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Viral Hemorrhagic Septicemia Virus (Vhsv) Great Lakes Strain University of Wisconsin Milwaukee UWM Digital Commons Theses and Dissertations May 2013 Viral Hemorrhagic Septicemia Virus (Vhsv) Great Lakes Strain Ivb: Viral Detection, Mechanisms of Infection, and Host-Virus Interactions in the Yellow Perch (Perca Flavescens) Wendy Joy Olson University of Wisconsin-Milwaukee Follow this and additional works at: https://dc.uwm.edu/etd Part of the Aquaculture and Fisheries Commons, Biology Commons, and the Virology Commons Recommended Citation Olson, Wendy Joy, "Viral Hemorrhagic Septicemia Virus (Vhsv) Great Lakes Strain Ivb: Viral Detection, Mechanisms of Infection, and Host-Virus Interactions in the Yellow Perch (Perca Flavescens)" (2013). Theses and Dissertations. 323. https://dc.uwm.edu/etd/323 This Dissertation is brought to you for free and open access by UWM Digital Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UWM Digital Commons. For more information, please contact [email protected]. VIRAL HEMORRHAGIC SEPTICEMIA VIRUS (VHSV) GREAT LAKES STRAIN IVB: VIRAL DETECTION, MECHANISMS OF INFECTION, AND HOST-VIRUS INTERACTIONS IN THE YELLOW PERCH (PERCA FLAVESCENS) by Wendy Joy Olson A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Sciences at The University of Wisconsin-Milwaukee May 2013 ABSTRACT VIRAL HEMORRHAGIC SEPTICEMIA VIRUS (VHSV) GREAT LAKES STRAIN IVB: VIRAL DETECTION, MECHANISMS OF INFECTION, AND HOST-VIRUS INTERACTIONS IN THE YELLOW PERCH (PERCA FLAVESCENS) by Wendy Joy Olson The University of Wisconsin-Milwaukee, 2013 Under the Supervision of Professor Drs. Frederick Goetz & Ava Udvadia Abstract Viral hemorrhagic septicemia virus (VHSV) is one of the most devastating and problematic viral fish diseases to plague the European aquaculture industry, and due to its pathogenicity, disease course, mortality rates, and wide host range, remains one of the most pathogenic viral diseases of finfish worldwide. A new freshwater strain of viral hemorrhagic septicemia virus IVb (VHSV-IVb) in the Great Lakes has been found capable of infecting a wide number of naive species, and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. In this study, the yellow perch, Perca flavescence , one such species documented in several fish kills affiliated with VHSV, was used as a research model to elucidate host-virus interactions to better understand the mechanisms of viral infection. A direct comparison of viral infection kinetics and net mortality among yellow perch stocks derived from distinct genetic and geographic regions found large variation in susceptibility to the disease, suggesting that genetic variance within a population can play a significant role in survival after infection with VHSV-IVb. Upon investigation of the early acute innate immune ii response after exposure to the VHS virus, a significant up-regulation of Mx expression in the liver, as well as the inflammatory response genes IL-1β and SAA, in all three tissues sampled, head kidney, spleen, and liver, was directly correlated to viral load indicating the role of these genes in the initial stages of infection. Viral load increased most rapidly in the head kidney and spleen, suggesting that potential down-regulation of Mx in these tissues may represent a viral strategy to increase replication. Finally, when the pathology and distribution of the virus were monitored in different tissues of adult fish exhibiting a low level of VHSV infection over time, the finding of significant viral load in the gills and blood suggest that the sampling of these tissues may offer a more accurate, non-lethal alternative to viral screening from the currently, more traditional and lethal cell culture analysis of the head kidney/spleen. A significant viral load in the brain, however, particularly in the later stages of infection, also suggests that latent virus may remain in the brain neurons undetected in asymptomatic carriers of the virus, classifying the VHS virus as neurotropic, as well as a hemorrhagic. iii TABLE OF CONTENTS CHAPTER 1 - BACKGROUND & INTRODUCTION ................................................1 MAIN OBJECTIVES................................................................... ..........................16 CHAPTER 2 - COMPARATIVE SUSCEPTIBILITY AMONG THREE STOCKS OF YELLOW PERCH TO VHSV-IVB.........................................................................21 MATERIALS & METHODS..................................................................................24 RESULTS................................................................................................................28 DISCUSSION..........................................................................................................35 