Comparison of four methods to quantify Equid herpesvirus 1 load by real-time polymerase chain reaction in nasal secretions of experimentally and naturally infected horses.
Abstract: The objective of the current study was to compare the performance of 4 methods to quantify Equid herpesvirus 1 (EHV-1) by real-time polymerase chain reaction (PCR) in nasal secretions from experimentally and naturally infected horses. Nasal secretions were collected on the challenge day and daily thereafter for 13 days from 4 experimentally infected horses. Additional nasal swabs were collected from 30 horses with clinical signs consistent with natural EHV-1 infection. Absolute quantitation of EHV-1 target molecules was performed using standard curves for EHV-1 and equine glyceraldehyde-3-phosphate dehydrogenase, and DNA yield, and was expressed as EHV-1 glycoprotein B (gB) gene copies per million nucleated nasal cells, EHV-1 gB gene copies per entire swab, EHV-1 gB gene copies per 1 microl of purified DNA, and EHV-1 gB gene copies per 1 ng of template DNA. The study results showed that all 4 calculation methods yielded comparable results between experimentally and naturally infected horses, and that the different methods were significantly correlated with each other. Reporting of quantitative results for EHV-1 viral load in nasal swabs collected from infected horses constitutes an important advance in both the research and diagnostic fields, allowing one to determine the infectious risk of affected horses, disease stage, or response to antiviral therapy. However, protocols that normalize the PCR results against a preselected volume of DNA or nasal secretions are likely to be more prone to variations than protocols that calculate the load for the entire swab, incorporate a housekeeping gene, or use a constant amount of extracted DNA.
Publication Date: 2009-11-11 PubMed ID: 19901285DOI: 10.1177/104063870902100611Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research study aims to compare four different methods of measuring the load of Equid herpesvirus 1 (EHV-1), a virus affecting horses, via real-time polymerase chain reaction (PCR) in the nasal secretions of both artificially and naturally infected horses.
Overview of the Methodology
- Nasal secretions were collected daily for 13 days from four horses that were intentionally infected with EHV-1.
- Additionally, nasal swabs were collected from 30 other horses showing signs of natural EHV-1 infection.
- The EHV-1 load was determined by quantifying the number of EHV-1 target molecules (virus-specific genetic material) via standard curves for EHV-1 and equine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) — a common housekeeping gene.
- The absolute quantity of the EHV-1 load was expressed in four different ways: EHV-1 glycoprotein B (gB) gene copies per million nucleated nasal cells; EHV-1 gB gene copies per entire nasal swab; EHV-1 gB gene copies per 1 microliter of purified DNA; and EHV-1 gB gene copies per 1 nanogram of template DNA.
Findings from the Study
- The results suggested that all four calculation methods yielded similar results for both intentionally and naturally infected horses.
- The various methods employed were significantly correlated with each other, suggesting that they could reasonably be used interchangeably to quantify EHV-1 load.
- The approach of providing quantitative results for EHV-1 viral load offers progress in both research and diagnostic areas. It helps determine the infectious risk of affected horses, their disease stage, or their response to antiviral therapy.
Implications and Limitations
- However, the study also suggests protocols that normalize the PCR results against a predetermined DNA volume or nasal secretions may be more subject to variations. This is in comparison to those that calculate the load for the entire swab, incorporate a housekeeping gene, or use a constant amount of extracted DNA.
- Regardless, this work poses significant advancements, offering a more targeted and detailed look at EHV-1 infections across varied scenarios.
Cite This Article
APA
Pusterla N, Hussey SB, Mapes S, Leutenegger CM, Madigan JE, Ferraro GL, Wilson WD, Lunn DP.
(2009).
Comparison of four methods to quantify Equid herpesvirus 1 load by real-time polymerase chain reaction in nasal secretions of experimentally and naturally infected horses.
J Vet Diagn Invest, 21(6), 836-840.
https://doi.org/10.1177/104063870902100611 Publication
Researcher Affiliations
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. npusterla@ucdavis.edu
MeSH Terms
- Animals
- Herpesviridae Infections / diagnosis
- Herpesviridae Infections / genetics
- Herpesviridae Infections / veterinary
- Herpesvirus 1, Equid / genetics
- Herpesvirus 1, Equid / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / genetics
- Horse Diseases / virology
- Horses
- Male
- Nasal Mucosa / virology
- Nose / virology
- Orchiectomy / veterinary
- Polymerase Chain Reaction / methods
- RNA, Viral / blood
- Viral Load / veterinary
Citations
This article has been cited 5 times.- Pusterla N, Barnum S, Young A, Mendonsa E, Lee S, Hankin S, Brittner S, Finno CJ. Molecular Monitoring of EHV-1 in Silently Infected Performance Horses through Nasal and Environmental Sample Testing.. Pathogens 2022 Jun 24;11(7).
- Price D, Barnum S, Mize J, Pusterla N. Investigation of the Use of Non-Invasive Samples for the Molecular Detection of EHV-1 in Horses with and without Clinical Infection.. Pathogens 2022 May 13;11(5).
- Nielsen SS, Alvarez J, Bicout DJ, Calistri P, Canali E, Drewe JA, Garin-Bastuji B, Gonzales Rojas JL, Gortázar C, Herskin M, Michel V, Miranda Chueca MÁ, Roberts HC, Padalino B, Pasquali P, Spoolder H, Ståhl K, Calvo AV, Viltrop A, Winckler C, Carvelli A, Paillot R, Broglia A, Kohnle L, Baldinelli F, Van der Stede Y. Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infection with Equine Herpesvirus-1.. EFSA J 2022 Jan;20(1):e07036.
- Stasiak K, Dunowska M, Rola J. Outbreak of equid herpesvirus 1 abortions at the Arabian stud in Poland.. BMC Vet Res 2020 Oct 6;16(1):374.
- Doubli-Bounoua N, Richard EA, Léon A, Pitel PH, Pronost S, Fortier G. Multiple molecular detection of respiratory viruses and associated signs of airway inflammation in racehorses.. Virol J 2016 Nov 29;13(1):197.
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