RNase footprinting and nitrocellulose filter-binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3’(+) stem loop (SL) RNA of West Nile virus (WNV) (2). Base substitutions in the major eEF1A binding site or adjacent areas of the 3’(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative affect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3’ SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, mutations that increased the efficiency of eEF1A binding to the 3’ SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3’ SL facilitates viral minus-strand initiation. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3’ end of the genome and the RC. eEF1A bound with similar efficiency to the 3’ terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue RC in infected cells. These results suggest that eEF1A plays a similar role in the RNA replication of all flaviviruses.

The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.

Resistance to flavivirus-induced disease is conferred by a single gene that encodes oligoadenylate synthetase (Oas) 1b (Oas1b). Oas1b is not a functional synthetase suggesting its anti-flavivirus mechanism is RNase L-independent and that it may be mediated by interactions with other host cell protein(s). A yeast two-hybrid screen was used to identify host cell binding partners of Oas1b. Candidate partners were confirmed by yeast co-transformation and co-immunoprecipitation analyses. Oxysterol binding protein-related 1L (ORP1L) and ATP binding cassette subfamily F 3 (ABCF3) were found to interact with Oas1b. RNAi knockdown studies suggested that ORP1L and ABCF3 form a tripartite complex with Oas1b that is critical for the flavivirus-induced disease resistance mechanism. Stresses including oxidation, nutrient starvation, and viral infections often induce the formation of stress granules (SGs) in eukaryotic cells. In response to stress, eIF2α kinases phosphorylate eIF2α leading to stalled 48S pre-initiation complexes and SG formation. West Nile virus (WNV) Eg101 infections were previously shown not to induce the formation of SGs. Infections with viruses of other natural WNV strains, as well as a WNV lineage 1/2-based infectious clone (W956IC) were analyzed and only W956IC infections were found to induce SGs. eIF2α kinase knockout MEFs were used to show that the W956IC-induced SGs were PKR-dependent. WNV chimeras were made by inserting Eg101 genes into the W956IC backbone. Chimeras replacing NS5 or NS1 and NS5 or NS1 and NS3 and NS4a reduced SG formation as well as early viral RNA synthesis similar to Eg101 infections. W956IC infections but not Eg101 infections were shown to produce exposed viral dsRNA at early times after infection. The data suggest that natural WNV infections evade the cell SG response by suppressing the amplification of viral RNA until cytoplasmic membranes have been remodeled to protect replication complexes from detection. It was previously reported that WNV Eg101 infections inhibited the formation of arsenite-induced SGs. The ability of other natural WNV strain infections to inhibit SG formation by arsenite (HRI), DTT (PERK), W956IC co-infection (PKR), and heat shock treatments was assessed. WNV infections only inhibited arsenite-induced SG formation suggesting that WNV infections specifically suppress the response to oxidative intermediates.

This thesis describes the West Nile virus (WNV) epidemic in horses by exploring all aspects: sub-clinical infection, development of clinical disease and case fatality. All of the collected data were then compiled to create predictive risk maps of WNV infection for the province of Saskatchewan. During the 2003 season, 133 clinical cases were documented with laboratory testing. Week of onset of clinical signs, gender, and coat color were significant predictors of whether the horse died or was euthanized due to severity of clinical signs. Studies of the serological response to vaccination and natural infection were examined to interpret the lab results from over 1100 samples taken from approximately 875 horses in 2003. A serologic study involving 212 horses on 20 farms determined the prevalence of sub-clinical infection (55.7% (95%CI, 44.9% to 65.8%)) and identifed risk factors for infection. The study found risk of infection was highest in the Grasslands ecoregions compared to the Boreal Transition ecoregion. A case control study looked at risk factors for development of clinical disease. The study followed 23 case farms and control farms with a total of 300 horses sampled. This was the first field study to show that vaccination was efficacious in preventing the development of clinical signs. The inclusion of horse surveillance data in the Saskatchewan Health WNV Integrated Surveillance Initiative was useful; however, it was discontinued due to time constraints, logistics, and declining monetary resources. Since West Nile Virus is a mosquito-borne disease it is highly influenced by environmental changes, spatially and temporally. Discriminant analyses were used to partition Saskatchewan rural municipalities (RM) into categories of risk of infection with WNV based on acquired horse data and different environmental and meteorological data derived from both satellites or climate stations. The result was the creation of yearly predictive risk maps defining low to high risk of infection with WNV for each RM. The 2003 epidemic provided a novel opportunity to study an important zoonotic disease emerging in a new environment. The information gathered will further the knowledge base upon which decisions for prevention of infection and clinical disease are made.

