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Because little is known about juvenile American White Pelicans (Pelecanus erythrorhynchos) this study was conducted to gather more information on disease, general ecology and growth of American White Pelicans from hatching to fledging. In July 2011, American White Pelican regurgitate samples from North and South Dakota sub-colonies were collected/analyzed in preparation for a captive trial. Nutrient content compared between the colonies was found to be significantly different. Concentrations of Immunoglobulin Y and A in regurgitate samples were significantly different between colonies. A captive trial began 29 May 2012 and ended 30 July 2012, in which 16 American White Pelicans were hand raised from hatching to fledging. During the captive trial, various growth parameters, intake and fecal output were examined to determine the effect of the parasite Bolbophorus damnificus in 8 infected and 8 non-infected (parasite free) pelicans. Growth data collected on B. damnificus infected (n = 8) American White Pelicans was compared to previously mentioned parasiteree pelicans (n = 8) to determine effects of the parasite. There were no differences between groups for culmen length (P= 0.214), tarsal length (P = 0.306), body weight (P = 0.884) or intake (P = 0.963). There was also no effect of the parasite on body temperature. Towards the end of the captive trial, several pelicans both on (n = 16) and off (n = 11) trial became naturally infected with West Nile Virus. Clinical symptoms ranged from lethargy and/or wing droop to total paralysis. Progression of disease is detailed in two well-defined case studies with additional information included on clinical signs, physiological parameters, and a review of the pathology of disease for other infected birds.

Scavenging bird of prey have been abundant throughout the Mediterranean region for years, establishing a food acquisition relationship with human activities, their characteristics make them especially suitable for the recognition of dangerous environmental conditions. The West Nile Virus (WNV) considered the most widespread arbovirus has been previously discovered in our 3 species of studio. The analyses from feather follicles and serum samples obtain from different regions in Spain shown a higher seroprevalence against West Nile virus or cross-reacting flaviviruses in the northern part of the country and in the Canary archipelago. Our results showed a general seroprevalence of 19.8% (34 out of 172), a prevalence of 19,8% in Egyptian vultures, a prevalence of 27.1% (13 out of 48) in Griffon vultures and no prevalence in Cinereous vultures. Differences were observed for the body condition off Griffon and Egyptian vultures when anthropized and not anthropized areas were compared, being significantly worse in anthropized areas for Griffon vultures and significantly better for Egyptian vultures. The exposure to the virus of the nestlings increased with the age, showing a higher tend in Egyptian vultures than in Griffon vultures. Our results confirm the circulation and a higher seroprevalence of West Nile virus or cross-reacting flaviviruses than in previous studies.

