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Archive: 1999 Session - Appendix 24

1999 Session of the Research Group of the Standing Technical Committee of EuFMD



Improved SVD diagnosis : an overview of the EU research project PL96-1545 on SVD.[1]


Kris De Clercq[2]
[1] Manuscript based on the Consolidated Progress Reports of the project made by the Co-ordinator and all Partners.
[2] Chairman of the Research Group of the Standing Technical Committee of the European Commission for the Control of FMD.



The goals of this project are to investigate and develop reagents and methods which will improve the diagnosis of swine vesicular disease (SVD). A particular interest is the resolution of the singleton reactor (SR) problem. The project has focused on three specific objectives:

To improve virus detection.
Development of accurate and versatile ELISA and PCR techniques for the detection of small traces of SVD virus in nasal swabs, faeces and tissues. Optimisation of in-situ hybridisation and in-situ PCR techniques to detect virus in post mortem organs, and characterisation of SVD virus isolates to identify the origin of new outbreaks, patterns of spread and viral evolution.

To improve serology.
Development of novel and simple ELISA based on recombinant and synthetic antigens, as well as novel techniques to detect SVD virus-specific cellular immune response and serological methods including isotype (IgG/IgM) analysis of the sera to study the evolution of the disease.

To study viral pathogenesis.
The application of the above techniques will be applied to study the duration of SVDV infection as well as the identification of cells and organs involved in viral persistence.




The Options


The options for disposal of animal carcasses on a routine basis e.g. casualty animals, pets etc. are generally prescribed by law in most countries. Directive 90/667/EC on animal disposal confirms the acceptable disposal routes as a treatment or processing plant; or burning; or burial.
For small scale disposal problems these regulations are generally acceptable, but it is not always possible or safe for the environment for these methods to be used on a large scale such as large scale animal disease epizootics like those mentioned in the introduction. While many countries have guidelines on what to do with carcasses slaughtered in contagious disease emergencies, it is not clear if there are any scientific studies demonstrating the safety of these protocols for the environment.
The only literature that comes close to approaching this problem is that of agricultural engineering, where the engineers have contemplated the environmental safety of disposing of the regular and often large numbers of mortalities associated with intensive poultry and pig production. However, this literature generally prescribes measures that appear to be insufficient in dealing with highly contagious animal disease and would have huge difficulties in scaling up from 5% of a herd capacity to full herd capacity. (I have listed some of these methods in the Other Potential Options section below) This leaves us with the three generally prescribed methods of carcass disposal, rendering, burning and burying, and the advantages and disadvantages of these methods are discussed below. In order to stimulate discussion and some lateral thinking, I have also listed some non-standard solutions to this problem in the section titled Other Potential Options below.



Tasks and Results


Task 1. Development of protocols for SVD antigen detection using MAbs and polyclonal antibodies.
Standardisation and validation of simple and rapid tests based on a selected panel of Mabs Three pairs of catcher/tracer Mabs were selected during the first year as best combinations for antigen detection in pathological materials and in infected cells cultures. These combinations allow the detection of all antigenic variants of SVDV occurred to date, as well as the possibility to classify the SVDV isolates into one of the 4 known antigenic groups, directly from the pathological sample. An additional Mab (2A12) has been selected as a universal detector, to be used in the protocol for the identification of SVDV in pathological samples.



