. These procedures take 4 days to confirm a negative sample. The most sensitive system for FMD virus isolation is through use of primary calf thyroid (CTY) cells. Thus, calf thyroid glands must be continually sourced each week and their supply can be particularly problematic during an outbreak. The development of fluorogenic reverse transcription polymerase chain reaction (RT-PCR) procedures hold particularly high promise for their application to FMD and other vesicular virus diagnosis and are currently the subject of a separate DEFRA-funded project proposal SE1121 ‘Application of RT-PCR for the diagnosis of FMD and other vesicular diseases’. The indications are that definitive diagnosis may be achieved directly on a suitable sample by real time RT-PCR without recourse to prolonged cell culture passage for amplification and isolation of the virus. The dependence on primary CTY cells for diagnostic use would thus be reduced. However, in the case of a primary outbreak, virus would normally need to be isolated in cell culture for stringent virus strain characterisation studies and the availability of an alternative cell system (i.e. a permanent cell line) of equal sensitivity to CTY cells would be desirable. Current RT-PCR procedures take twice as long the ELISA to complete. The experience of the UK 2001 FMD outbreak showed that close to 90% of positive samples were defined by ELISA on epithelial suspensions. Consequently, such samples as can be tested by ELISA would be first done so. Those samples yielding a negative ELISA result and those not suitable for examination by ELISA (e.g. blood, probang and milk samples), then tested by RT-PCR.

The antigen detection ELISA’s use polyclonal antisera. Each batch is of finite supply and successive batches may have different characteristics. The potential of monoclonal antibodies (mabs) to replace polyclonal reagents needs to be more fully explored and to accomplish this, more mabs need to be sourced or produced and characterised.

The consequence of the UK Governments adoption of a 24/48 hour culling policy for animals on infected/contiguous premises towards the end of March 2001 meant that the majority of subsequent outbreaks were confirmed by clinical diagnosis alone. However, subsequent laboratory investigations on samples received from such premises would suggest that such classification of animal disease status by clinical judgement could sometimes be flawed. Nevertheless, if the period between sample collection and laboratory receipt (normally 0.5 to 1 day) is also considered then the limitations of the described diagnostic procedures of ELISA combined with virus isolation in cell culture are apparent. The combined procedures are too slow for use in emergency situations. Rapid, on farm diagnosis will be required in the future to quickly investigate the clinical suspicion of disease to ensure that the presence of the virus is quickly identified and then that it is speedily eliminated. Candidate tests to facilitate this are those using battery operated PCR technology (for virus detection) and pen-side (immunodiffusion) tests and other novel approaches (biosensors, micro-arrays) (for both antigen and antibody detection). Immunodiffusion tests will require the development of mabs and genetically expressed viral antigens to standardise the methods. Pen-side tests for disease surveillance and for testing animals to ensure freedom from infection before licensed movement are additionally required. Diagnostic techniques based on the micro-array principal have the potential to diagnose many different conditions at once, and testing for an exotic virus such as FMDV could thereby be incorporated into a diagnostic screening assay for other common pathogens, to improve the detection of primary cases. Thermographic imaging could be a convenient method to screen groups of farm animals to identify those with relatively high or low body temperatures or areas of inflammation. This could allow large numbers of animals to be screened quickly with minimal handling and restraint, in order to identify any that have inflamed feet or pyrexia. Such animals would then be more closely examined and if appropriate, samples would be taken from them to test for the presence of virus.

Scientific objectives of the project

The project has the following objectives :

SID 5 (Rev. 3/06) Page 5 of 22

1. Evaluate FMD mabs and recombinant integrins for inter- and intra-typic reactivity for use in the indirect sandwich ELISA for antigen detection, in chromatographic strip test devices, in piezo-electric biosensors and in the competition ELISA for serology. Produce further mabs if gaps exist in the collection.

2. Develop prototype pen-side tests for FMD, SVD and VS diagnosis.

3. Field validate FMD and other vesicular disease pen-side strip test devices.

4. Further evaluation of new cell lines and integrin transfected cells for FMD and other vesicular virus diagnosis.

5. Develop prototype micro-array test for vesicular viruses.

6. Evaluation of the application of thermographic imaging to the detection of animals affected by FMD.

Objective 1

The collection of FMDV monoclonal antibodies (mabs) has been catalogued and is summarised in the table below :

Source Immunogen No. Source Immunogen No.

  Virus Strainof mabs   Virus Strain of mabs

In-house FMDV C1 Oberbayern 39Brescia, Italy FMDV Asia 1 3

FMDV A22 IRQ 24/64 37 FMDV A 4FMDV A24 Cruzeiro 40 FMDV C 1FMDV A5 5 FMDV O1 Lausanne 6FMDV C1 Noville 7 FMDV O Italy 93 1FMDV SAT 1 BOT 1/68 5 FMDV O1 Manisa 7FMDV SAT 1 17 FMDV O UK 2001 8FMDV SAT 2 RHO 1/48 6 FMDV A5 Parma 3FMDV SAT 2 7 FMDV A Modena 84 3FMDV SAT 2 BOT 38 FMDV A Albania 96 1FMDV SAT 3 ZIM 4/81 4 FMDV C Brescia 64 13FMDV Asia 1 PAK 1/54 10 FMDV Asia 1 NEP 29/97 9SVDV UKG 27/72 11 FMDV SAT 1 BOT 1/68 9SVDV ITL 1/92 5 FMDV SAT 2 ZIM 5/81 7SVDV ITL 9/93 4 SVDV ITL/91 11VSV Indiana 1 (Ind C) 14

VSVIndiana 2 (Argentina) 2

VSVIndiana 3 (Alagoas) 2

VSV New Jersey 12

Bayer, Germany FMDV O1 Manisa 6 Turkey FMDV O1 Manisa 10

FMDV A22 IRQ 24/64 10FMDV A Iran 3 India FMDV O 17FMDV A Albania 8 FMDV Asia 1 11FMDV SAT 1 Zim 4

FMDV Asia 1 Shamir 5Madrid, Spain FMDV SPA 7/79 3

BVI, Botswana FMDVSAT 1 BOT 01/77 5 South Africa FMDV SAT 2 7

FMDV SAT 2 ZIM 07/83 3

FMDVSAT 2 BOT 04/80 4 Canada FMDV 40

  FMDV SAT 3 ZIM 09/81 4        

SID 5 (Rev. 3/06) Page 6 of 22

These mabs are being evaluated through characterisation of their type-specificity and intra-typic reactivity and consequently, their potential for use in antigen detection ELISA’s for diagnosis and for selection for lateral flow device (LFD) incorporation for pen-side tests (see objective 2). Examples of these types of characterisation studies are illustrated by the two tables below on results achieved with some of the type C mabs :

