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Archives of virology1995; 140(8); 1483-1491; doi: 10.1007/BF01322675

Detection of equine arteritis virus (EAV) by polymerase chain reaction (PCR) and differentiation of EAV strains by restriction enzyme analysis of PCR products.

Abstract: A polymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1,000 times by performing nested PCR enabling the detection of RNA at a level of 0.5-5 PFU. Differentiation among the virus strains and the live vaccine was achieved by cutting the PCR-amplified products from three sets of primers with six restriction endonucleases. Using this procedure it was possible to distinguish among the seven EAV strains used.
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Publication Date: 1995-01-01 PubMed ID: 7661700PubMed Central: PMC7087138DOI: 10.1007/BF01322675Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research has developed a test using the polymerase chain reaction, or PCR, that can both detect and differentiate between seven different strains of equine arteritis virus, a disease that affects horses. This new method is not only more sensitive than previous tests, it can also tell the difference between various strains of the virus and a live vaccine.

Methodology

  • The researchers identified and used a gene (ORF6) that codes for a specific protein (membrane protein M) in the equine arteritis virus. They developed a PCR-based assay, a type of laboratory test, that uses primers specific to this gene.
  • They collected viral RNA from cell cultures that were infused with each of the seven EAV strains, as well as from the live vaccine, Arvac.
  • Four sets of primers were used in the PCR reaction to detect the virus and enhance the test’s sensitivity.

Use of Nested PCR

  • The researchers used a technique called nested PCR, which increased the sensitivity of detection by 100 to 1,000 times.
  • This method enabled them to detect the virus at a quantity as low as 0.5-5 Plaque-forming units (PFUs), a measure of virus quantity.

Differentiation of Virus Strains

  • The PCR products from three sets of primers were cut with six different restriction endonucleases, types of enzymes that cut DNA at specific sequences.
  • This process allowed them to differentiate among the various EAV strains and the vaccine.
  • The result was a technique that could not only detect the presence of the equine arteritis virus but also distinguish between different virus strains.

Conclusion

  • Overall, the researchers were able to distinguish all seven EAV strains by using this PCR-based method.
  • This study contributes significant progress towards better detection and differentiation methods for equine arteritis virus, which could eventually lead to improved treatment and prevention strategies.

Cite This Article

APA
Sekiguchi K, Sugita S, Fukunaga Y, Kondo T, Wada R, Kamada M, Yamaguchi S. (1995). Detection of equine arteritis virus (EAV) by polymerase chain reaction (PCR) and differentiation of EAV strains by restriction enzyme analysis of PCR products. Arch Virol, 140(8), 1483-1491. https://doi.org/10.1007/BF01322675

Publication

Archives of virology
ISSN: 0304-8608
NlmUniqueID: 7506870
Country: Austria
Language: English
Volume: 140
Issue: 8
Pages: 1483-1491

Researcher Affiliations

Sekiguchi, K
  • Epizootic Research Station, Equine Research Institute, Japan Racing Association, Tochigi-Ken.
Sugita, S
    Fukunaga, Y
      Kondo, T
        Wada, R
          Kamada, M
            Yamaguchi, S

              MeSH Terms

              • Base Sequence
              • DNA Primers
              • DNA Restriction Enzymes
              • Equartevirus / classification
              • Equartevirus / genetics
              • Equartevirus / isolation & purification
              • Molecular Sequence Data
              • Open Reading Frames / genetics
              • Polymerase Chain Reaction
              • RNA, Viral / analysis
              • RNA, Viral / genetics
              • Sensitivity and Specificity
              • Viral Matrix Proteins / genetics

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              Citations

              This article has been cited 5 times.
              1. Balasuriya UB, Go YY, MacLachlan NJ. Equine arteritis virus. Vet Microbiol 2013 Nov 29;167(1-2):93-122.
                doi: 10.1016/j.vetmic.2013.06.015pubmed: 23891306google scholar: lookup
              2. Echeverría MG, Díaz S, Metz GE, Serena MS, Panei CJ, Nosetto E. Genetic typing of equine arteritis virus isolates from Argentina. Virus Genes 2007 Oct;35(2):313-20.
                doi: 10.1007/s11262-007-0081-4pubmed: 17294142google scholar: lookup
              3. Gagnon CA, Dea S. Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes. Can J Vet Res 1998 Apr;62(2):110-6.
                pubmed: 9553709
              4. Kheyar A, Martin S, St-Laurent G, Timoney PJ, McCollum WH, Archambault D. Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus. Clin Diagn Lab Immunol 1997 Nov;4(6):648-52.
                doi: 10.1128/cdli.4.6.648-652.1997pubmed: 9384283google scholar: lookup
              5. Gilbert SA, Timoney PJ, McCollum WH, Deregt D. Detection of equine arteritis virus in the semen of carrier stallions by using a sensitive nested PCR assay. J Clin Microbiol 1997 Aug;35(8):2181-3.
                doi: 10.1128/jcm.35.8.2181-2183.1997pubmed: 9230412google scholar: lookup
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