Appendix 79

Appendix 79

POSTER SESSION

Chairperson: Dr Nilay UNAL Rapporteur : Dr Stéphan ZIENTARA Dr Georgi GEORGIEV

At the Open Session of the EUFMD Research Group Meeting 17 posters were presented. These posters deal with -Epidemiology (1) -FMDV structure (1) -FMD vaccines (2) -Pathogenesis (4) -Diagnosis (molecular diagnosis-7) (serological diagnosis-2)

Recommendations of the Poster Session

The reporting group would like to underline the fact that the decision of the FMD Research Group to organise such a poster session was a good initiative.

On the basis of the presented posters in this session the reporting group makes following recommendations:

- necessıty to reinforce the surveillance of FMD around the world in particular in Africa - develop studies on the structure of the FMDV proteins in order to better understand their fonctions and develop antiviral molecules - develop reagents (recombinant antıbodies or monoclonal antibodies) for diagnosıs or studies on pathogenesis - develop and reinforce studies on the pathogenesis of FMD and the interactions virus\cells - develop studies on the immune response against FMDV in susceptible species - develop studies on the molecular diagnosis of FMDV (real-time PCR, loop mediated amplification, rolling circle amplification) - develop tools for rapid and reliable diagnostic method for serological diagnosis of FMD - necessity of comparative studies, validatıon and standardisatıon new diagnostic methods

Summary of the posters presented

Identification of FMDV replication in cells within the foot and tongue epithelia

S. Durand1*, C. Murphy1, S. Alexandersen1,2 . 1 Pirbright Laboratory, Institute for Animal Health, Ash Rd, Woking, Surrey, GU24 0NF, UK. 2 Present address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK4771 Kalvehave, Denmark.

This study deals with identification of FMDV replication sites by in situ hybridisation in pigs.

In situ hybridization (ISH) has been used to detect FMDV RNA in tissues from infected pigs. A digoxigeninlabelled RNA probe corresponding to a coding part of the RNA-dependent RNA polymerase (3D) genomic region was prepared.

Results indicate that the basal cell appears to be the cell type demonstrating the highest signal for the detection of the FMDV positive sense RNA in both tongue and foot epithelium. The detection FMDV positive sense RNA showed very strong signal in basal cells (especially in foot lesions). Mouth lesions showed in general less signals than in foot lesions.Although the stratum spinosum cells show more signs of cytolysis than the basal cells, the FMDV RNA signal in the stratum spinosum cells was more diffuse and less concentrated. These results are strongly suggesting that the basal cells could be the early replication site of FMDV in vivo.

Laser Micro-Dissection studies of FMDV infection in pigs

R. Ahmed1*, S. Durand1, Z. Zhang1, M. Quan1, C. Murphy1 and S. Alexandersen2 1Institute for Animal Health, Pirbright, Woking, Surrey, GU24 ONF, U.K. 2Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK-4771 Kalvehave, Denmark

The objective of this study was to isolate and quantify foot-and-mouth disease virus (FMDV) in the different epithelium cell-types in order to observe potential differences in FMDV RNA distribution in specific tissues.

Laser Micro-Dissection (LMD) was carried out on frozen sections from selected tissues of infected pigs. After RNA extraction the samples were tested for FMDV and 18S ribosomal RNA (as a RNA marker) by real time quantitative RT-PCR.

The data suggest that there is more virus detected in the epithelium of skin, especially the stratum basal cells, than in tongue and soft palate. The differences in virus level in epitelia may be a contributing factor to the formation of vesicles in certain tissues.

The pathogenesis of FMD in young lambs

E. Ryan1*, S. Durand2, J. Brownlie1, S. Alexandersen2,3 1 The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK. 2 Pirbright Laboratory, Institute for Animal Health, Ash Rd, Woking, Surrey, GU24 0NF, UK. 3 Current address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK4771 Kalvehave, Denmark.

This poster presents the first study on the pathogenesis of FMD in young lambs using quantitative PCR and in situ hybridization (ISH).

The pathogenesis of FMD was characterised by measuring viral RNA levels in daily serum and nasal swab samples and in post-mortem tissue samples, measured by quantitative RT-PCR.

FMDV in lambs initially dermotrophic, can became myocardiotropic and myotropic with fatal results.

FMDV RNA detection in pig samples by conventional and Real Time RT-PCR methods

I. Avalos*, A. Izquierdo, A. Llorente, A. Canals and E. Blanco Centro de Investigación en Sanidad Animal (CISA-INIA). Valdeolmos, Madrid 20130. Spain.