Specificity of reaction of FMDV type C1 Oberbayern mabs

Mab   FMDV serotype  

    O1 M

anis

a

A22

IRQ

24/

64

C1 N

ovill

e

C1 O

berb

ayer

n

C3 R

esen

de

SAT

1 R

HO

12/

78

SAT

1 B

OT

1/6

8

SAT

2 Z

IM 0

7/83

SAT

3 Z

IM 4

/81

Asi

a 1

CA

M 9

/80

Asi

a 1

Sham

ir

SVD

V U

KG

27/

72

C1 Oberbayern NF2.20.C6.D10 N N Y N N N N N NC1 Oberbayern NF2.20.C6.E9 N N Y N N N N N NC1 Oberbayern NF2.20.D11.B3 N N Y N N N N N NC1 Oberbayern NF2.20.E8.C6 N N Y N N N N N NC1 Oberbayern NF2.20.E11.A9 N N Y N N N N N NC1 Oberbayern NF2. 20.E11.B4 N N Y N N N N N NC1 Oberbayern NF2.20.E11.C10 N N Y Y N N N N N NC1 Oberbayern NF2.20.E11.D4 N N Y Y N N N N N NC1 Oberbayern A4.D12 N N Y Y Y/L N N N N N NC1 Oberbayern C2.A3 N N Y Y N N N N N N NC1 Oberbayern D7.G2 N N Y Y Y/L N N N N N NC1 Oberbayern E2.B4 N N Y Y N N N N N N NC1 Oberbayern A4.D12 N N Y Y Y/L N N N N N NC1 Oberbayern C2.A3 N N Y Y Y/L N N N N N NC1 Oberbayern D7.G2 N N Y Y Y/L N N N N N NC1 Oberbayern E2.B4 N N Y Y Y/L N N N N N NC1 Oberbayern E11.A9 N N Y Y N N N N N   N N

Y, reaction equivalent to polyclonal antiserum reactionN, no reaction

Intra-typic reactivity of FMDV type C1 Oberbayern mabs (expressed as a percentage - mab versus polyclonal)

Mab   Type C FMDV

    IND

51/

79

C B

resc

ia

PHI 7

/84

C1 D

etm

old

C C

ZE

3/8

9

C1 N

ovill

e

C1 O

berb

ayer

n

C3 R

esen

de

C2 9

97

C4 T

DF

C2 P

ando

C N

EP

35/9

6

C5 A

RG

/69

C P

arag

uay/

69

C B

AN

1/9

2

C P

HI 9

/94

C IT

L 2

/89

C1 Oberbayern NF2.20.C6.D10 89 71 0 111 64 36 78 0 48 37 0 0 0 12 0 104C1 Oberbayern NF2.20.C6.E9 97 76 0 101 78 38 80 0 50 46 0 0 0 12 0 111C1 Oberbayern NF2.20.D11.B3 104 79 0 120 71 39 84 0 48 49 0 0 0 9 0 107C1 Oberbayern NF2.20.E8.C6 125 96 0 122 87 49 94 0 64 60 0 0 0 13 0 112C1 Oberbayern NF2.20.E11.A9 105 89 0 113 77 50 93 0 49 52 0 0 0 8 0 102C1 Oberbayern NF2. 20.E11.B4 98 83 0 115 70 43 93 0 46 54 0 0 0 8 0 103C1 Oberbayern NF2.20.E11.C10 116 93 0 118 77 68 89 0 54 55 0 25 0 0 8 0 109C1 Oberbayern NF2.20.E11.D4 107 93 0 120 73 65 90 0 44 51 0 23 0 0 8 0 105C1 Oberbayern A4.D12 12 31 0 68 38 67 76 28 75 50 20 38 36 2 0 81C1 Oberbayern C2.A3 11 60 0 59 50 47 57 12 68 64 10 33 27 1 0 100C1 Oberbayern D7.G2 39 70 28 41 86 26 45 22 0 14 77 19 16 26 64 84C1 Oberbayern E2.B4 10 41 0 56 56 49 59 11 70 64 13 35 35 1 0 102

SID 5 (Rev. 3/06) Page 7 of 22

C1 Oberbayern A4.D12 10 32 0 53 42 55 69 18 88 50 23 40 48 2 0 99C1 Oberbayern C2.A3 13 61 0 64 82 62 68 14 76 79 30 49 44 1 0 114C1 Oberbayern D7.G2 22 85 20 43 103 22 52 24 0 0 89 24 21 36 67 86C1 Oberbayern E2.B4 17 66 0 69 81 75 77 18 94 86 27 53 47 1 0 113C1 Oberbayern E11.A9 53 28 0 50 30 40 65 0 30 36   16 0 0 59 0 88

A prototype FMD virus antigen detection ELISA has been developed using an integrin αvβ6 recombinant protein as a ligand in combination with type-specific mabs as detectors. The following is the Abstract of the published paper presenting the results :

Ferris et al - AbstractRecombinant integrin v6 was evaluated as a capture ligand in a sandwich ELISA for the detection and serotyping of foot-and-mouth disease (FMD) virus. Our routinely applied method employs seven serotype-specific rabbit polyclonal antibodies as capture ligands and seven serotype-specific guinea pig polyclonal antibodies as detecting reagents. The recombinant integrin bound FMD virus of all seven serotypes but not that of another vesicular disease, swine vesicular disease (SVD). Considerable heterotypic cross-reactions were evident when using the integrin capture ligand in combination with guinea pig detecting antibodies but totally type-specific reactions resulted when serotype-specific monoclonal antibodies (mabs) were used instead of the guinea pig reagents. The specificity of reaction of the integrin capture/mab detector combination was superior to that of our routinely employed rabbit/guinea pig ELISA and offers an improvement for test interpretation. As a universal trapping reagent for all FMD virus serotypes the v6 recombinant protein also has the potential for application in other test procedures for viral identification (e.g. pen-side chromatographic strip-tests, biosensors, immunocapture RT-PCR, antigenic characterization procedures and monoclonal antibody profiling of emerging field virus strains) and in antibody detection assays employed for the diagnosis of FMD.

The principal of the antigen detection ELISA design has thus been demonstrated to be successful. However, the choice of the mabs for diagnostic purpose may not necessarily be ideal and others continue to be evaluated for selection.