Task 2. Development and validation of highly sensitive and specific RT-PCR methods for SVD detection in animal samples and tissues.
Direct RT-PCR will be used for differential diagnosis of several swine diseases (SVD, FMD, VS) in a single assay by using a combination of specific primers. Results are published in :
Vangrysperre W. and De Clercq K. (1996). Rapid and sensitive polymerase chain reaction based detection and typing of foot- and mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related viruses. Archives of Virology 141, 331-344.
J.I. N“Àez., E. Blanco, T. Hernöndez, C. G…mez-Tejedor, M.J. MartŒn, J. Dopazo and F. Sobrino. A RT-PCR assay for the differential diagnosis of vesicular viral diesases of swine. J. Virol. Methods. (1988) 227-235.
Improvement of sensitivity will be achieved by nested PCR (Lin, F., Mackay, D.K.J. and Knowles, N.J., 1997. Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction. J. Virol. Methods, 65, 111-121.) and PCR-ELISA (Callens, M. and De Clercq, K., (1999). Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection. J. Virol. Meth. 77, 87-99.).
Improvement of PCR protocols and Immune PCR will be used to avoid Taq polymerase inhibitors present in faeces. In-situ PCR will be used directly on tissue samples. The results will be compared with those obtained with MAb-ELISA Ag detection test and virus isolation (VI).
All VI, ELISAÎs and PCR tests were validated on material from experimental infected pigs (Mackay, D.K.J. and Wilsden G., EU Community Reference Laboratory for SVD: Standardisation Exercise in SVD virus, antigen and genome detection. Report to be presented at the EU Community Reference Laboratory for SVD meeting in November 1999).
The PCR-ELISA has been optimized and validated by working on the detection of the virus genome in epithelia, throat swabs and faeces samples collected from outbreaks or from experimentally infected pigs. It was found that this assay is more sensitive than direct RT-PCR and than virus isolation in a variety of clinical samples.
Next to this the value of immunohistochemistry and in-situ hybridization was validated.
Mulder WA, van Poelwijk F, Moormann RJ, Reus B, Kok GL, Pol JM, Dekker A (1997). Detection of early infection of swine vesicular disease virus in porcine cells and skin sections. A comparison of immunohistochemistry and in-situ hybridization. J Virol Methods, 68(2):169-75.



Task 3. Establishment of a SVDV bank and characterisation of isolates
3.1. Collection of SVDV isolates.

The collection of old and recent SVDV isolates is being continued. It has been updated with more than 50 new Italian isolates, collected during 1998 and January 1999. In addition, the isolates France 1/74, Italy 3/77, Italy 5/77, Greece 1/79, Belgium 2/79, Portugal 25/95, Portugal 3/95, Taiwan 119/97, Taiwan 2/98 were included in the collection, as well as and old isolate from Russia, named O72, received from the Institute in Vladimir. Five more SVDV strains were received as inactivated antigens from Poland, which will be antigenically characterised since agreement to receive them also as infectious viruses was not obtained.



3.2 Molecular characterisation and epidemiology of SVDV isolates.
Antigenic profiling of nearly 100 SVD viruses corresponding to Italian isolates since 1995 (from R1205, January 1995 to R1292, January 1999) has been determined using the three panels of monoclonal antibodies. In addition, two isolates from Portugal (1995) and two recent isolates from Taiwan (1997, 1998) were characterised.
The molecular characterisation and sequence of part of VP1 was also done. The comparison of these sequences with those of recent and historic strains of SVDV show that all Italian isolates from 1996 and 1997 fell into the single genetic group defined as Group IV in the paper of Brocchi et al. (1998). This group contains all of the viruses isolated from Italy since 1993 and is distinct from viruses isolated in the country during the period 1988-1992. This group also contains the viruses responsible for the outbreaks of SVD which occurred in the Netherlands, Portugal and Spain during the early 1990's. Isolates of SVD were also received from Taiwan which were responsible for outbreaks in December 1997 and January 1998. These viruses, together with viruses from Hong Kong from the late 80's and early 90's formed a separate subgroup within Group IV. These data suggest that the recent European SVD viruses have a common origin with viruses from the Far East. The first virus of this group to be recovered in Europe was from Romania in 1987 (ROM/1/87). The link between this isolate and the Far East remains obscure.
Brocchi E, Zhang G, Knowles NJ, Wilsden G, McCauley JW, Marquardt O, Ohlinger VF, De Simone F (1997). Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-94. Epidemiol Infect, 118(1):51-61.
Zhang G, Haydon DT, Knowles NJ, McCauley JW (1999). Molecular evolution of swine vesicular disease virus. J Gen Virol, 80 ( Pt 3):639-51.