The main aim of the present study was to assess the suitability of different RT-PCR protocols for the FMDV RNA detection and/or quantification in samples collected from pigs experimentally infected.

FMDV RNA was detected in blood samples of infected pigs only until 3 d.p.i The biopsy samples were all positive, only kidney samples were negative. The quantification of viral RNA in these samples, indicated that the lymph nodes have the greater viral RNA concentration, while the liver are the samples with less quantity of FMDV.

The results obtained in this study suggest that pharingeal swabs are samples more suitable to perform the FMD diagnosis at least during the first 10 days of infection.

Immunogenicity and protection conferred by synthetic peptides that reproduce B and T epitopes of FMDV in pig

I. Avalos1*, A. Canals1, C. Cubillos1, H. Serrano1,2, E. Borras3, D. Andreu3, F. Sobrino 1,4 and E. Blanco1 1Centro de Investigación en Sanidad Animal (CISA-INIA). Valdeolmos, Madrid 20130. Spain. 2Dpto.Biologia. Universidad de Puerto Rico, Arecibo (P.Rico-USA) 3Dpto.Química Orgánica. Universidad Pompeu Fabra, Barcelona. Spain 4Centro de Biología Molecular “Severo Ochoa”, Madrid. Spain

The authors analysed the immunogenic potential conferred in pigs afforded by 3 synthetic peptides which included B (sites A and D) and T (protein 3A) cell epitopes of FMDV, and the protection induced after a challenge with an homologous strain.

Significant neutralization antibodies and lymphoproliferative responses were detected in the three groups of pigs immunised. The result show that this peptide TB maintain the immunostimulatory ability of B- and T-cell components. Although the vaccination protocol followed with these primers were not enough to prevent the FMD, the clinical findings and the low detection of viral RNA in swabs of the immunised pigs are consistent with a partial protection and with a restricted viral transmission by the respiratory via.

Diagnostic differences between real-time RT-PCR and virus isolation tests on cattle probang samples following infection with FMDV

C. Doel1, Z. Zhang1 J. Bashiruddin and S. Alexandersen1,2 1Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey, GU24 0NF, U.K. 2Present address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK4771 Kalvehave, Denmark

This report highlights an elevated incidence of mismatches between Real Time PCR and virus isolation with probang samples taken between 6 and 21 days post-infection (dpi) from experimentally infected cattle.

For the period 1 to 5 dpi there was a 5% mismatch between PCR and virus isolation tests. For the period 6 to 21 dpi, about 77% (52/68) of the samples were positive by RT-PCR compared to about 29% (20/68) by virus isolation. In 50% (34/68) of samples the RT-PCR and virus isolation results did not agree. The greatest disagreement of 75% (12/16) was on 7 dpi.

The possible technical and biological reasons for these discrepancies between real-time RT-PCR and virus isolation such as implications of the quality of probang samples, the cell cultures, automated extraction, the presence of antibodies and stage of the disease are discussed.

A novel, but simple molecular method for detection of FMDV

J.P. Dukes1, D.P. King1 and S. Alexandersen2 1Division of Epidemiology, Pirbright Laboratory, Institute for Animal Health, Pirbright, Woking, Surrey GU24

ONF; Juliet.dukes@bbsrc.ac.uk

2 Current address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK4771 Kalvehave, Denmark.

This study describes loop mediated amplification (LAMP) which is a novel technique that amplifies specific nucleotide sequences under isothermal conditions Unlike PCR, a denatured template is not required and the amplified product of LAMP can be visualised with the naked eye.

LAMP may prove a cost-effective method for the detection of FMDV genomes at the pen-side, in the field and by developing countries.

Towards the construction of an O1 Manisa chimeric vaccine

V. Fowler1, E. Rieder2, T. Henry2, D. Paton1and P. Barnett1 1Pirbright Laboratory, Institute for Animal Health, Ash Rd, Woking, Surrey, GU24 0NF, UK. 2Plum Island Animal Disease Center, Agricultural Research Service, Greenport, New York 11944.

The first stage in the construction of a vaccine with improved marker characteristics, based on the widely used vaccine strain, O1 Manisa is described.

A full -length cDNA clone of O1 Manisa was constructed using an exchange-cassette strategy based on an existing A12 full-length cDNA clone.

