Equine Herpesvirus type 1 (EHV-1) is a worldwide threat to the health of horses. It can cause mild respiratory disease, abortions and deaths of newborn foals as well as a potentially fatal neurologic disorder known as Equine Herpesvirus Myeloencephalopathy (EHM). The virus is maintained in populations by stress-induced periodic reactivation of virus in long-term latently infected horses and transmission of the reactivated virus to susceptible individuals. In horses, peripheral blood leukocytes (PBLs) are thought to be an important site for EHV-1 latent genomes. Since the Unfolded Protein Response (UPR) is a cellular response to a variety of stressors that has been linked to reactivation of herpes simplex virus in humans, a virus closely related to EHV-1, I tested the hypothesis that latent EHV-1 relies on the UPR as a pluripotent stress sensor and uses it to reactivate lytic gene expression.
Since little work has been done in defining the UPR in horses, I first successfully developed a quantitative real-time polymerase chain reaction (RT-qPCR) assay to detect and quantitate transcripts for selected UPR genes in equine dermal (E.Derm) cells and PBLs. Activation of the UPR was achieved in both cell types using thapsigargin and a difference in gene expression after activation of the UPR in two equine cell types was found. A nested PCR assay to detect and distinguish latent EHV-1 and EHV-4 was evaluated and the sensitivity of the technique to detect EHV-1 was determined. I discovered that the nested PCR technique was not sensitive enough to detect the estimated one latent viral genome in 50,000 PBLs.
Lytic EHV-1 infection was characterized by single step growth curve in E.Derm cells and consistent detection of temporal EHV-1 gene expression by RT-qPCR was achieved. The relationship between EHV-1 gene expression and UPR gene expression during lytic infection was investigated. While EHV-1 infection had no effect on UPR gene expression, activation of the UPR appeared to decrease the expression of EHV-1 genes temporarily and reversibly during the first 4 h after infection. Finally, detection of EHV-1 in PBLs from horses presumed to be latently infected by co-cultivation with E. Derm cells permissive to EHV-1 infection was attempted. To detect viral DNA, PBLs were stimulated with thapsigargin or interleukin 2 (IL-2) which was previously reported to induce reactivation of latent EHV-1. I was not able to reproduce previously published experiments of reactivation in vitro of latent EHV-1 by stimulation with IL-2, and virus reactivation did not occur after stimulation of PBLs with thapsigargin.
In summary, a RT-qPCR assay to measure the expression of equine UPR genes was developed and activation of the UPR by treatment of E.Derm cells and PBLs with thapsigargin was successfully achieved. A difference in gene expression after activation of the UPR in two equine cell types was found. In contrast to what has been reported for other alphaherpesviruses, there appears to be no, or only little, interaction between the UPR and EHV-1 during viral infection. Detection of latent EHV-1 genomes in PBLs was not achieved by using a nested PCR, as this technique was not sensitive enough to detect the estimated one latent viral genome in 50,000 PBLs. Finally, latent EHV-1 was not detected in presumed latently infected PBLs or reactivated by triggering the UPR in equine PBLs.
| Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2013-06-1138 |
| Date | 2013 June 1900 |
| Contributors | Lohmann, Katharina, Misra, Vikram |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text, thesis |
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