Task 4. Development and validation of isotype differential ELISA
New isotype specific ELISA.

Quantitative determination of IgG and IgM may be of great value to study the evolution of the disease and as a diagnostic tool to support virus detection. Since the presence of IgM evidences recent infection its determination has great potential for the identification of clinically unapparent disease. The serological methods for the assessment of different isotypes (IgM and IgG) in swine sera are validated during the yearly serosurveillances for SVDV.
Chenard, G., Bloemraad, M., Kramps, J.A., Terpstra, C., Dekker, A. 1998. A monoclonal antibody-based ELISA to detect antibodies directed against swine vesicular disease virus. Journal of virological methods 75; 105-112.
An IgA specific ELISA is in development. This will contribute to the knowledge of the mucosal immunity to SVDV, and could be used to detect epitopes not recognised by other antibody isotypes.



Task 5. Development of a technique to detect the production of cytokines by T lymphocytes in response to SVDV.
Animals that have been exposed to or infected with SVDV have T lymphocytes sensitised to SVD antigens. A booster response is observed in vivo following a second infection or antigen contact. This booster response can be reproduced in vitro . When sensitised PBMC (peripheral blood mononuclear cells) from infected pigs are exposed to the virus, SVDV specific T cells become activated and release cytokines. A swine cytokine detection ELISA for some of the cytokines will be developed. A parallel approach will be used by developing a semi-quantitative oligonucleotide sorbent assay (ELOSA). Detection of cytokine mRNA in cell lysates will be carried out by PCR using specific sense and antisense primers for the individual cytokines. The cDNA products will be revealed in a ELOSA using cytokine-specific capture (biotin) and reporter (fluorescein) primers. Primers for basal cell marker actin will be included to control sample to sample variation in the quantity of mRNA and variation in the RT and PCR reactions.
The results so far indicate that when the pigs are infected with SVDV, the profiles of the cytokines produced (Interferon gamma and IL2) are those that promote the development of the Th1 immunity and an inflammatory response (TNF).
A disadvantage of the test is its dependence on the availability of fresh blood samples from infected pigs.



Task 6. Development and validation of ELISA using recombinant and synthetic antigens.
Identification of viral epitopes recognised by sera of infected animals is essential to select the antigens to be used in a rational design of diagnosis methods.
Dekker, A., Leendertse, C.H., Poelwijk, F., Rebel, J.M.J., Moormann R.J.M. 1999. Chimeric SVD viruses produced by fusion PCR: A new method for epitope mapping. Journal of General Virology (submitted).

Antigenic characterisation will be achieved by using different approaches:


6.1. Phage Display analysis of monoclonal and polyclonal antibodies.
A phage library consists of a vast array of clones each of which expresses a unique peptide sequence as part of one of the major phage structural proteins. By careful affinity selection using the receptor (SVDV antibody), specific phage can be isolated and the nature of the inserted peptide sequence determined using conventional molecular biology techniques. Phage display combinatorial libraries are useful tools for antibody specificity analysis. In addition to studying linear epitopes in the absence of protein sequence information, they can identify mimotopes of antibodies directed against conformational protein features. Purified antibodies (polyclonal antisera and Mabs) will be covalently linked to paramagnetic beads and used as selection media for the phage libraries. Specific phage clones isolated from antibody matrices will undergo further rounds of affinity selection. Clones which have a high specificity will be sublconed, propagated in E. coli and purified using standard protocols. High affinity phage clones (as assayed by ELISA and western blot analysis) will be subjected to DNA sequencing in order to determine the identity of the synthetic oligonucleotide inserts. These phage will be propagated in bulk to provide antigen sources for further ELISA development.
The sequences found correspond to peptides which are capable to mimic viral epitopes, thus potentially useful for detect antibodies against SVDV. Using polyclonal SVDV antisera, the process yielded some clones which differentiated positive and negative antisera.



