This thesis assesses the water quality of the River Nile around the city Khartoum in Khartoum State, Sudan Republic, and investigates eventual influences of the city on the River Nile by analysis of the following parameters: temperature, pH, and conductivity, and Adsorbable Organic Halogen (AOX), cadmium (Cd), lead (Pb), chromium (Cr), Total Organic Carbon(TOC) and Nitrate (NO3− ). A survey of the area is also included. It was concluded that the city Khartoum added small but legible concentrations of cadmium, lead, chromium and TOC to the river Nile. However, the resulting concentrations were all within acceptable levels. Also, the observed results showed that the Blue and White Nile, which merge together upstream on the outskirts of Khartoum, had concentrations of AOX resp. chromium, which were not suitable for drinking water.

West Nile virus (WNv) is an arbovirus and is transmitted by infected mosquitoes after feeding on the blood of birds carrying the virus. The Canadian WNv national surveillance system has just completed its tenth year of operation. The thesis is to evaluate the surveillance system and analyze multi-year human data. The evaluation includes the use of multiple lines of complementary methods such as the US CDC surveillance guidelines, Canadian Evaluation Framework, document review and a survey. Logistic and Poisson regressions were used for data analyses. WNv has become endemic in most parts of Canada since the virus occurred in 2001. The virus activity is peak around August. High numbers of human cases with WNv neurological syndrome identified pose a significant health concern due to the long term sequelae among affected patients. WNv national surveillance met its main objectives and there is a continual need for the surveillance.

West Nile Virus (WNV) first appeared in the western hemisphere in 1999, and has since spread across the United States and into Mexico and the Caribbean. It has been hypothesized that WNV has spread rapidly via migratory birds, and that various avian species may facilitate viral amplification during winter months. The goals of this research were to determine the role of American Tree Sparrows (Spizella americana) in the spread of WNV during their igrations and to determine the role of the Northern Cardinal (Cardinalis cardinalis) in winter survivorship and subsequent spring amplification of WNV. Additional wintering avian species were sampled to provide a general survey of the prevalence of WNV in winter in south-central Kansas. Blood samples were taken from the brachial vein of migratory and wintering birds captured using mist nets at four wintering feeding stations at the Wichita State University Field Station. Some samples were taken from retrapped birds within a single winter to determine if winter transmission occurs. Some birds were resampled in consecutive winters to monitor seroconversion rates. Analysis of serum samples were performed, in triplicate, using an epitope-blocking ELISA. The current study was conducted during the consecutive winters of 2003-04 and 2004-05. It was concluded that resident species had an increased incidence of WNV exposure when compared to that of migratory species. This difference suggests that migratory species may not have as important a role in the dissemination of WNV as first hypothesized. Also, minimal, if any, winter transmission occurs on communal feeding grounds. Viral amplification during the winter was not demonstrated, although one individual seroconverted during a single winter. / Thesis (M.S.)--Wichita State University, Dept. of Biological Sciences / Includes bibliographical references (leaves 35-39)

The 1,2,5-Thiadiazolidin-3-one 1,1-dioxide scaffold was used in the design and synthesis of inhibitors of Dengue Virus and West Nile Virus proteases and human tryptase. The scaffold was successfully used in the synthesis of potential inhibitors of Dengue Virus and West Nile Virus proteases. Inhibitors of Human tryptase synthesized based on the 1,2,5-Thiadiazolidin-3-one 1,1- dioxide scaffold were shown to be effective mechanism-based inhibitors of the enzyme. / Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry. / "July 2006." / Includes bibliographic references (leaves 64-66).

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.

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.

<p>This thesis assesses the water quality of the River Nile around the city Khartoum in Khartoum State, Sudan Republic, and investigates eventual influences of the city on the River Nile by analysis of the following parameters: temperature, pH, and conductivity, and Adsorbable Organic Halogen (AOX), cadmium (Cd), lead (Pb), chromium (Cr), Total Organic Carbon(TOC) and Nitrate (NO3− ). A survey of the area is also included. It was concluded that the city Khartoum added small but legible concentrations of cadmium, lead, chromium and TOC to the river Nile. However, the resulting concentrations were all within acceptable levels. Also, the observed results showed that the Blue and White Nile, which merge together upstream on the outskirts of Khartoum, had concentrations of AOX resp. chromium, which were not suitable for drinking water.</p>