"Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) is an Alloherpesvirus (Order Herpesvirales) capable of causing severe morbidity and mortality in lake trout (Salvelinus namaycush). After several decades of absence, EEDV re-emerged in the Great Lakes basin and was the causative agent of substantial mortality events in fingerling and two year old lake trout in 2012 and 2017 respectively. This resurgence highlighted the challenges associated with managing an infectious disease when many questions remain regarding its biological and pathological properties. As a result, several studies were designed in order to advance our knowledge of viral targets, disease progression, and the availability of research models and diagnostic assays. As EEDV cannot be propagated in vitro, I first aimed to determine the in vivo viral exposure dose required to cause clinical disease consistent with that seen in natural outbreaks of EEDV. Results revealed that 103 viral copies per mL of immersion bath water is not a sufficient dose to produce clinical disease, while 106 viral copies per mL of water can produce up to 100% mortality. Utilizing this predetermined dose range, I then assessed the temporal course of an EEDV infection by determining the sequential distribution of virus and identification of specific viral target tissues and cells using quantitative PCR and in situ hybridization assays. Following exposure of naïve juvenile lake trout to EEDV, the virus first targeted the epidermis of the skin and fins followed by the epithelial lining of primary and secondary gill lamellae and eventually infection of endothelial cells and monocytes resulting in viremia and disseminated infection of multiple visceral organs. However, viral titers remained significantly higher among external tissues compared to visceral organs throughout the study. Subsequently, in order to elucidate a more comprehensive understanding of the pathologic changes associated with EEDV infection, I next examined sequential gross and pathological alterations in lake trout tissues. After an extended incubation period, severe pathology was first observed grossly and microscopically in the cutaneous epithelium followed by the hematopoietic organs and vessels during the later viremic stage of disease. Following these advancements in our understanding of EEDV-lake trout interactions and associated pathology, my focus shifted to expanding future experimental and diagnostic capabilities. First, I produced two novel cell lines of lake trout origin. With the limited number of commercially available aquatic cell lines, and none originating from lake trout, diagnosis of, and research into lake trout specific immunology or disease pathology, has been hampered. The successful production of primary cultures from adult liver, yearling fin, yearling gonad and fry body cells of lake trout origin, contributes to what was previously a void in salmonid tissue culture options. Without an established in vitro model of EEDV replication to date, there was a need for a diagnostic assay that was not only sensitive and specific, but also time and cost effective, leading to the development of a loop mediated isothermal amplification (LAMP) assay for the quantitative diagnosis of EEDV in fish tissues. This assay is highly specific for the EEDV glycoprotein gene, is cost effective, and has the potential for commercialization and use in field conditions. The end result of this dissertation is the uncovering of new insights on EEDV ecology, and a significant advancement in our understanding of EEDV-lake trout interactions."--Pages ii-iii.
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