CHAPTER 3- EXPRESSION KINETICS OF KEY GENES IN THE EARLY INNATE IMMUNE RESPONSE TO VHSV-IVB INFECTION................................44 MATERIALS & METHODS..................................................................................48 RESULTS................................................................................................................53 DISCUSSION..........................................................................................................63 CHAPTER 4 - VIRAL DISTRIBUTION AND PATHOLOGY OF VHSV-IVB) IN ADULT YELLOW PERCH...........................................................................................75 MATERIALS & METHODS..................................................................................79 RESULTS................................................................................................................84 DISCUSSION..........................................................................................................93 CHAPTER 5-FINAL DISCUSSION............................................................................108 CURRICULUM VITAE................................................................................................117 iv LIST OF FIGURES CHAPTER 1 - INTRODUCTION & OBJECTIVES.....................................................1 Figure 1-1. (Photo image ) VHS-induced fish kill.............................................................2 Figure 1-2. USDA APHIS List of VHS susceptible/regulated species.............................3 Figure 1-3. VHSV genome and generalized life-cycle......................................................5 Figure 1-4. Juvenile pike exhibiting clinical signs of VHS...............................................6 Figure 1-5. Clinical signs of VHSV...................................................................................7 Figure 1-6. Phylogenetic relation among VHS isolates...................................................11 Figure 1-7. VHS-IVb distribution in the Great Lakes.....................................................12 Figure 1-8. Yellow perch distribution in North America................................................14 CHAPTER 2 - COMPARATIVE SUSCEPTIBILITY AMONG THREE STOCKS OF YELLOW PERCH TO VHSV-IVB.........................................................................21 Figure 2-1. (Photo image )Clinical signs of VHSV-IVb infection in yellow perch.........30 Figure 2-2. Cumulative % survival for each population of VHS exposed fish...............31 Figure 2-3. Kinetics of VHSV-IVb infection..................................................................33 Figure 2-4. Linear regression between qRT-PCR vs. plaque assay.................................34 CHAPTER 3- EXPRESSION KINETICS OF KEY GENES IN THE EARLY INNATE IMMUNE RESPONSE TO VHSV-IVB INFECTION................................44 Figure 3-1. (Photo image )Clinical signs of VHSV-IVb infected yellow perch..............53 Figure 3-2. Daily mortalities recorded in VHSV infected yellow perch.........................54 v Figure 3-3. Linear regression analysis of viral load over time........................................55 Figure 3-4. Increase in Viral load over time....................................................................56 Figure 3-5. Mx gene expression over time......................................................................58 Figure 3-6. Linear regression analysis of Mx expression vs. Viral load.........................59 Figure 3-7. IL-1β gene expression over time...................................................................61 Figure 3-8. Linear regression analysis of IL-1β expression vs. Viral load......................62 Figure 3-9. SAA gene expression over time....................................................................64 Figure 3-10. Linear regression analysis of SAA expression vs. Viral load.....................65 CHAPTER 4 - VIRAL DISTRIBUTION AND PATHOLOGY OF VHSV-IVB) IN ADULT YELLOW PERCH...........................................................................................75 Figure 4-1. Cumulative % survival for each population of VHS exposed fish...............87 Figure 4-2. Viral titer over time in low-dose VHSV-injected fish by plaque assay........88 Figure 4-3. Viral titer over time in high-dose VHSV-injected fish by plaque assay.......90 Figure 4-4. Viral titer over time in high-dose VHSV-injected fish by qPCR..................92 Figure 4-5. Linear regression analysis of VHSV in blood vs. Brain...............................93 Figure 4-6. DIC & Fluorescence image of VHSV-associated erythrocytes....................95 Figure 4-7. Fluorescence image of a VHSV-associated leukocyte..................................96
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