An International Patent Application following the UK Patent Application of June 2004 -Improved FMDV Test – and based on the use of integrins as a ligand for FMD diagnosis was filed in June 2005. However, the Institute is unlikely to defend the patent as it does not appear at present that there is a good commercial prospect for FMDV diagnostics, and the costs associated with taking patents to grant would not easily be repaid and so the intention is to maintain the international patent application unto to the end of its natural life (23 Dec 2006) in order to allow some further investigation of its potential.

The solid phase competition ELISA (SPCE; using polyclonal antisera) for the measurement of antibodies to FMDV has been evaluated for serotypes other than the already validated type O assay. The extension of validation for types A, C and Asia 1 has largely been accomplished but for the 3 SAT serotypes a serious limitation has been the paucity of suitable SAT sera of known status to examine. Sera collected during a field study in Zimbabwe were examined by the SPCE and results of the study have been accepted for publication in The Veterinary Record : Sammin et al - Abstract

During 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 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.

Panels of varying numbers of field sera or from experimentally infected animals (and collected at sequential intervals) have been obtained from Drs Herve Coupier, BVI, Botswana, Wilna Vosloo, Onderstepoort, South Africa and Alfonso Clavijo, Winnipeg, Canada for further SAT SPCE test validation. SPCEs have been established based on inactivated concentrated antigens that have been obtained from Merial and using suitable rabbit and guinea pig polyclonal antisera available in the WRL’s reagent collection which most likely match the SAT virus strains used by Merial for antigen production. The results

SID 5 (Rev. 3/06) Page 8 of 22

generated from the SPCE have been compared with those arising from the virus neutralisation test (VNT) and the liquid phase blocking assay (LPBE) but have yet to be fully analysed. On initial inspection it would appear that the SAT SPCEs may be useable at a much lower cut-off PI value than for those defined for the other serotypes. Whether this is impression is true remains to be confirmed but should become evident once sufficient numbers of sera from individual animals of known negative status have been tested to allow for calculation of test specificity. To this end a large collection of negative sera (bovine, ovine and porcine) has now been obtained from VLA, Weybridge for SPCE test study.

Objective 2

Numerous attempts at Pirbright and by our commercial collaborator, Svanova Biotech, Sweden, have resulted in failure to revive the FMDV C1 hybridoma, the mab from which was the basis for the LFD that had been successfully developed for FMDV antigen detection (pen-side tests). Considerable efforts have been expended in identifying alternative candidates as replacements for use in such LFDs and in the examination of subsequent prototype devices.

The following table is an example of the type specificity of reaction achieved with some mab examples in the conventional antigen ELISA against FMDVs of each serotype and illustrates a degree of reaction against either 4 or 7 FMDV serotypes :

Specificity of reaction (ELISA optical density values) of selected FMDV mabs to illustratepan-reactivity of their FMDV reaction

Mab Immunogen FMDV serotype  

    O1 M

anis

a

O1 B

FS 1

860

O U

KG

12/

2001

A5 A

llier

A22

IRQ

24/

64

A24

Cru

zeir

o

C1 N

ovill

e

C3 R

esen

de

SAT

1 B

OT

1/6

8

SAT

1 T

155

/71

SAT

1 R

HO

12/

78

SAT

2 Z

IM 0

7/83

SAT

2 K

183

/74

SAT

3 Z

IM 4

/81

SAT

3 B

EC

1/6

5

Asi

a 1

Sham

ir

Asi

a 1

CA

M 9

/80

SVD

V U

KG

27/

72

5F10 Asia 1 1.9 0.7 0.7 1 2 0.8 0.3 0.2 0.6 0.8 1.6 0.1 1.6 0.9 02A4 Asia 1 1.9 1.6 0.7 0.8 2.6 1.1 0 0.4 0 0 0 0 1.8 1 05H5 A 1.9 1.5 1.1 0.8 2.5 1.1 0 0.3 0 0 0 0 1.2 0.8 02C12 A 1.2 1.6 1.6 0.9 1.7 1.6 1.6 013A6 SAT 1 1.3 1.7 1.6 1.1 1.8 1.6 1.4 06F6 C 0.9 2.1 2.5 0.4 0.8 1.8 1.2 04A3 C 1.9 0.5   0.7 1.1   2.2 0.5 1.1 0.4   1.4 0.4 1.8 0.2 1.8 0.8 0Polyclonal   2.5 1.3   1.2 1.8   2.7 1.3 2.1 0.6   2.5 1.2 2.3 0.5 2.1 1.5 1.8

The following table is a summary of the test evaluations of the prototype LFDs produced by Svanova and evaluated in the laboratory using epithelial suspensions (and some cell culture antigens) of FMD virus (plus SVDV and negative) sample submissions :

Svanova FMD LFD test results summarised as number of positive test reactions out of total number tested of each FMDV serotype plus SVDV and negative samples and compared to antigen ELISA

Virus ELISA FMDV mabtype 2C12 15F7 13A6 75405 C1 1D6 4D1 5B6 5H11 4A3 2A4 1F10 5H5

    (A22)(Asia 1) (SAT1) (A24)     (A Modena) (C)      

O 42/46 15/38 21/38 20/38 34/38 42/47 4/11 0/11 1/11 1/11 7/11 20/22 19/22 20/22A 22/26 5/20 11/20 11/20 19/20 22/25 2/5 0/5 1/5 2/5 1/5 16/16 15/16 15/16C 11/12 1/12 4/12 7/12 12/12 4/8 0/1 0/1 0/1 0/1 0/1 4/6 5/6 5/6SAT 1 14/16 2/16 9/16 11/16 16/16 11/16 0/2 0/2 0/2 0/2 1/2 7/9 5/9 6/9SAT 2 18/19 3/19 13/19 14/19 18/19 10/19 2/4 0/4 0/4 0/4 3/4 0/11 3/11 2/11SAT 3 6/6 0/6 3/6 3/6 6/6 1/5 nd nd nd nd nd 1/4 2/4 1/4Asia 1 18/19 2/15 8/15 10/15 15/15 19/19 0/2 0/2 1/2 0/2 1/2 10/12 12/12 11/12SVDV 7/7 1/7? 0/7 3/7 7/7 2/4 nd nd nd nd nd 0/2 0/2 0/2Negative 0/18 3/8 2/8 3/8 7/8 2/18? 0/3 0/3 0/3 0/3 0/3 0/11 1/11 0/11

SID 5 (Rev. 3/06) Page 9 of 22

The specificity of test reaction was found to be a problem with some of the LFDs using mabs 2C12, 15F7, 13A6 and 75405, while others (1D6, 4D1, 5B6, 5H11 and 4A3) performed poorly against a wide selection of divergent FMDV strains. More recently, other mabs (2A4, 1F10 and 5H5) have yielded more promising results, particularly against the more prevalent viruses of the O, A, C and Asia 1 serotype group, although it is true to state that both the degree and sensitivity of reaction against those of the SAT virus group is lower. Svanova are now attempting to generate LFDs using gold particles instead of latex beads to coat with mabs in the expectation that the sensitivity of reaction will be increased. We are also attempting to select either an existing SAT mab or mabs or to prepare more SAT mabs to create a mab cocktail to increase the pan-reactivity of test reaction. The progress of these studies will be reported on in next year’s annual report.

