Graduate Thesis Or Dissertation


Host-pathogen Interactions of Respiratory RNA Viruses and the Application of Recombinase Polymerase Amplification for Viral Diagnostics Public Deposited

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  • Humans and viral disease are inextricably intertwined. Viral disease plays an immeasurable role in human life, from the disease and economic burden associated with every facet of contending with human viral disease, to managing the consequences of viral disease in organisms important to our food supply, economy, and entertainment. The studies within this dissertation encompass crucial areas of viral research: host-pathogen interactions and diagnostics. Chapters 2 and 3 of this dissertation describe both the study and manipulation of viral-pathogen interactions. The next two chapters describe the application of recombinase polymerase amplification (RPA) for the detection of two prominent viral pathogens in need of improved diagnostic strategies. In Chapter 2, a host-viral pathogen interaction was exploited in an effort to develop a method for increasing influenza virus in the context of cell culture-based viral propagation for vaccine production purposes. PPMO antisense technology was employed to target and suppress the expression of the host gene Interferon alpha (IFNα), which is mainly involved in innate immune response against viral infection, in chicken embryo fibroblast (DF-1) cells. Suppression of IFNα by PPMO resulted in significantly reduced levels of IFNα protein in treated wells measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMO increased the ability of the influenza virus to replicate in DF-1 cells. Over a three-fold increase in viral production was observed in PPMO treated wells compared to those of untreated controls, which was observed to be independent of the initial viral input. Our results indicate that the use of PPMOs to target host protein expression can result in increased production of influenza virus; a technology that could be used on its own for improvement of vaccine production strategies or as a screening tool for subsequent permanent alterations in cell culture lines that would have similarly increased influenza virus production.In Chapter 3, host-viral pathogen interactions were examined in an attempt to understand an aspect of the host response to Respiratory Syncytial Virus (RSV) infection. The host gene, Myeloid Cell Leukemia 1 (Mcl-1), is upregulated early in RSV infection and is thought to have anti-apoptotic function. Mcl-1 knockout and wild type (WT) mouse embryonic fibroblast (MEF) cells were used to characterize the viral response to the absence of the host protein Mcl-1. The lack of Mcl-1 caused MEF cells to become highly permissive to RSV infection and resulted in extremely high levels of RSV compared to viral replication in WT MEF cells. Mcl-1 knockout cells also exhibited uncharacteristic morphology during RSV infection with increased and enlarged syncytial formation. Interestingly, apoptosis, which Mcl-1 helps regulate, was not induced in knockout cells until late in infection. The work presented in Chapter 3 provides evidence that Mcl-1 upregulation in RSV infection would not be beneficial to the virus, rather Mcl-1 upregulation is most likely an antiviral strategy and suggests a possible function for Mcl-1 separate of apoptosis regulation.In Chapter 4, we have developed a quick, sensitive, and adaptable recombinase polymerase amplification (RPA) diagnostic assay to detect a significant human pathogen, dengue virus (DENV). Dengue is considered the most important arbovirus worldwide and the World Health Organization has listed improved diagnosis as a key step in the fight to reduce the burden of DENV. We demonstrate that our DENV2 specific RT-RPA assay is sensitive and specific, as it is able to amplify DENV2 with as little as 50 copies per reaction within 20 minutes at a constant temperature, and was able to amplify both laboratory and clinical isolates strains. Our results provide justification for future development of RPA as a diagnostic strategy for detection of DENV in a clinical setting at point-of-care, thus eliminating the need for a costly thermocycler. In Chapter 5, RPA is applied to the herpes virus, cyprinid herpes virus 3 (CyHV-3). This DNA virus has had a considerable impact on koi and common carp that leads to devastating economic losses to both fishery and koi hobbyist. One problem with current diagnostics is the inability to reliably detect latently infected fish, capable of acting as carriers to nascent fish populations. The RPA assay to detect CyHV-3 was specific and sensitive, yielding results in approximately 20 minutes, and was able to detect the virus in latently infected koi more efficiently than a real-time PCR assay, when directly compared. RPA products were detected by a simple colorimetric lateral flow assay that could allow for detection outside of the diagnostic lab, allowing for sensitive and accurate surveillance and early diagnosis of CyHV-3 in the laboratory and field.Overall, the studies herein provide valuable knowledge about viral diseases. The data collected provides insights into the characterization of host-pathogen interactions of RSV. These insights are informative for the disease pathogenesis of this significant pathogen but may also apply to closely related viruses. In addition, a methodology is described in a new format that could prove to be valuable for influenza vaccine prevention strategies, as well as for any vaccine produced in cell culture. This work also describes the application of an isothermal amplification strategy for detection of two viral diseases that are in desperate need of improved diagnostics. RPA as a diagnostic tool is easily adaptable and can improve the speed, sensitivity, and resource consumption of viral diagnostics leading to the ability to detect viral disease at point-of-care or in low resource settings.
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