Browsing by Author "DeWitte-Orr, Stephanie"

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COVID-19: Where we've been, where we're going
(Balsillie School of International Affairs, 2022-02-24) DeWitte-Orr, Stephanie; Whiteside, Alan
This paper offers a brief history of viral pandemics and the science of the COVID-19 virus. After reviewing COVID-19’s economic, social, psychological and political impacts, we touch on the implications for health care. Finally, we look at the need for a global, integrated approach that requires new multi-disciplinary thinking, and what this means for policy makers.
DsRNA-mediated antiviral immunity in fish cells: visualization, sensors, and innate immune responses
(University of Waterloo, 2018-06-15) Poynter, Sarah J.; Dixon, Brian; DeWitte-Orr, Stephanie
The global aquaculture industry is a multibillion dollar business that is threatened by pathogens, including a wide array of aquatic viruses. Currently there are no antiviral treatments available to combat viral outbreaks, and as such viral infection can cause vast economic loss. Two important species for aquaculture include rainbow trout (Oncorhynchus mykiss), destined for human consumption, and fathead minnow (Pimephales promelas), a common species grown for bait purposes. Double-stranded (ds)RNA is a potent immunomodulating molecule produced during viral replication; dsRNA treatment induces a robust antiviral state that makes host cells refractive to viral replication. This thesis explored dsRNA-induced innate antiviral pathways from two angles. Firstly, the differences in dsRNA-induced responses between virally-produced dsRNA, synthesized dsRNA with natural sequence variation, and synthesized dsRNA with a homogenous sequence were analyzed in rainbow trout cells. The dsRNA, regardless of source, was sensed at the cell surface by a common receptor in rainbow trout cells and induced an innate immune response and antiviral state against two aquatic viruses, viral hemorrhagic septicemia virus and infectious pancreatic necrosis virus; the virally-produced and lab-synthesized dsRNA molecules with natural sequence variation produced the most similar responses. The second angle of approach was to better understand the host’s response to exogenous dsRNA treatment. To this end, novel dsRNA sensors were identified and characterized in rainbow trout. This included class A scavenger receptors, the purported surface receptors for dsRNA, including MARCO, SCARA3, SCARA4, and SCARA5, as well as two novel cytoplasmic dsRNA sensors from the DExH/D-box family, DDX3 and DHX9. The receptors found in rainbow trout contained the same conserved domains that are found in their mammalian counterparts, a first indication of conserved functionality. Two MARCO variants were identified and found to bind to two gram-negative and one gram–positive bacteria, but surprisingly not to dsRNA. Rainbow trout DDX3 and DHX9 are both functional in their ability to bind to dsRNA. The culmination of these findings was the development of a dsRNA molecule with sequence variation that can act as a potent antiviral therapy in vitro in fathead minnow cells. The findings from this thesis demonstrate the importance of ‘natural’ dsRNA as an innate immune signalling molecule and its potential to function as a prophylactic antiviral therapeutic for fish.
The therapeutic application of Pituitary Adenylate Cyclase-Activating Polypeptide and double stranded (ds)RNA as immunostimulants and antiviral agents in crustaceans
(University of Waterloo, 2022-05-16) Monod, Emma; Dixon, Brian; DeWitte-Orr, Stephanie
Currently valued at 28 billion USD per year, the global shrimp market is growing faster than any other aquaculture industry. The initial fast-paced global industry expansion led to many catastrophic viral shrimp pandemics. White spot syndrome virus (WSSV), a DNA virus with global distribution, is a major causative agent of massive shrimp die-offs worldwide. Shrimp farmers are limited in their ability to manage and reduce disease burden due to the absence of effective antiviral treatments. The present study focuses on the application of therapeutics in crustaceans with the goal of inducing a general immune response to control viral infection. Double-stranded (ds)RNA has been well characterized in vertebrates as an innate immune stimulant and potent inducer of the antiviral response through type I interferons and interferon stimulated genes. Pituitary adenylate cyclase activating polypeptide (PACAP) is a highly conserved, multifunctional, cationic neuropeptide with antimicrobial properties. In vertebrates PACAP has been shown to regulate pro- and anti-inflammatory cytokine production through cAMP signalling cascades and to interfere directly with viral protein transcription. In this study, wild caught Ontario crayfish were injected with 10 µg of High molecular weight (HMW) Polyinosinic:polycytidylic acid (synthetic dsRNA), a low (6 µg) and high (12 µg) dose of PACAP-38 and a combination of 10µg Poly(I:C) HMW and 12µg PACAP-38. The safety of these treatments was first established through exposure of primary hemocytes in vitro to concentrations of PACAP between 1.95 and 250nM and Poly(I:C) HMW between 0.0076 and 10µg/mL which did not reduce cell metabolism or membrane integrity. These therapeutics were then delivered to crayfish by both intramuscular and ventral sinus injection. Following treatment for 6-, 24-, 48- and 168-hours immune system activation was measured using functional markers of cellular and humoral responses, including the total number of hemocytes, lectin activity, nitric oxide concentration and activated phenoloxidase function. Following in vivo stimulation by ventral sinus injection, animals treated with Poly(I:C) HMW, PACAP and the PACAP+HMW combination had a higher total number of hemocytes and a significant increase in hemagglutination and phenoloxidase activity between 6 and 168 hours. PACAP treatment for 6 hours resulted additionally in a significant increase in the hemolymph concentration of nitric oxide. This work establishes the immunostimulatory role of dsRNA and PACAP-38 in crustaceans which will benefit the shrimp aquaculture industry by contributing to the development of effective broad-spectrum immune therapies.