Another FMD LFD (Biosign) produced by Princeton BioMeditech Corporation, New Jersey, USA has also been evaluated, with results summarised below :

Biosign FMD LFD results summarised as number of positive test reactions out of total number tested of each FMDV serotype plus SVDV and negative samples and compared to results achieved from antigen ELISA

Virus ELISA Biosigntype    O 40/41 37/42A 18/21 18/21C 10/11 9/11SAT 1 10/12 0/12SAT 2 13/14 0/14SAT 3 5/5 0/5Asia 1 16/16 12/16SVDV 5/5 0/5Negative 0/13 0/13

This device would thus appear to work specifically for the detection of type O, A, C and Asia 1 FMD virus strains, although it does not detect FMD viruses of the SAT virus group. The intention is to extend the evaluation of this device using more FMD viruses and to validate further its specificity by examining more negative samples (both from naïve animals and using suspensions of submitted samples shown to be ELISA/virus isolation/PCR negative).

Prototype LFDs for FMDV antigen detection have also been received from Indian Immunologicals but have been found to have poor specificity of test reaction.

Prototype VSV and SVD LFDs have been received from Svanova. VSV and SVD epithelial suspensions have now been prepared for LFD test evaluation and the results will be reported on in next year’s annual report.

Objective 3

It has not been possible to undertake any work against this objective as suitable LFDs have either yet to be produced for field validation or not fully validated in the laboratory.

Objective 4

A variety of cell lines have been obtained to investigate their sensitivity to FMD (plus SVD and VS, where appropriate) virus replication and namely, MAX (SV40 immortalised swine kidney), bovine turbinate, bovine kidney, new-borne swine kidney, new-borne pig trachea, pig ileum, LLC-pig kidney cell lines, a human carcinoma cell line (SW) and Athos, a procine thyroid cell line in comparison with our routinely employed primary bovine thyroid (BTY) cells and pig kidney cell line (IB-RS-2). Epithelial suspensions of samples containing FMDV or SVDV plus cell culture grown antigens were titrated in the respective cell cultures and the results are summarised in the following Table :

Comparative titrations of epithelial suspensions (and cell culture grown antigens, cc) of FMD, SVD and VS viruses in cell cultures (titres in log10 TCID50/ml)

SID 5 (Rev. 3/06) Page 10 of 22

                             

Cell culture type FMDV SVDVVSV-NJ

  O B

RA

1/9

2

O U

AE

2/20

03

O T

AW

9/9

7

O1

BFS

186

0 cc

O1

Kau

fbeu

ren

cc

A IR

N 7

/97

C3

Res

ende

SAT1

TA

N 4

4/99

SAT2

ER

I 9/9

8

SAT3

BEC

1/6

5

Asi

a 1

MA

Y 1

4/92

UK

G 2

7/72

UK

G 2

7/72

cc

cc

IB-RS-2 5.95 5.95 6.45 3.7 6.45 6.45 5.45 6.2MAX (SV40 immort 5.45 5.2 5.45 <3.2 6.2 6.7 5.2 5.7swine kidney) BTY 6.45 NT NT NT 7 4.95 7.45 7.2 5.45 6.2 NT NT NTBovine turbinate 5.7 NT NT NT 5.2 3.2 5.95 5.95 3.7 4.95 NT NT NT(P16-19) BTY 7.45 6.7 <2.2 NT 7.45 5.2 7.45 6.7 6.2 6.95 NT NT 2.45?IB-RS-2 6.45 6.95 6.2 NT 6.45 3.95 7.45 6.7 5.95 6.2 4.45 8.2 3.2NSK (Newborn swine kidney) 3.2 5.2 5.2 NT 5.95 4.2 5.7 6.2 4.45 5.7 3.45 7.7 2.7NPTr (Newborn pig trachea) 4.7 4.7 4.95 NT 5.45 2.95 6.45 6.2 4.2 5.2 3.45 7.7 2.45 BTY 6.7 6.2 <2.2 8.95 6.45 4.7 6.7 7.2 6.45 6.45 NT NT <1.2BK 97 (bovine kidney) 6.45 5.45 <2.2 >8.2 6.2 4.45 6.95 6.7 5.95 5.95 NT NT 2.45 BTY 6.7 6.95 <2.2 8.95 6.95 4.7 6.7 7.2 6.45 6.95 NT NT <1.2IB-RS-2 NT NT 6.2 >8.2 NT NT NT NT NT NT NT NT ?BHK-21 NT NT NT NT NT NT NT NT NT NT NT NT 4.2MDBK <2.2 <2.2 <2.2 >8.2 <2.2 <2.2 <2.2 <2.2 <2.2 <2.2 NT NT <1.2MDBK Clone 1 <2.2 <2.2 <2.2 <2.2 2.45 <2.2 <2.2 <2.2 <2.2 <2.2 NT 0 <1.2 BTY 6.2 8.45 6.95 6.45MDBK <2.2 7.2 <2.2 <2.2MDBK Clone 1C6 <2.2 8.45 <2.2 <2.2MDBK Clone 1G8 <2.2 7.95 <2.2 <2.2MDBK Clone 1F10 7.45 <2.2MDBK Clone 1F6 7.45 <2.2 BTY 6.95 9.2MDBK Clone 1G8 <2.2 7.2MDBK 1G8 D4 <2.2 6.2MDBK 1G8 E5 <2.2 7.45 BTY 7.2 8.2BK 5.45 7.7BK B7 5.2 8.2BK 3B11 <2.2 7.7 BTY 6.95 8.7BK97 p12 6.2 8.2BK p20 4.95 8.2BEK p8 6.7 8.7BK B7 4.2 8.45 BTY 6.45RS 5.45 7.95Pig Ileum IPI-21 3.7 <3.2 LLC-PK1 (DMEM) 5.2 3.45 1.7 6.95LLC-PK1 (med 199) 5.95 2.95 <1.2 7.2RS 6.7 6.7 2.2 7.95

