Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.

West Nile Virus (WNV) has emerged as a major cause of viral encephalitis. Since its outbreak in the United States 27,000 people have presented with clinical WNV disease resulting in 1074 fatalities. WNV causes a range of disease from mild febrile illnesses to severe and fatal encephalitis. To date, there are currently no therapeutic agents or vaccines available to treat WNV infection in humans. In order to address this, a better understanding of the mechanisms responsible for viral neuroinvasion and neuropathology are required. Using a range of in vitro and in vivo studies, we have investigated the routes by which WNV enters the CNS. Virus replication was observed in the brain microvascular endothelial cells in mice that succumbed to WNV encephalitis. Moreover, we demonstrated that infection of a polarized HBMEC with WNV induced apoptosis. Microarray analysis of WNV-infected HBMEC’s revealed that WNV elicited the expression of cytokines that have been shown to contribute to permeablization of the BBB. These findings suggest that WNV can enter the CNS through the BBB via multiple mechanisms. Real-time RT-PCR performed on WNVinfected HBMECs identified two host genes involved in the host cellular anti-oxidant response that were differentially regulated during viral infection. Furthermore, the addition of the antioxidant, N-acetylcysteine, restored cell viability and decreased viral replication, indicating that oxidative stress contributes to WNV-induced pathogenesis. The current state of knowledge regarding the pathogenesis of WNV encephalitis is based on studies that have defined the role of systemic immune responses to WNV. Limited investigations have been undertaken to determine the contribution of brain cells in the defence, or damage to the brain once WNV has gained access to the CNS. Real-time RT-PCR results in conjunction with in vivo CBA assay data, suggested several candidate host genes that could contribute to the pathogenesis of WNV. Thus, it is necessary to further define the mechanisms of WNV induced pathogenesis as this will aid in the development of targeted strategies to prevent neurological infection and mitigate neurological diseases in affected individuals.

Thesis (M.S.), Biology--University of Central Oklahoma, 2007. / Includes bibliographical references (leaves 27-35 ).

Thesis (Ph.D.)--York University, 2006. Graduate Programme in Mathematics and Statistics. / Typescript. Includes bibliographical references (leaves 126-132). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR19847

Thesis (Ph. D.)--Georgia State University, 2007. / Title from file title page. Margo Brinton, committee chair; W. David Wilson, Teryl Frey, committee members. Electronic text (120 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Nov. 20, 2008. Includes bibliographical references.

Thesis (M.A.)--State University of New York at Binghamton, Dept. of Geography, 2006. / Includes bibliographical references.

Mémoire (M.Sc.B.)--Université du Québec à Chicoutimi, 1990. / Document électronique également accessible en format PDF. CaQCU

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 98-108). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 98-108). Also available on the Internet.

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisors: Jack B. Gingrich and Douglas W. Tallamy, Dept. of Entomology & Wildlife Ecology. Includes bibliographical references.