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Equine arteritis: international disease control by improved methods of virus detection and characterisation

Contract nr: FAIR-CT98-4123
Project nr: 4123
Project type: SC
Starting date: 01/01/1999
Duration: 36 months
Total cost: 1,298,084 EUR
EC Contribution: 946,486 EUR
Scientific Officer: Isabel MINGUEZ-TUDELA
Research topic: Animal health
Acronym: INTERNATIONAL CONTROL OF EAV

Background:
Equine viral arteritis is of growing concern because of the increase in the international movement of horses, with the concomitant increased risk of spreading infectious diseases. Recent experiences have demonstrated that introduction of the virus into EU Member States can enormously disrupt the local equine industry. Prevention and control of this disease depends principally on identification of carrier stallions. A significant proportion of stallions that become infected with equine arteritis virus (EAV) become long-term shedders of the virus in semen. Although clinically normal, such animals readily infect mares by natural service or when their semen is used for artificial insemination. Detection of such stallions relies on laboratory tests, but is problematic because:

1) vaccination may cause stallions to sero-convert without necessarily having been exposed to wild-type virus;
2) testing for shedding in stallions using current tests is laborious and slow;
3) not all shedding stallions appear to initiate clinical outbreaks;
4) the disease is often sub-clinical, so many countries do not attempt to control its spread.

Objectives:
To deal with these difficulties, the following research is proposed:

1) standardisation of newly developed RT-PCR/sequencing methods for rapid detection and characterisation of viruses in semen;
2) investigation of the virulence determinants of EAV isolates as a predictors of clinical significance;
3) a thorough investigation of immune responses as a means of differentiating carrier stallions from naive or vaccinated animals.

Description:
To characterise the pathogenicity of EAV and to find virulence markers, genetic differences were investigated in virulent and avirulent variants of the virus. Biological clones were obtained and the plaque size was estimated in correlation to virulence in vivo. For further identification of virulence markers, recombinant cDNA clones were generated, on the basis of a full-length infectious clone (obtained from the University of Leiden). Phylogenetic analysis and molecular epizootiology of EAV was made by comparative sequencing of viruses from two continents. The genetic stability of the virus was followed in 20 persistent carrier stallions in Austria, Italy and Sweden. In order to develop and harmonise the diagnostic methods, virus neutralisation (VN), virus isolation (VI) and RT-PCR assays were applied in ring tests. To provide safety for PCR, an internal control (mimic) has been developed. Sensitive assays have been applied to measure EAV-specific antibody responses and cellular immunology. Baculovirus vector systems have been obtained to express the structural proteins of EAV, in order to study the role of structural proteins in immunological protection of infected horses.

Current situation/results:
Comparative sequence analysis of the structural protein-coding region of EAV revealed minor differences between virulent and avirulent strains, indicating that the virus is genetically very stable in this part of the genome. The observation was confirmed by sequence analysis of the serial samples collected from persistent shedder stallions. In vitro and in vivo studies with the large and small plaque variants showed that the plaque size is seemingly not correlated to virulence, but further studies using chimeric recombinant cDNA clones will hopefully shed light on this issue.
Phylogenetic analyses resulted in genetic grouping on new virus variants. Viruses were grouped in the European and American genogroup in correlation to their origin. The "exceptions" turned out to represent horses transported between the continents. Interestingly, a South African donkey isolate represented a totally new genotype of EAV. A database for EAV will be established, based on the large sequence collection of over 100 isolates of EAV from all over the world, incorporating clinical data as origin, date of isolation and breed of the infected horse.

The immunological reagents have proved suitable for measuring antibodies in equine serum and to detect and quantitate subisotype antibody responses. Cell lines were established as suitable targets to measure cellular immunity. It was found that, as with immune responses to other viral pathogens, the early (post-acute) responses to EAV show a broad specificity, whereas convalescent (recall) responses are more restricted. The baculovirus constructs allow the expression of EAV structural proteins. The expressed proteins are useful tools in virus diagnosis and in studies of structural proteins in protection against the disease.

Future actions:
The whole structural protein-coding region of the biological variants will be cloned and sequenced. Chimeric cDNA constructs will be tested for infectivity and used to generate new recombinant viruses. The in vivo characteristics of the recombinant viruses will be studied. Further samples will be collected from the persistent carrier stallions to see the genetic stability of the virus. In order to study the genetic evolution, the quasispecies distribution will also be investigated. An EAV sequence database will be available through password-protected accession on the website of the project. To improve the direct detection of EAV, various RT-PCR methods will be compared, the most reliable assay will be selected and adapted to automated procedures. Internal controls will ensure the reliability of diagnosis. Cellular immunity will be investigated to confirm the identity as well as the antigen-specificity of effector cells. To study immunological protection provided by viral structural proteins, recombinant baculoviruses will be used to express the structural proteins.

Website: http://www.eaveu.org/


Coordinator
Sándor BELAK
The National Veterinary Institute
Department of Virology
Biomedical Center
Box 585
S-751 23 Uppsala
Tel.: +46 18 67 41 35
Fax: +46 18 47 14 520
E-mail: Sandor.Belak@svavir.uu.se


Partners

  • Duncan HANNANT
    Animal Health Trust
    Lanwades Park, Kentford
    UK-CB8 7UU Newmarket
    Tel.: +44 1638 75 10 00
    Fax: +44 1638 75 07 94
    E-mail: duncan.hannant@aht.org.uk

  • David James PATON
    Veterinary Laboratories Agency
    (An Executive Agency of the Ministry of Agriculture, Fisheries and Food)
    Woodham Lane
    UK-KT15 3NB Addlestone
    Tel.: +44 1932 35 72 85
    Fax: +44 1932 35 72 39
    E-mail: dpaton.cvl.wood@gtnet.gov.uk

  • Gian Luca AUTORINO
    Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana
    Via Appia Nuova 1411
    I-00178 Roma Capannelle
    Tel.: +39 06 79 09 94 00
    Fax: +39 06 79 34 07 24
    E-mail: glautorino@rm.izs.it

  • Norbert NOWOTNY
    University of Veterinary Sciences
    Vienna University of Veterinay Medicine
    Veterinaerplatz 1
    A-12100 Vienna
    Tel.: +43 1 250 77 23 04
    Fax: +43 1 250 77 23 90
    E-mail: norbert.nowotny@vu-wien.ac.at

 
 
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