SID 5 (Rev. 3/06) Page 11 of 22

BTY 6.7 3.2 2.95 BTY 6.2 7.7CT23 ≤2.2 7.45RS B p22 6.2 7.95RS B p72 5.2 7.95LLC-PK1 p6 ≤2.2 7.45? Procine kidney cell lineLK98 p5 4.95 8.45 Lamb kidney cell lineBEK p8 4.7 7.7BK97 p3 5.95 7.95BK97 p17 5.2 7.95MDBK p161 ≤2.2 7.7MDBK (BDVF) ≤2.2 7.45 SW (Human carcinoma) MK ND ND ≤1.2SW (Human carcinoma) B3 ≤2.2 ≤2.2 ≤1.2SW (Human carcinoma) B6 ≤2.2 7.7 4.6SW (Human carcinoma) B8 ≤2.2 3.2 ≤1.2BTY 6.95 8.45 7.2 Athos p89 (Clicks/RPMI) 5.95 6.45 Athos = pig thyroid cell lineAthos p90 (RPMI) 6.45 6.45Athos p90 (DMEM) 5.95 6.2RS B p30 5.7 5.45BTY 6.2 7.2

The conclusions are that the majority of these cell lines either do not recognise wild-type FMDV, have inferior sensitivity to our current cell cultures, which are routinely used for FMD/SVD diagnosis (i.e. primary calf thyroid and IB-RS-2 cells) or else the progress of a cytopathic effect takes too long or is difficult to recognise. However, a further ‘cell line’ of bovine kidney (BK) cells has only marginally inferior sensitivity for FMDV than primary calf thyroid cells is proving useful for propagating virus for virus strain characterisation studies.

Attempts to generate new cell lines can be summarised by detailing a paper - Characterisation of integrin expression in cell cultures used for the diagnosis of Foot-and-Mouth disease, King, D.P., Burman, A., Gold, S., Shaw, A.E., Reid, S.M., Jackson, T. and Ferris, N.P. – which is in preparation for journal submission.

AbstractThe ability to successfully propagate field isolates of foot-and-mouth disease virus (FMDV) is an important aspect of laboratory activities relating to the diagnosis and control of disease outbreaks. Unfortunately, the most sensitive and established cell systems for the isolation of FMDV possess undesirable characteristics such as limited life-span, poor or variable analytical sensitivity or contamination with other viral agents. These factors place limitations on their use and can hinder the logistics of routine FMDV diagnostics. The long term objective of this project is to engineer replacement cell lines with desirable characteristics suitable for routine in-vitro passage of FMDV. In an attempt to understand the surface determinants that are required for FMDV sensitivity, this study used real-time RT-PCR and flow-cytometry to measure the expression of integrin molecules by a panel of cell cultures. The most sensitive cell cultures were primary bovine thyroid cells and IB-RS-2 cell line which had highest levels of αvβ6and αvβ8 respectively, confirming the importance of these heterodimers as receptors for the virus. In contrast, expression of αvβ3 did not appear to correlate with sensitivity to FMDV in the cell cultures selected. A stable MDBK cell line generated after transfection with a plasmid containing bovine α6 demonstrated increased expression of cell surface αvβ6. Unfortunately, this line showed no increased sensitivity to a field isolate of FMDV, although binding of FMDV was enhanced compared with parental MDBK cells. These findings indicate that cellular attributes in addition to cell surface expression of the receptor for the virus may play a critical role in determining the sensitivity of cells to field isolates of FMDV.

IntroductionA variety of laboratory-based tools including isolation of virus (VI) in sensitive cell cultures can be used to provide an objective diagnosis of FMD from suspect clinical material. In terms of diagnostic sensitivity and speed, the performance of VI has been recently superseded by molecular methods such as RT-PCR. However, since the ability to successfully grow field isolates of FMDV is likely to be a crucial element of future vaccine production for emerging variants and will also support studies to assess the antigenic relationships of FMDV strains in reference laboratories, it is anticipated that there will still be a requirement to perform VI on field isolates of FMDV in the foreseeable future.

SID 5 (Rev. 3/06) Page 12 of 22

Unfortunately, many of the established cell systems for the propagation of FMDV possess disadvantages that hinder their routine use. In particular, the most sensitive cell cultures are primary bovine thyroid cells (BTY) which typically contain a mixture cell types: slowly proliferating sensitive cell populations can be overgrown by cells (such as fibroblasts) that are insensitive to FMDV. Furthermore, the requirement to regularly source thyroid glands for the preparation of BTY cells is expensive, labour intensive and requires skill and experience to produce suitable BTY cell monolayers. Alternative stable cell lines such as IB-RS-2 (a permanent porcine kidney cell line) have variable analytical sensitivity towards porcinophilic and other field isolates of FMDV, and are also persistently infected with classical swine fever virus (in house sequence data from RT-PCR) which can restrict their use in some laboratories. Although other cell lines are available for FMDV culture, it has been recently shown that many of these have lower sensitivity for field isolates of FMDV (Ferris et al., 2006).

The long term objective is to engineer replacement cell lines with desirable characteristics suitable for routine in-vitro passage of FMDV. A previous study attempted to immortalise BTY and piglet kidney (PK) cell cultures using oncogene transfection to generate stable cell lines that were sensitive to FMDV (Ferris et al., 2002). A number of immortalised cell lines proved stable upon repeated cell culture passage and many supported the growth of FMDV. However, none of these immortalised lines exhibited either the degree of sensitivity or the specificity for all virus serotypes and strains as shown by BTY and IB-RS-2 cell cultures. In light of these findings, the immediate goal of this current study was to understand the cell surface determinants that underpin susceptibility to infection by FMDV. Field strains of the virus utilise αv integrin heterodimers (particularly αvβ6, αvβ8, αvβ3, and to a lesser extent αvβ1) as cellular receptors. Novel real-time RT-PCR assays (utilising novel cDNA sequence data obtained for porcine integrin chains) were developed and used together with flow-cytometery to assess integrin expression in a variety of cell cultures that can be used for the propagation of the virus.

Materials and MethodsCloning and sequencing of porcine integrin subunits

The nucleotide sequence of porcine βv, β1, β3, β5, 6 and β8 were determined by RT-PCR and cDNA cloning. The design of the PCR primers (results not shown) was based on nucleotide similarities between the available GenBank sequences for other species. Full length coding sequences of porcine 6 and 8 were obtained as two overlapping fragments using separate RT-PCRs for the 5’ and 3’ ends. Briefly, template RNA was extracted from porcine tongue tissue or IB-RS-2 cells using Trizol® reagent, cDNA was synthesised and PCR products were separated by electrophoresis. Amplicons of the correct size were excised from the agarose gels and purified and cloned, after which three independent clones containing the PCR inserts were sequenced and assembled.