After this step, the major antigenic site, the G-H loop of VP1 (residues 132-159), will be replaced with the corresponding region of alternative serotypes (A24 Cruzeiro and C3 Resende) in order to enhance the marker characteristics of the vaccine.

Production of monoclonal antibodies against the three SAT serotypes of FMDV, helpful as standardised reagents in diagnostic assays

S. Grazioli*, D. Gamba and E. Brocchi

Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy

The authors have developed panels of MAbs specific for each of the three SAT serotypes.

FMDV SAT 1; out of 35 MAbs raised against type SAT 1, strain Botswana 1/68, 27 neutralise virus infectivity; 10 of them do not recognise in WB isolated viral proteins, suggesting they identify conformation-dependent epitopes, while 17 are WB-positive and presumably react with VP1. FMDV SAT 2; 22 MAbs were raised against type SAT 2, strain Zimbabwe 5/81. Five MAbs with neutralising activity recognise a linear epitope in WB, 13 further neutralising MAbs, negative in WB, detect conformational epitopes. Out of 5 non-neutralising MAbs, 3 react as conformation-dependent MAbs, while one clearly positive in WB recognised a linear epitope, presumably located on VP2. FMDV SAT 3; only 2 weak positive MAbs specific for type SAT 3 were produced. All MAbs from the three panels are type-specific, except one that cross-reacts with the seven FMDV serotypes.

Sets of MAbs specific for FMDV type SAT 1 and SAT 2 are available.

Analysis of possible genetic differences in selected tissues from cattle with acute and persistent FMDV infection

J. Horsington1, J.B. Bashiruddin1, Z. Zhang1, C. Doel1 and Soren Alexandersen1,2 1Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey, GU24 0NF, U.K. 2Present Address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK4771, Kalvehave, Denmark.

This paper explores the primary structure of some areas of the genome to gauge its variability in tissues from several disease stages.

Cattle were exposed to FMDV type O UKG/34/2001 and tissue samples from epithelial lesions during the acute stage and the dorsal soft palate of carrier animals were collected. Parts of the IRES, leader, 2B, 2C, 3C and 3D genomic areas derived from the epithelium and nasopharyngeal tissues were sequenced. Some genetic variability was seen in the virus from different stages of disease. Whole genomic sequencing of these RNAs is currently underway and will allow a broader assessment of the viral genetic heterogeneity within infected animals.

A ring-test for the laboratory detection of FMDV by RT-PCR and virus isolation

D.P. King*, S.M. Reid*, G.H. Hutchings, A.E Shaw, N.P. Ferris*, and D.J. Paton

Institute for Animal Health, Pirbright, Surrey, GU24 0NF, UK.

The paper describes organisation the ring test which will provide valuable comparative data on the relative performance of virus isolation (VI) and the different RT-PCR methods.

The IAH, Pirbright Laboratory will co-ordinate a proficiency panel for FMDV virological testing.

Purification of RNA dependent RNA polymerase and its predicted three-dimensional structure

K. Sakamoto, S. Ohashi, R. Yamazoe, K. Morioka, K. Yoshida Department of Exotic Disease, National Institute of Animal Health, Kodaira, Tokyo, 187-0022, Japan.

The authors describe the purification of FMDV RNA dependent RNA polymerase, 3D protein in order to determine the predicted three-dimensional structure.

The 3D protein was purified by affinity chromatography with Ni Sepharose.

In near future the authors have planned to determine the structure of the crystallized 3D protein by X ray analysis.

Analytical validation of One-Step real-time RT-PCR for FMDV RNA

S. Van Borm, N. Goris, M. Staes, I. Musch and K. De Clercq

CODA-SERVA, Department of Virology, Groeselenberg 99, 1180 Ukkel, Belgium

This poster describes the use of a real-time Reverse Transcriptase PCR (real-time RT-PCR) for the detection of serial dilutions of FMDV RNA, purified from the supernatant of virus productions on cell cultures.

Viral RNA from 82 different strains (29 strains for serotype A, 10 for serotype Asia and 43 for serotype O) was extractedand Real-Time RT-PCR was performed.

This work shows that the discussed technique is able to detect a broad range of FMDV subtypes and serotypes.