The study of the evolution and phylogeny of West Nile virus (WNV) has been an important area of research since the introduction of WNV in 1999. However, genome sequencing of isolates from the Southeastern part of the United States has been somewhat limited. To determine how WNV has evolved at a more localized level, ten isolates from Florida and Georgia from 2003-2012 were completely sequenced using Illumina's next-generation technology. In addition, a phylogenetic comparison of both the complete genome and select partial genomes was completed to ensure consistency among the results. This study further demonstrated the dominance of the North American WN02 genotype within the Southeastern United States. In addition, phylogenetic analyses revealed the continued presence of genetic variance in 2012 with the finding of a new group within the North American clade. In conclusion, WNV has continued to evolve within the Southeastern US.

This thesis describes aspects of the ecology of West Nile virus (WNV) including factors contributing to the distribution of WNV, possible future distribution, and effects of WNV on host abundance in the Canadian prairies provinces: Alberta, Saskatchewan, and Manitoba. Using mosquito surveillance data collected between 2005 and 2008, models integrating abiotic and biotic factors were constructed to predict the weekly and monthly scales of WNV infection rate in Culex tarsalis, which is the primary vector of WNV in the Canadian prairies. At the weekly scale, the WNV infection rate in Cx. tarsalis increased with increasing Cx. tarsalis abundance and mean temperature lagged from 1 to 8 weeks, but decreased with an increasing mean precipitation lagged from 2 to 6 weeks. Furthermore, precipitation was a ‘distorter variable’ which altered the association between Cx. tarsalis abundance and the WNV infection rate. Study at the monthly scale showed that higher mean temperature and time lagged mean temperature elevated were associated with increased numbers of Cx. tarsalis and higher WNV infection rates. However, increasing precipitation was associated with higher abundance of Cx. tarsalis and lower WNV infection rate. In addition, this study found that increased temperature fluctuation and wetland land cover were associated with decreased WNV infection rate in Cx. tarsalis. Climate change could drive dramatic alterations in the spatial and temporal distribution and overall incidence of vector-borne diseases. The constructed models and biological thresholds were used to predict the distribution of Cx. tarsalis and WNV infection rate in the prairie provinces under a range of potential future climate and habitat conditions. In the current endemic regions, the projected WNV infection rate under the median outcome scenario in 2050 was 18 times higher than under current climate conditions. Seasonal occurence of Cx. tarsalis infected with WNV extended from June to August to include May and September. Moreover, models predicted northward range expansion for Cx. tarsalis and WNV. The declines of susceptible bird abundance caused by WNV may further influence the bird community composition and, in turn, affect the incidence of WNV through a dilution effect. The North American Breeding Bird Survey data was used to evaluate the effect of WNV on the abundance of selected birds in the Canadian prairies, as well as the effects of bird community composition on the WNV risk. There was no significant decline in bird abundances of selected birds following the emergence of WNV. These findings suggest that the effect of WNV on selected bird abundance and bird community composition is insignificant. In addition, there is no evidence to support the association between bird community composition and WNV infection rate in Cx.tarsalis in the Canadian prairies. Lastly, findings in this thesis and current knowledge were integrated to create a decision making flowchart for the prevention of WNV infection in the prairie provinces.

The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.

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.

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.