Development of real-time RT-PCR assays to measure integrin expression

Real-time RT-PCR assays were designed to detect bovine and porcine integrin subunits (results not shown). These assays were designed to target conserved regions of the in-house sequences for porcine integrin sub-units that were shared with sequence data for available bovine and ovine integrin chains held in the GenBank database. All assays spanned predicted intron/exon junctions. For each assay, standard curves were used to quantify mRNA copy number for the individual integrin transcripts.

Characterisation of Cell lines by real-time RT-PCR

The real-time RT-PCR assays were used to measure integrin mRNA expression in a variety of established cell cultures. These cell systems comprised bovine thyroid cells, IB-RS-2, bovine kidney (BK), bovine embryonic kidney (BeK), porcine kidney (LLC-PK) and lamb kidney (LK) cells. Briefly, cell suspensions were prepared by trypsination of cultures in 25 cm3 flasks. Cell concentration was estimated using a hemocytometer. Total RNA was extracted from the samples using Trizol®

reagent and RT set up and PCR was performed using the MagNA Pure LC with amplification in an Mx4000TM Multiplex Quantitative PCR System (Stratagene).

Characterisation of Cell lines by flow-cytometry

After trypsinisation, cell suspensions were incubated with the mouse anti-integrin monoclonal antibodies. After washing with FACS buffer, a secondary PE-conjugated antibody (goat anti-mouse IgG2A) was added and incubated. Cells were fixed in paraformaldehyde and level of integrin expression was determined (FACS-Calibur).

Sensitivity of cell lines for FMDV

The ability of each of the cell lines to detect a titration series of two O serotype FMDV viruses (UAE 2/2003 and O1 BFS 1860) was determined and the relative titre of the virus detected by these different cell lines was expressed as 50% tissue culture infective doses (TCID50/ml).

Transfection of cell lines with bovine beta 6 construct

Low passage cultures of Madin-Darby Bovine Kidney (MDBK) and bovine embryonic kidney cells (BEK) were used in the transfection experiments. An expression construct bovine β6 was prepared in pcDNA6 (Invitrogen). Plasmid pcDNA-6 was linearised by restriction-enzyme digestion with Sal I. Adherent MDBK and BEK cell cultures were transfected with

SID 5 (Rev. 3/06) Page 13 of 22

different concentrations (2 µg - 500 ng) of plasmid using Lipofectamine 2000 in OPTI-MEM media. After overnight culture, cells were typsinised and washed with fresh media and the selectable marker (blastacidin). After 7-14 days growth, surviving cells were cloned twice by single cell cloning in the presence of the selectable marker to generate homogeneous cell populations. After selection of cell clones with blasticidin, cell surface expression of the integrin heterodimer αvβ6 was determined by flow cytometry.

ResultsCell surface expression of integrinsAs expected, these data indicated that BTY cells expressed high levels of β6 integrin. In contrast, IB-RS-2 cells had higher expression of β8 sub-unit, a finding that which was corroborated by flow-cytometry confirming that high levels of β6 (and possibly β8) expression is a characteristic of effective cells for FMDV diagnosis. Additional data from engineered human SW480 cell expressing β3, β6 and β8 also showed that the β6 cell line was most sensitive to FMDV.

Comparative sensitivity of cell lines for FMDVTable 1 shows that both bovine thyroid and renal swine cell lines have the greatest sensitivity for a representative field isolate of FMDV (UAE 2/2003). Other cell lines tested did not have as high sensitivity against this isolate. Two of these cell lines (CT23 and MDBK) were completely insensitive (under these conditions), although the isolate BFS 1860 was able to grow in these cells indicating that they are not deficient in FMDV replication machinery

Table 1: Comparative sensitivity of cell cultures to FMDV. Values shown are TCID50/ml.

CELL CULTURE FMDV IsolateO UAE 2/2003* O1 BFS 1860†

Primary Bovine Thyroid BTY 6.20 7.70Porcine Kidney IB-RS-2 (p22) 6.20 7.95Porcine Kidney IB-RS-2 (p72) 5.20 7.95Porcine Kidney LLC-PK1 (p6) ≤ 2.20 7.45Bovine Kidney BK97 (p3) 5.95 7.95Bovine Kidney BK97 (p17) 5.20 7.95Bovine embryonic kidney BeK 4.70 7.70Madin-Darby bovine kidney MDBK ≤ 2.20 7.70Lamb kidney LK98 4.95 8.45Engineered Calf thyroid CT23 ≤ 2.20 7.45

*Field isolate (clinical epithelium) of FMDV†Cell culture adapted isolate O1 BFS 1860 can enter cells via an integrin-independent (heparin sulphate) pathway.

Generation of a stable cell line with increased expression of αVβ6Using the bovine β6 plasmid, 25 MDBK-derivative cell lines were generated. However, only 2 of these contained the full-length β6 transcript (determined by PCR) and only one line (1G8) demonstrated stable cell surface expression of αvβ6 (Figure 1). A high level of αvβ6 expression was maintained for over 1 month with this cell line in the presence of 10 µg/ml blasticidin. Preliminary experiments showed that FMDV bound more readily to the 1G8 cell line compared with parental MBDK cells (data not shown). To date, all attempts to transfect BEK cells with bovine β6 have been unsuccessful

V6 expression (10D5)

MDBK wild-type

MDBK.1G8

Presence of full length 6 CDS by PCR

1G8

1D10

1F6

1C9

1F10

3A3

Pla

smid

- 2.7 kb

V6 expression (10D5)

MDBK wild-type

MDBK.1G8

Presence of full length 6 CDS by PCR

1G8

1D10

1F6

1C9

1F10

3A3

Pla

smid

- 2.7 kb

SID 5 (Rev. 3/06) Page 14 of 22

Figure 1: Cell surface expression of bovine v6 integrin on the 1G8 MDBK cell line detected by flow-cytometry. The presence of full length 6 transcript in the 1F10 and IG8 cell lines was shown by PCR analysis (see agarose gel).

The sensitivity of the MDBK.1G8 cell line for FMDV was determined (as described for other cell lines - above). Unfortunately, these experiments showed no increased sensitivity of any of these lines to a field isolate of FMDV (Table 2), although binding of FMDV was enhanced with the 1G8 cell line compared with control MDBK cells.