Production of a single-chain variable fragment antibody against foot-and-mouth disease virus

Yong-Joo Kim, Jee-Yong Park, Soo-Jeong Kye, Jong-Hyeon Park, Jae-Ku Oem, Kwang-Nyeong Lee, Nam-In Jo Foreign Animal Disease Research Division, National Veterinary Research and Quarantine Service, Anyang 6 dong 480, An Yang, Kyeong gi, 430-824, Korea

[Yong-Joo Kim; Tel: 82-31-467-1858, Fax: 82-31-449-5882, E-mail: kimyj@nvrqs.go.kr]

This poster describes the construction of a scFv antibody from the murine monoclonal antibody (mAb) 16A11, specific for foot and mouth disease virus (FMDV) 0/SKR/2000, and its active form produced in Escherichia coli.

The sequencing results showed that the VH gene of constructed scFv was composed of germline VH1S6DFL16.2-JH2 and gremlin VK1-135-JK1 for the VL gene and the expressed scFv protein (about 30kDa) was detected on SDS-PAGE and immunoblotting. ELISA results showed that scFv retained almost the same antigen affinity and specificity as its parent mAb.

The scFv antibody against FMDV has been successfully constructed and produced, which lays the foundation for further study and applications.

The Development of an indirect ELISA for the detection of antibodies to the non-structural protein 3ABC of the Foot and Mouth Disease virus; The use of a polyclonal conjugate that allows a multispecies detection of antibodies

K. Persson, A. Nordengrahn, C. Decker and M. Merza

Svanova Biotech AB, Uppsala Science Park, SE-751 83 Uppsala, Sweden

This communication presents an indirect ELISA for the detection of antibodies to the 3ABC antigen of FMDV in the sera of Bovine, Swine, Ovine and Caprine utilising a newly devised polyclonal conjugate.

The bovine sera showed, as expected, a sensitivity and specificity of 100% using a PP cut-off value of 49.7. When testing the sheep sera, the ELISA showed a sensitivity of 89.2 and a specificity of 100% at a cut-off PP of 30. When testing the swine sera a sensitivity of 95.8 and a specificity of 100% were obtained at a cut-off PP of 30.

By using the test system presented in this communication, i.e. using the 3ABC protein as a coating antigen and a multi-species conjugate as the detection antibody one can overcome the problems of variations in the antibody response to the NS proteins.

Complexity of foot-and-mouth disease outbreaks in Kenya

A.K Sangula1*, H.O Oyas2, G.R. Thomson3 1Foot-and-Mouth Disease Laboratory, Embakasi, P.O.Box 18021, 00500 Enterprise Road, Nairobi, Kenya. 2 Veterinary Research Laboratories, Kabete, P.O. Kabete, Nairobi, Kenya. 3African Union (AU) Inter African Bureau for Animal Resources (IBAR), P.O. Box 30786, Nairobi, Kenya.

This paper provides a description of the viral diversity and epidemiological complexity of FMD in Kenya.

Five FMDV serotypes (O, A, C, SAT 1 and SAT 2) were recorded in outbreaks in various parts of Kenya in the year 2003 and the first half of the year 2004. Serotype O caused majority outbreaks followed by SAT 2, SAT 1 and A while serotype C was responsible for only one outbreak. Vaccine strains show varying immunological relationships to field strains.

These studies have indicated that FMDV serotypes in Kenya belong to different lineages and genotypes. Serotype SAT 2 exhibits the highest intratypic variation. This is complicated by the fact that the extent of transmission of FMD between wildlife and domestic livestock has not been investigated in any detail.

Rolling Circle Amplification for detection of FMDV

Mik Staes, Frank Vandenbussche, Steven Van Borm, Nesya Goris and Kris De Clercq CODA-SERVA, Department of Virology, Groeselenberg 99, 1180 Ukkel, Belgium

The authors describe the use of the rolling circle amplification technique (RCA) for the detection of FMDV. RCA was shown to allow detection of cDNA, prepared from viral RNA which was purified from the supernatant of virus productions on cell cultures.

First, the RCA technique was optimized on a synthesized template with the exact sequence of a 29 bp region in the 3D gene of the FMDV RNA. In a next step, RCA was optimized on double stranded cDNA generated from the viral RNA after a reverse transcription step which was followed by normal PCR amplification.

Starting from purified FMDV RNA, RCA allows detection after cDNA generation of the target sequence. In future experiments, serial dilutions of the purified RNA will be tested. Moreover, RCA will be optimized either with the viral RNA directly as a template, or after a single reverse transcription step. Finally, the technique will be validated on spotted saliva and field samples.