6.2. Synthesis of synthetic SVDV peptides.
A collection of overlapping 20-mer peptides from the major SVDV structural (VP1, VP2, VP3, VP4) and non-structural (3A, 3B, 3C) proteins will be prepared by Participant 2 and used to identify viral linear epitopes. Peptides will be used as ELISA antigens or, potentially, as T cell antigens for future exploration of cytokine-based SVD assays.
Some of the peptides selected in the peptide scanning were tested for serodiagnosis of SVD. The best results were obtained with a combination of peptides, obtaining a sensitivity of 88 %, where 100% of seropositive and symptomatic animals (n: 12 ) were reactive, and 75 % seropositive, asymptomatic animals were reactive (n:12). None of the negatives tested gave reaction (n:12). While this result presents an improvement with respect to the former P1-ELISA, the lack of reactivity found in some of the asymptomatic pigs still remains being a problem.



6.3. Development of new ELISA
During the project cloning is planned of cDNA from the four structural viral proteins, VP1, VP2, VP3 and VP4, and some non structural (NS) proteins, particularly the polymerase (3D) in E. coli vectors such as GST, pRSET for production of GST-fusion and poly Histidine-fusion proteins respectively, and baculovirus. The detection of a response to the NS proteins can be used to differentiate animals which have been infected from those that have been vaccinated. Structural SVDV proteins will be cloned and expressed as polyprotein (P1) and each single protein separately (VP1, VP2, VP3, VP4). The purified products will be distributed to other contract partners for evaluation as ELISA antigens.
Recombinant SVDV structural proteins. The sensitivity of the recombinant SVDV P1-based ELISA described as satisfactory for the group of symptomatic pigs, was low (approximately 50%) for the group of asymptomatics. The results indicate that the expression of recombinant P1 lacks important antigenic sites that are otherwise present in conventional SVDV antigens (virions) used in classic VNT and blocking ELISA.
M. A. Jimenez-Clavero, E. Escribano-Romero, J. M.Sönchez-VizcaŒno and V. Ley. (1998) Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus. Virus Research. 57. 163-170.
S. K. Nijhar, D. K. J. Mackay, E. Brocchi N. P. Ferris, R. P. Kitching, N. J. Knowles Identification of neutralizing epitopes on a European strain of swine vesicular disease virus. Journal of General Virology (1999), 80, 277Ë282
Recombinant SVDV non structural proteins. Recombinant non-structural (NS) proteins of SVD virus were analysed using an indirect ELISA. A positive result (OD >0.6 ODU) was definitive evidence of previous infection with SVD virus but a negative result did not differentiate between sera from infected pigs, naive pigs, or SRÎs.



Task 7. Analysis of the "singleton reactor" (SR) phenomenon.

7.1. Development of new methods to reduce the occurrence of SR.

A major problem with current serological tests for SVD is the identification of SR sera. These are sera which are positive for antibody to SVD yet which come from pigs which have never had contact with the virus. About 0.3 % of sera analysed in routine surveillance programme result in singleton reactions, 0.25% with a low neutralisation titre, and 0.07% with a high titre. Although this is only a small percentage, a high number of farms have to be revisited. Based on EU regulation there should be a complete stand still for transport of animals and animal products on these farms. This results in severe economic consequences for the farms involved. The factors which give rise to singleton reactor sera are unknown. It may simply be that the sera contain high levels of non-specific antibody, particularly IgM, which result in false positive reactions in serological tests. Alternatively it may be that the animals have been infected with an agent, particularly a virus, which shares epitopes with SVD virus. In this regard it is relevant to note that SVD virus is antigenically very similar to Coxsackie B5 virus, and to other Coxsackie viruses. It is therefore possible that singleton reactor animals result from infection of pigs with one of these cross reacting viruses.
A library of synthetic peptides was analysed in order to determine an association between SR reactivity with some of the peptides. The results shown that choosing a group of 2 selected peptides, it is possible to reduce the SR reactivity about 40 %. This panel of peptides will be further validated with more SR sera.
It has previously been shown that the majority of reactivity in SR sera is due to antibody of IgM class. This phenomenon has been further investigated by Partner 6, by examining the effect of pre-treatment of sera with 2 mercaptoethanol (2 ME), a reducing agent which dissociates IgM.
The number of SR can be reduced by analysing the laboratory results of 3 different tests.
De Clercq K. (1998). Reduction of singleton reactors against swine vesicular disease virus by a combination of virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA. Journal of Virological Methods, 70, 7-18.