Table 2: Sensitivity of engineered cell lines to FMDV (data not shown)

CELL CULTURE FMDV IsolateO UAE 2/2003 O1 BFS 1860

Primary Bovine Thyroid BTY 6.20 8.50Parent MDBK wt MDBK ≤ 2.20 7.20Bov.β6 transfected cell line MDBK.1G8 ≤ 2.20 8.00Bov.β6 transfected cell line MDBK.1C6 ≤ 2.20 8.50

DiscussionThe aim of this project is to engineer cell lines with characteristics suitable for the routine propagation of field isolates of FMDV. In these experiments, two separate approaches involving the introduction of bovine β6 or SV-5v transgenes into 2 bovine cell lines have been investigated. Successful introduction of the plasmid coding sequences and stable expression of the targeted protein in derivative cell lines was achieved using pcDNA6 and pEF-IRES for bovine β6 and SV-5v respectively. Although this initial characterisation data was encouraging, subsequent experiments have shown that none of the lines show increased sensitivity to infection by field isolates of FMDV.

ReferencesFerris NP, Hutchings GH, Moulsdale HJ, Golding J and Clarke JB (2002) Sensitivity of primary cells immortalised by oncogene transfection for the detection and isolation of foot-and-mouth disease and swine vesicular disease viruses. Vet Microbiol. 2002 Feb 4;84(4):307-16.

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

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the following accession numbers: DQ786569 (porcine beta 8), DQ786570 (porcine beta 6) and DQ786571 (porcine beta 5 fragment).

Generation of transfected cells lines that stably express the V protein of simian virus-5 (SV-5)An alternative approach used to try to increase the sensitivity to FMDV was to transfect cells to express the V-protein of the paramyxovirus, simian virus-5 (SV-5). SV-5v inhibits the response of type I and type II interferons due to interaction with the signalling protein, STAT1. Similar experimental protocols (as described above for α6) with plasmid (pEF-IRES-SV-5v Didcock et al., 1999: provided as a kind gift from Prof. R. Randall, University of St Andrews) with G418 (Invitrogen) as the selectable marker, linearised with Nde I) were used to attempt to generate stable MDBK and BEK cell lines expressing SV-5v. Resulting cell lines were characterised by PCR and confocal microscopy to detect SV-5v. Briefly, cover slips of adherent cells from each of the engineered cell lines were prepared by overnight culture in 24-well plates. Cells were fixed in 4% paraformaldehyde and washed twice in PBS. After permeabilising the cells with 0.1% Triton-X, primary monoclonal antibody (anti-V5-tag, Serotech) was added for 60 minutes. After further washes in PBS, the presence of SV-5v was detected using an Alexa488 conjugated secondary antibody visualised on a confocal microscope (Leica TCS SP2 confocal microscope). DAPI stain was used to locate cell nuclei. 11 MDBK (cells derived from bovine 6 expressing line 1G8) and 3 BEK stable cell lines were characterised. The presence of SV-5v coding sequences was confirmed by PCR in 7/11 MDBK (1G8) and 3/3 BEK cell lines. Confocal microscopy performed on 2 selected MDBK and 2 BEK cells lines demonstrated predominately intra-nuclear expression of SV-5v (Figure 4), similar to that previously reported for other cell systems (Young et al., 2003)

SID 5 (Rev. 3/06) Page 15 of 22

BEK wild-type control

BEK.B7 cell line

DAPI SV5-v

Merge

DAPI SV5-v

Merge

Figure 2: Intranuclear expression of SV-5v shown by confocal microscopy. Presence of SV-5v is predominantly located in the nucleus of cells (matches with DAPI staining: similar staining patterns were shown in a previous study Young et al., 2003).

Expression of SV-5v protein was predominately localised to the nucleus of MDBK and BEK cells. Type I and Type II interferons are produced in response to virus infection and mediate the establishment of an antiviral state. The aim of this approach was to attempt to increase the sensitivity of the cell cultures to FMDV by abolishing any anti-viral activity of interferons (including any bystander affect in neighbouring cells). Similar approaches have been recently used to generate cell lines for the detection of a variety of slow-growing wild-type viruses and vaccine candidate viruses (Young et al., 2003). However, neither MDBK cells nor BEK cells expressing SV-5v showed any evidence of increased sensitivity to field isolates of FMDV. These findings are possibly not unexpected, since FMDV rapidly shuts off cap-dependent translation, thereby limiting the role of interferon-mediated activity in infected in-vitro culture systems.

Possible future approaches to generate cell lines: Continue with experiments to transfect different parent cell lines with β6 and β8 integrin subunits. (Collaborative

project with Intervet BV may generate BHK cells expressing porcine αVβ6). Cloning of the sensitive cell components of BTY mixture using a cell sorter to select high expressers of αVβ6 Immortalisation of sensitive cells using telomerase or oncogene systems. (This work is planned in a current project

by T. Jackson and cells will be provided to us for FMDV sensitivity characterisation).

Objective 5

Introduction and ObjectivesUnambiguous viral identification with degenerate PCR is often complicated by the existence of highly homologous relatives. Most importantly, when the goal is to detect and identify an unknown agent, even the broadest multiplexed PCR is inherently biased. The capacity of microarrays to perform numerous assays on the same sample material increases this experimental range by many orders of magnitude. Microarrays enable broad-spectrum detection, or when strain level fingerprinting is necessary for source-tracking based on multiple genetic signatures. A first-generation microarray consisting of probes covering 150 viral ‘species’ has been developed by a consortium of the Veterinary Laboratories Agency and the Institute for Animal Health laboratories at both Compton and Pirbright. The WRL at Pirbright is validating the microarray’s ability to serotype and subtype veterinary viruses using Foot and Mouth Disease Virus (FMDV) as a model.

Material and methodsPhylogenetic trees were reconstructed (using MEGA2) from capsid gene sequence data available both in the public domain and generated in-house (N. Knowles, pers. com.). From these phylograms, clades or ‘genogroups’ were inferred. For FMDV, a ‘consensus’ or stereotyped sequence for each genogroup was calculated and used as the basis to design ~300 70 base oligonucleotide probes using UFOHunter Software (http://cgi.uc.edu/cgi-bin/kzhang/UFOHunter.cgi). Oligos were synthesised commercially and spotted to Epoxy-coated glass slides using a Migrogrid II robot (Biorobotics) using previously published protocol1. Total RNA was extracted from cell-cultured viruses (FMD O UKG/35/2001, A10 HOL/42, O TAW/3/97) and from epithelium of sheep 2dpi with O UKG 12/2001 (RNeasy, Qiagen and MagNa Pure LC Total Nucleic Acid Isolation Kit Roche), reverse transcribed (MultiScribe reverse transcriptase Applied Biosystems) amplified and labelled with Cy3 and hybridised1. Slides were scanned using a Hewlett Packard ScanArray Lite, fluorescence was quantified using ScanArray Express 2.0. Data were normalised to total fluorescence and median signal:noise ratios were calculated for each hybridisation.

