Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo.
Abstract: Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 10 cellular β actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 10 cellular β actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells may be influenced by cellular stages of differentiation.
Copyright © 2017 Elsevier B.V. All rights reserved.
Publication Date: 2017-04-25 PubMed ID: 28455133DOI: 10.1016/j.jviromet.2017.04.015Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article presents a study on the development of a quantitative PCR (qPCR) assay to assess the levels of equine herpesvirus 5 (EHV-5) infectivity and its replication in horse cells and tissues. By evaluating various sample types, including equine cell cultures, blood lymphocytes, and nasal swabs, the study explores the viral load in both lab-based (in vitro) and live horse (in vivo) contexts.
Development of the qPCR Assay for EHV-5 Quantification
- The primary goal of the study was to develop a quantitative PCR (qPCR) assay. This assay was intended to measure the viral copy number of EHV-5—an infection linked with pulmonary fibrosis in horses—in equine cell cultures, blood lymphocytes, and nasal swabs.
- PCR primers and a probe were engineered to target the E11 gene of the EHV-5 genome. The sensitivity, repeatability of the assay were established, and its specificity was confirmed by examining multiple isolates of EHV-5 and DNA from other equine herpesviruses.
Application of the qPCR Assay in Cell Cultures
- The qPCR assay was used in a series of experiments involving equine primary respiratory cell cultures and equine dermal cell cultures.
- The EHV-5 virus was introduced in three types of cell cultures: mature respiratory epithelial cells (fully differentiated four-week old), immature respiratory epithelial cells (newly seeded), and equine dermal cells.
- Data was collected by sampling cells and supernatants daily over a 14-day period. Observations showed that EHV-5 concentrations remained stable in mature respiratory and dermal cell cultures. However, an increase in EHV-5 copies was noted in the immature respiratory cells over the 14-day period.
Application of the qPCR Assay in Horses
- Besides the lab-based experiments, the qPCR assay was applied to collect blood lymphocytes and nasal swabs from horses experimentally infected with equine herpesvirus 1 (EHV-1).
- Before EHV-1 infection, EHV-5 was found in the lymphocytes and nasal swabs of 76% and 84% of test horses respectively. However, after the EHV-1 infection, EHV-5 was found in the lymphocytes of 52% of the horses, demonstrating a significant decrease. Meanwhile, the amount of EHV-5 in nasal swabs was not significantly different from pre-infection levels.
Conclusions
- This research concluded that qPCR is a reliable technique for exploring viral load in both in vitro and in vivo samples.
- It was also noted that the replication of EHV-5 in equine epithelial cells might be influenced by the cells’ stage of differentiation.
Cite This Article
APA
Zarski LM, High EA, Nelli RK, Bolin SR, Williams KJ, Hussey G.
(2017).
Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo.
J Virol Methods, 248, 44-53.
https://doi.org/10.1016/j.jviromet.2017.04.015 Publication
Researcher Affiliations
- Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University,784 Wilson Rd., East Lansing, MI, 48824 United States. Electronic address: zarskili@msu.edu.
- Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University,784 Wilson Rd., East Lansing, MI, 48824 United States. Electronic address: highem@msu.edu.
- Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University,784 Wilson Rd., East Lansing, MI, 48824 United States. Electronic address: rknelli@msu.edu.
- Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University,784 Wilson Rd., East Lansing, MI, 48824 United States. Electronic address: bolins@dcpah.msu.edu.
- Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University,784 Wilson Rd., East Lansing, MI, 48824 United States. Electronic address: will1273@dcpah.msu.edu.
- Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University,784 Wilson Rd., East Lansing, MI, 48824 United States. Electronic address: husseygi@cvm.msu.edu.
MeSH Terms
- Animals
- DNA Replication
- DNA, Viral / genetics
- Epithelial Cells / virology
- Gammaherpesvirinae / genetics
- Gammaherpesvirinae / isolation & purification
- Gammaherpesvirinae / physiology
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / isolation & purification
- Horse Diseases / virology
- Horses
- Lymphocytes / virology
- Nose / virology
- Real-Time Polymerase Chain Reaction / methods
- Respiratory System / virology
- Viral Load
- Virus Replication
Citations
This article has been cited 2 times.- Badr C, Souiai O, Arbi M, El Behi I, Essaied MS, Khosrof I, Benkahla A, Chabchoub A, Ghram A. Epidemiological and Phylogeographic Study of Equid Herpesviruses in Tunisia. Pathogens 2022 Sep 5;11(9).
- Zarski LM, Weber PSD, Lee Y, Soboll Hussey G. Transcriptomic Profiling of Equine and Viral Genes in Peripheral Blood Mononuclear Cells in Horses during Equine Herpesvirus 1 Infection. Pathogens 2021 Jan 7;10(1).
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