Task 8: Determine the duration of SVD infection and identify carrier animals
It has been tried to reproduce the establishment of persistence by repeating an experiment performed during the first year. However, persistence was not established as neither virus nor viral RNA could be detected beyond 21 days post infection in any of the animals. Only 3 out the 10 in-contact pigs in each group developed clinical disease. The majority of the remainder developed subclinical disease characterised by birus excretion and short term seroconversion to moderate titre. These results indicate that persistence is a rare phenomenon in SVD which probably depends to particular, as yet undefined, factors in both the virus and the host. The epidemiological significance of the carrier state in SVD remains unresolved.



Additional tasks performed


Pathogenesis of SVD: Virulence of recent field isolates of SVDV
Strains of SVDV differ in their virulence and completely avirulent strains have been isolated in Japan. Such avirulent strains are extremely useful as potential vaccine strains. The development of an attenuated or inactivated vaccine might be of considerable value to the EU for use in situations where conventional eradication measures prove ineffective. Reports from the field indicated that some of the recent isolates of SVDV from Italy produced only mild or subclinical infection in the field. An experimental infection was therefore carried out to determine the virulence of such a field strain under controlled conditions. The severity of the clinical signs was less than that caused by classic European strains from the 1970's, such as UKG/27/72, but the strain was not suitable for use as the basis of an attenuated vaccine strain.
Virulence studies are also carried out in another collaborative study:
Kanno T, Inoue T, Mackay D, Kitching P, Yamaguchi S, Shirai J (1998). Viruses produced from complementary DNA of virulent and avirulent strains of swine vesicular disease viruses retain the in vivo and in vitro characteristics of the parental strain. Arch Virol 143(6):1055-62.




Dr. V. Ley
CISA-INIA. Valdeolmos

  • Partner 1.
    Contract manager and co-ordinator: Dr. Victoria Ley
    Centro de Investigaci…n en Sanidad Animal (CISA)-INIA.
    Valdeolmos, 28130 Madrid. Spain
  • Partner 2.
    Contract manager: Dr. Alastair Douglas
    Virology Department, Department of Veterinary Science, Queen's University Belfast,
    Stoney Road, Stortmont. Belfast BT4 3SD. Northern Ireland. UK
    Tel: 44 1232-525639. Fax: 44-1232-525773
  • Partner 3.
    Contract manager: Dr. Emiliana Brocchi
    Department of Virology, Instituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia (IZSLE), Via Antonio Bianchi, 7. Brescia, Italy
    Tel. and Fax: 39-30-2290310.
  • Partner 4.
    Contract manager: Dr. Kris De Clerq
    Veterinary and Agrochemical Research Centre (VAR)
    Groeselenberg 99. B- 1 180 Ukkel (Brussels).
    Tel. 32-2-3754455 Fax: 32-2-3750979
  • Partner 5.
    Contract manager: Dr. Aldo Dekker
    Department of porcine and exotic virus diseases, Institute for Animal Science
    and Health, (ID-DLO). Houtribweg 39. 8200 AB Lelystad, Flevoland, The Netherlands.
    Tel. 31-320-238238 Fax: 31-320-238668
  • Partner 6.
    Contract manager: Dr. D. Mackay
    Institute for Animal Health. Pirbright laboratory.
    Ash Rd. Pirbright, Surrey GU24 ONF, UK.
    Tel.44-1483-232441 Fax: 44-1483-232448