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Journal of clinical microbiology1994; 32(3); 658-665; doi: 10.1128/jcm.32.3.658-665.1994

Detection of equine arteritis virus following amplification of structural and nonstructural viral genes by reverse transcription-PCR.

Abstract: A reverse transcription (RT)-PCR assay was developed for the detection of equine arteritis virus (EAV) in cell culture supernatant and in horse semen. Four different sets of oligonucleotide primers complementary to sequences located in the 3' end of the polymerase gene (open reading frame [ORF] 1b) and to sequences representing the entire ORFs 3, 4, and 7, which encode for nonstructural (ORFs 3 and 4) or viral nucleocapsid (ORF 7) proteins, were compared for their abilities to amplify the targeted EAV sequences by the RT-PCR procedure. The sensitivities of the RT-PCR for amplification of EAV sequences located in the 3' end of ORF 1b and ORF 4 were 2 median tissue culture infective doses (TCID50s) of viral particles in the EAV-infected cell culture supernatant for both ORFs and 20 and 200 TCID50s of viral particles, respectively, in virus-containing horse semen. The sensitivities were much lower when primers complementary to ORFs 3 and 7 were used in the RT-PCR, with a minimum detection limit of only 2 x 10(4) TCID50s of viral particles in virally infected cell culture supernatant, as determined by analyzing the resulting RT-PCR products on ethidium bromide-stained agarose gels. The specificities of the RT-PCR assays for all primer sets tested were confirmed when the amplified cDNA products of the expected size reacted positively with the corresponding virus-specific digoxigenin-labeled cDNA probes in the chemiluminescence assays. Although the sensitivity of the RT-PCR for amplification of ORF 3 and 7 sequences was lower, all sets or primers were capable of amplifying several cell culture-adapted EAV field isolates when the virus was present in high enough quanities in the test sample. When horse semen samples were analyzed for the presence of EAV by the RT-PCR with primers specific to the ORF 1b 3' end and ORF 4 sequences and by virus isolation in cell cultures, there was 100% concordance among the assays. The RT-PCR assay targeting the 3' end of ORF 1b and/or ORF 4 EAV RNA may be an alternative to conventional methods for the diagnosis of EAV infection in horses.
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Publication Date: 1994-03-01 PubMed ID: 8195375PubMed Central: PMC263103DOI: 10.1128/jcm.32.3.658-665.1994Google Scholar: Lookup
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Summary

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This research article discusses the development of an assay to detect the presence of equine arteritis virus (EAV) in cell culture and horse semen using reverse transcription-PCR methods.

Understanding the EAV Detection Procedure

  • An assay was generated using reverse transcription-PCR (RT-PCR), which is a laboratory technique that combines reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR).
  • Four sets of oligonucleotide primers designed to complement sequences in the polymerase gene (ORF 1b) and sequences representing the complete ORFs 3, 4, and 7, which encode for nonstructural (ORFs 3 and 4) or viral nucleocapsid (ORF 7) proteins, were compared to determine their effectiveness at amplifying target EAV sequences.

Sensitivity and Specificity of the RT-PCR for EAV detection

  • The sensitivities of the RT-PCR for amplifying EAV sequences in ORF 1b and ORF 4 were estimated using measures of viral concentration known as median tissue culture infective doses (TCID50s).
  • When using primers complementary to ORFs 3 and 7, the detection limit of the RT-PCR was lower, only able to detect significantly greater viral concentrations in the infected cell culture supernatant.
  • The specificity, or the ability of the test to correctly determine the presence of the virus, of the RT-PCR assays for all primer sets were confirmed by observing that the amplified cDNA products of the expected size interacted positively with corresponding virus-specific digoxigenin-labelled cDNA probes in chemiluminescence assays.

Comparative Analysis & Potential Applications

  • Although the sensitivity of ORF 3 and 7 sequence amplification was lower, all primer sets were able to amplify several cell culture-adapted EAV field isolates if the virus was present in high enough quantities in the test sample.
  • When horse semen samples were analyzed for EAV using RT-PCR with primers specific to ORF 1b 3′ end and ORF 4 sequences, as well as by virus isolation in cell cultures, there was 100% agreement amongst the assays.
  • These findings suggest that the RT-PCR assay targeting the 3′ end of ORF 1b and/or ORF 4 EAV RNA could be an alternative to conventional methods for diagnosing EAV infection in horses.

Cite This Article

APA
St-Laurent G, Morin G, Archambault D. (1994). Detection of equine arteritis virus following amplification of structural and nonstructural viral genes by reverse transcription-PCR. J Clin Microbiol, 32(3), 658-665. https://doi.org/10.1128/jcm.32.3.658-665.1994

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 32
Issue: 3
Pages: 658-665

Researcher Affiliations

St-Laurent, G
  • Université du Québec à Montréal, Département des Sciences Biologiques, Québec, Canada.
Morin, G
    Archambault, D

      MeSH Terms

      • Animals
      • Arterivirus Infections / diagnosis
      • Arterivirus Infections / veterinary
      • Base Sequence
      • DNA Primers / genetics
      • DNA, Viral / genetics
      • Equartevirus / genetics
      • Equartevirus / isolation & purification
      • Genes, Viral
      • Horse Diseases / diagnosis
      • Horses
      • Male
      • Molecular Sequence Data
      • Open Reading Frames
      • Polymerase Chain Reaction / methods
      • Polymerase Chain Reaction / statistics & numerical data
      • RNA, Viral / genetics
      • Semen / microbiology
      • Sensitivity and Specificity
      • Viral Structural Proteins / genetics

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      Citations

      This article has been cited 17 times.
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      7. Abed Y, St-Laurent G, Zhang H, Jacobs RM, Archambault D. Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane envelope proteins expressed from recombinant baculovirus. Clin Diagn Lab Immunol 1999 Mar;6(2):168-72.
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