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Development and Application of a Quantitative PCR Assay to Study Equine Herpesvirus 5 Invasion and Replication in Equine Tissues in vitro and in vivo.

Development and Application of a Quantitative PCR Assay to Study Equine Herpesvirus 5 Invasion and Replication in Equine Tissues in vitro and in vivo.
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Zarski LM, High EA, Nelli RK, Bolin SR, Williams KJ, Hussey GS,


Zarski LM, High EA, Nelli RK, Bolin SR, Williams KJ, Hussey GS, (click to view)

Zarski LM, High EA, Nelli RK, Bolin SR, Williams KJ, Hussey GS,

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Journal of virological methods 2017 04 25() pii 10.1016/j.jviromet.2017.04.015

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 was 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 14 days. 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(6) 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(6) 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.

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