"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. We developed a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell culture, blood lymphocytes, and nasal swabs of horses. The PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old, fully differentiated (mature) and newly seeded (immature) primary equine respiratory epithelial cell (ERECs) cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 12-14 days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with EHV-1. The qPCR assay detected EHV-5 at concentrations around 104 intracellular genomes per cell culture in experimentally inoculated mature ERECs, and these values remained stable throughout 12 days. Intracellular EHV-5 copies detected in the immature cultures increased over 14 days and reached levels greater than 106 genomes per culture. EHV-5 was detected in the lymphocytes of 97% of horses and in the nasal swabs of 88% of horses both pre and post EHV-1 infection. 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."--Page ii.
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