SID 5 (Rev. 3/06) Page 16 of 22

Figure 1. Hybridisation profiles obtained from cell-cultured FMDV isolates. A & D = scanned image & fluorescence profile of A10 HOL/42; B & E = O UKG 12/2001; C& F = O TAW 3/97. White circles indicate fluorescing spots specific to the isolate used.

ResultsVisual inspection of scanned images showed serotype specific spots fluorescing when cell-culture viruses of different serotypes were hybridised (Fig 1A and B). Analysis revealed different hybridisation ‘profiles’ belonging to serotype A and serotype O (Fig 1D &1E). Two viruses belonging to different topotypes2 (O TAW 3/97 and O UKG12/2001) also showed differential spots fluorescing when scanned images were inspected visually (Fig 1B & 1C). Quantitative analysis of fluorescence also revealed different hybridisation ‘profiles’ (Fig 1E & 1F). Hybridisations of RNA isolated from the epithelia of sheep experimentally infected with O UKG 12/2001 showed different ‘spots’ fluorescing (Fig 2A & B), and quantitative analysis gave different profiles. By ‘normalising’ the data from the ‘infected’ with that of the ‘uninfected’ a much cleaner viral signature was produced (Fig 2C).

Figure 2 Hybridisation profiles obtained from RNA isolated from epithelia of uninfected sheep (A) and sheep infected with O UKG 35/2001 (B). C = Viral signature obtained from data ‘normalised’ to uninfected control. White circles indicate spots that fluoresce specifically in infected tissue.

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Discussion and ConclusionsThis first-generation microarray is capable of discriminating different serotypes of FMDV. Different topotypes within a serotype (15% diveregence in nucleotide sequence of VP1) also appear to give different ‘signatures’. Viral signatures are also detectable in ‘real’ samples from infected animals, and normalisation to an uninfected control reduces the majority of noise. A second-generation array has now been printed that includes oligos for all vesicular diseases and known picornaviruses in order to differentially diagnose FMD. This second-generation array has the capacity to distinguish 250 viral species from 29 virus families. Current work at Pirbright is focused on validating this array with further topotypes, and related picornaviruses.

References1. Burton, J.E., Oshota, O.J., North, E. , Hudson, M.J, Polyanskaya, N., Brehm, J., Lloyd G. and Silman, N.J.

Development of a multipathogen oligonucleotide microarray fordetection of Bacillus anthracis Molecular and Cellular Probes 19 (2005) 349–357

2. Knowles, N.J, and Samuel, A.R Molecular Epidemiology of Foot and Mouth Disease Virus. Virus Research 91 (2003) 65-80

Objective 6

Fever and the production of blisters on the feet and tongues are prominent clinical features of acute infection of ruminants and pigs with foot-and-mouth disease virus (FMDV). Inflamation and the resultant production of heat and generalised fever may be detectable by thermal imaging and a preliminary study was conducted to evaluate this.

Cattle, sheep and pigs were infected with FMDV (O/SKR/2000) and then monitored for clinical signs and temperature for 4 days. Thermal imaging with a recording camera, Thermoview model Ti30, 8-13um wavelength (Raytek) was used to monitor the surface temperatures of the feet of all in-contact animals once a day from 0 – 4 days after inoculation of donor animals. The average foot surface temperature (ASFT) and the hottest part of the foot or hottest foot surface temperature (HFST) were compared to rectal measurements of core body temperature.

Pigs consistently showed the largest increases in HFST after infection. The images of the pigs show noticeable changes through the course of the infection both by contact and inoculation. Pig, day 0, image 1 - shows cold feet around 19oC and rather warmer thighs 30oC.

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Thermal Image 1

Pig, day 1, image 2 - shows all feet cold with body surface temperature, 31.9oC.

Thermal Image 2

Pig, day 2, image 3 - shows all feet hot with body temperature, 33oC.

Thermal Image 3

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Pig, day 3, image 4 - shows hot pig, temperature up to 34oC.Thermal Image 4

Pig, day 4, image 5 - shows hot pig, temperature up to 33.8oC.Thermal Image 5

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Ruminants were less likely to present with hot feet that could be directly attributable to FMDV infection and hot feet were sometimes observed at day 0, e.g. thermal image 6 (cattle, day 0) shows hot right fore left inner claw and both legs with a maximum temperature of 26.5oC.

Thermal Image 6

The camera and the associated software for image analysis were simple to use. Areas of inflammation accompanied by heat showed clearly on the images. Although the progression of disease could be followed to some extent, the practical value of thermal imaging for the diagnosis of FMD seems limited in ruminants because of the variable temperatures detected prior to infection. In pigs there appeared to be a better correlation between progression of disease and hot extremities, although more measurements are needed, especially from uninfected animals. In the field, the camera might be used to help single out animals for examination and sampling.

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.Peer reviewed publications:

Ferris, N.P., Abrescia, N.G.A., Stuart, D.I., Jackson, T., Burman, A., King, D.P. and Paton, D.J. (2005). Utility of recombinant integrin αvβ6 as a capture reagent in immunoassays for the diagnosis of foot-and-mouth disease. Journal of Virological Methods 127, 69-79.

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).

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

Presentations:

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, 102.

King, D.P., Shaw, A.E., Reid, S.M., Hutchings, G.H., Jackson, T. and Ferris, N.P. (2004). Towards the development of engineered cell lines for FMDV diagnosis. 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 49, 316-320.

King, D.P., Reid, S.M., Shaw, A.E., Hutchings, S.M., Paton, D.J and Ferris, N.P. (2004). A ring test for the lab detection of FMDV by VI and 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

Nigel Ferris, Nicola Abrescia, David Stuart, Terry Jackson, Alison Burman, Donald King and David Paton (2004). Recombinant integrin αvβ6 as a capture reagent in immunoassays for the diagnosis 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 50, 321-330.

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.

Paton, D.J., Ferris, N.P., Knowles, N.J., Valarcher, J-F., Newman, B., King, D.P., Reid, S.M., Dukes, J. and Parida, S. (2005). 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.P., Knowles, N.J., Valarcher, J-F., Newman, B., King, D.P., Reid, S.M., Dukes, J. and Parida, S. (2005). 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.

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