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Archives of virology2006; 152(1); 137-149; doi: 10.1007/s00705-006-0810-3

Reverse transcriptase-polymerase chain reaction for the detection equine rhinitis B viruses and cell culture isolation of the virus.

Abstract: Equine rhinitis B virus (ERBV), genus Erbovirus, family Picornaviridae occurs as two serotypes, ERBV1 and ERBV2. An ERBV-specific nested reverse transcriptase-polymerase chain reaction (RT-PCR) that amplified a product within the 3D(pol) and 3' non-translated region of the viral genome was developed. The RT-PCR detected all 24 available ERBV1 isolates and one available ERBV2 isolate. The limit of detection for the prototype strain ERBV1.1436/71 was 0.1 50% tissue culture infectious doses. The RT-PCR was used to detect viral RNA in six of 17 nasopharyngeal swab samples from horses that had clinical signs of acute febrile respiratory disease but from which ERBV was not initially isolated in cell culture. The sequences of these six ERBV RT-PCR positive samples had 93-96% nucleotide identity with six other partially sequenced ERBV1 isolates and one ERBV2. ERBV was isolated from one of the six samples at fourth cell culture passage when it was shown that the addition of 20 mg/mL MgCl(2) to the cell culture medium enhanced the growth of the virus. This isolated virus was antigenically similar to ERBV2.313/75. Determination of the nucleotide sequence of the P1 region of the genome also indicated that the isolate was ERBV2, and it was therefore designated ERBV2.1576/99. This is the first reported isolation of ERBV in Australia. The study highlights the utility of PCR for the identification of viruses in clinical samples that may initially be considered negative by conventional cell culture isolation.
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Publication Date: 2006-08-24 PubMed ID: 16932985PubMed Central: PMC7086902DOI: 10.1007/s00705-006-0810-3Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

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 study investigates a new method for detecting equine rhinitis B viruses (ERBV) using a specific RT-PCR (reverse transcriptase-polymerase chain reaction) technique. The technique was able to successfully detect all ERBV1 and one ERBV2 strain, proving its effectiveness in identification of viruses that may not be initially identified by conventional cell culture isolation. The result also included the first reported isolation of ERBV in Australia.

Introduction of the Research Study

  • The Equine rhinitis B virus (ERBV) is part of the Erbovirus genus, belonging to the Picornaviridae family. There are two types of ERBV, known as ERBV1 and ERBV2. This study discusses the development of an ERBV-specific nested reverse transcriptase-polymerase chain reaction (RT-PCR) that is used to duplicate a product within certain regions of the viral genome.
  • The RT-PCR technique is designed to detect ERBV1 and ERBV2 isolates. This study involved testing this technique on 24 different ERBV1 isolates and a single ERBV2 isolate.

Results of the RT-PCR Method

  • The RT-PCR technique was successful in detecting all tested ERBV isolates. The detection limit for the prototype strain ERBV1.1436/71 was found to be 0.1 50% tissue culture infectious doses, making this technique highly sensitive.
  • Importantly, the RT-PCR was able to detect the presence of viral RNA in six out of 17 nasopharyngeal swab samples taken from horses presenting symptoms of acute febrile respiratory disease, even though the ERBV was not initially detectable in cell cultures. This demonstrates the potential superiority of the RT-PCR technique over more conventional detection methods.

Isolation and Comparison of the ERBV2 Strain

  • The ERBV was successfully isolated from one of the six positive samples at the fourth cell culture passage by adding 20 mg/mL MgCl(2) to the cell culture medium, which enhanced the growth of the virus. The isolated strain was antigenically similar to ERBV2.313/75.
  • Comparison of the nucleotide sequence of this isolate confirmed that it was ERBV2, indicating a further validation of the RT-PCR method.

Significance of the Study

  • This research represents the first reported isolation of ERBV in Australia, adding to the knowledge base regarding the global spread of the virus.
  • Overall, the study underscores the utility of the RT-PCR technique for detecting viruses in clinical samples which may initially be deemed negative using traditional cell culture isolation methods, potentially improving early detection and treatment of diseases caused by ERBVs in horses.

Cite This Article

APA
Black WD, Hartley CA, Ficorilli NP, Studdert MJ. (2006). Reverse transcriptase-polymerase chain reaction for the detection equine rhinitis B viruses and cell culture isolation of the virus. Arch Virol, 152(1), 137-149. https://doi.org/10.1007/s00705-006-0810-3

Publication

Archives of virology
ISSN: 0304-8608
NlmUniqueID: 7506870
Country: Austria
Language: English
Volume: 152
Issue: 1
Pages: 137-149

Researcher Affiliations

Black, W D
  • Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Australia.
Hartley, C A
    Ficorilli, N P
      Studdert, M J

        MeSH Terms

        • Amino Acid Sequence
        • Animals
        • Antibodies, Viral / blood
        • Antigens, Viral
        • Australia
        • Base Sequence
        • Cells, Cultured
        • DNA Primers / genetics
        • Erbovirus / classification
        • Erbovirus / genetics
        • Erbovirus / immunology
        • Erbovirus / isolation & purification
        • Gene Products, pol / genetics
        • Horse Diseases / diagnosis
        • Horse Diseases / immunology
        • Horse Diseases / virology
        • Horses
        • Molecular Sequence Data
        • Neutralization Tests
        • Picornaviridae Infections / diagnosis
        • Picornaviridae Infections / immunology
        • Picornaviridae Infections / veterinary
        • Picornaviridae Infections / virology
        • RNA, Viral / genetics
        • RNA, Viral / isolation & purification
        • Reverse Transcriptase Polymerase Chain Reaction / methods
        • Reverse Transcriptase Polymerase Chain Reaction / statistics & numerical data
        • Sensitivity and Specificity
        • Sequence Homology, Amino Acid
        • Serotyping

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        Citations

        This article has been cited 4 times.
        1. Woo PC, Lau SK, Choi GK, Huang Y, Wernery R, Joseph S, Wong EY, Elizabeth SK, Patteril NA, Li T, Wernery U, Yuen KY. Equine rhinitis B viruses in horse fecal samples from the Middle East. Virol J 2016 Jun 7;13:94.
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        2. Lu Z, Timoney PJ, White J, Balasuriya UB. Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses. BMC Vet Res 2012 Jul 25;8:120.
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        3. Mazzei M, Sorvillo B, Sgorbini M, Bindi F, Perelli A, Laus F. The Role of Viral Pathogens in Horse Respiratory Diseases: A Cytological and Molecular Approach Using Next-Generation Sequencing. Animals (Basel) 2024 Nov 21;14(23).
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        4. Stasiak K, Dunowska M, Rola J. Prevalence and Sequence Analysis of Equine Rhinitis Viruses among Horses in Poland. Viruses 2024 Jul 26;16(8).
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