Mosquito-borne viruses, including dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), are rapidly emerging, global pathogens. Though the number of people impacted by each virus varies, there have been thousands to millions of people infected. The focus of this thesis work centers around USUV and WNV; both have RNA genomes and belong to the Flaviviridae virus family. Both WNV and USUV were initially isolated in Africa and have since spread to Europe; interestingly, WNV has also spread globally and is considered endemic in the Americas. Similar to other flaviviruses, USUV and WNV are maintained in a mosquito vector-avian host transmission cycle, with spillover infection into humans. Human infections of WNV and USUV are usually asymptomatic, but in severe cases can cause neuroinvasive disease. WNV and USUV belong to the JEV serocomplex group, which indicates that antibodies produced against these viruses share a common antigen; the common antigen is hypothesized to be the envelope (E) protein on the outside of the virion. Neutralizing antibodies against both WNV and USUV have been found in birds and humans across Europe. In vitro cross-neutralization of WNV and USUV has been modeled experimentally and been observed in clinical settings. The neutralizing antibody response generated against WNV has been studied extensively in mouse models; however, there are few studies which examine the neutralizing antibody response generated against USUV. Whether prior WNV exposure protects against USUV disease is also unknown. The main goal of this thesis was to characterize how a primary flavivirus exposure would influence a secondary flavivirus exposure; specifically, we wanted to observe if WNV exposure would protect against USUV disease in vivo and generate a cross-neutralizing antibody response in vitro. For the WNV exposure, we used an attenuated vaccine strain of WNV that contains the WNV E gene (D2/WN-V3) developed by our collaborators. We hypothesized that treatment with D2/WN-V3 would protect against USUV infection. Two in vivo models were used: CD-1 mice and interferon alpha-beta receptor 1 deficient (Ifnar1-/-) mice. We discovered that sera from mice vaccinated with D2/WN-V3 neutralized both WNV and USUV in vitro. In the Ifnar1-/- model, we observed that vaccinated mice had higher survival rates and lower USUV viremia levels after USUV challenge. This work helps characterize the consequences of flavivirus antibody cross-neutralization in vitro and cross-protection in vivo. As the flavivirus field moves toward the goal of creating a pan-flavivirus vaccine, both cross-reactive antibodies and cross-protection need to be considered. / Master of Science / West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that were originally isolated in Africa during the 20th century. Both viruses are maintained through a transmission cycle between mosquito vectors and avian hosts. Mosquitos transfer the infectious agent (WNV or USUV) through feeding on a bird (usually a passerine species); once in the bird, the virus can replicate to high levels. Human infections of WNV and USUV from mosquitos can also occur, with symptoms ranging from mild febrile illness to severe encephalitis or meningitis. Over the past few decades, WNV and USUV have spread to Europe, most likely through infected migratory birds. Interestingly, mosquito surveillance studies in mainland Europe have found mosquitos that tested positive for both USUV and WNV. In Europe, antibodies for both viruses have been found in humans and birds, indicating a previous exposure to WNV, USUV, or both. The neutralizing antibody response is a critical immune defense against viral infections. Neutralizing antibodies bind strongly to the outside of the virion (virus particle), preventing the virion from interacting with and infecting the host cell. For WNV and USUV, one of the targets that neutralizing antibodies bind to is the outer envelope (E) protein of the virion. In clinical settings and experimental studies, cross-neutralization of WNV and USUV has been documented. During cross-neutralization, a serum sample containing neutralizing antibodies against WNV can also neutralize USUV, and vice versa. Although the neutralizing response against WNV has been characterized in humans and lab animal models such as mice, there is little research regarding the neutralizing response against USUV. Importantly, whether prior WNV exposure provides protection against USUV infection is currently unknown. The main goal of this thesis was to characterize the disease outcome and neutralizing response against USUV after a WNV exposure. For the WNV exposure, we used a vaccine strain of WNV that contains the E gene (D2/WN-V3) developed by our collaborators. We predicted that vaccinated mice would avoid USUV clinical signs of disease and generate neutralizing responses to WNV and USUV. To do this work, we used two laboratory mouse models: mice with an intact immune response system (CD-1) and mice with a stunted immune response (Ifnar1-/-). We discovered that serum from vaccinated mice did cross-neutralize WNV and USUV. In the Ifnar1-/- model, vaccinated mice had higher survival rates and lower levels of virus in blood after USUV infection compared to unvaccinated mice. Ultimately, this work highlights the importance of characterizing the immune response against similar viruses and will inform the development of human vaccines for both viruses.

West Nile Virus (WNV), a vector-borne disease continues to be a serious threat to public health in the United States, particularly in the Southwest region. While all the states in the U.S. experienced a decreasing trend of WNV disease in 2010, the state of Arizona experienced a sharp increase from 20 in 2009 to 166 cases the following year. This dissertation endeavored to develop forecasting models to predict future cases of disease and identify counties with increased propensity for WNV. Furthermore, this study aimed to identify environmental and economic factors that contributed to the increase in WNV cases in Maricopa County, Arizona. A spatiotemporal stochastic regression model was developed using Bayesian principles and was successful in calculating the annual mean cases of disease from 2003 to 2011 for all counties. The model was also able to predict future cases of disease by fitting historical data. The model-based inference identified counties in the southern region of Arizona as having an elevated propensity for disease compared to counties in the northern region. A Seasonal Autoregressive Integrated Moving Average (SARIMA) model was developed and effectively forecasted monthly cases of human WNV in Maricopa County, Arizona. By fitting the SARIMA model to monthly historical disease data from 2005 to 2011, the temporal model presented a decreasing trend of monthly incidence of disease for 2012. The impact of home foreclosures, climate variability, and population growth on the resurgence of human WNV disease cases in Maricopa County during the 2010 epidemic was investigated. These factors were found to have contributed to the resurgence of the disease by creating the optimal environmental conditions that allowed the amplification of mosquito populations, thus increasing the risk of disease transmission to humans. As spatiotemporal disease data become readily available, forecasting models can be an important and viable risk assessment tool for public health practitioners. Forecasting models allow the mobilization and distribution of limited resources to areas with elevated propensity for disease, and the timely deployment of intervention programs to reduce the overall risk of disease. / Graduation date: 2013

West Nile virus (WNV) is an emerging infectious disease that has widespread implications for public health practitioners across the world. Within a few years of its arrival in the United States the virus had spread across the North American continent. This research focuses on the development of a spatially explicit GIS-based predictive epidemiological model based on suitable environmental factors. We examined eleven commonly mapped environmental factors using both ordinary least squares regression (OLS) and geographically weighted regression (GWR). The GWR model was utilized to ascertain the impact of environmental factors on WNV risk patterns without the confounding effects of spatial non-stationarity that exist between place and health. It identifies the important underlying environmental factors related to suitable mosquito habitat conditions to make meaningful and spatially explicit predictions. Our model represents a multi-criteria decision analysis approach to create disease risk maps under data sparse situations. The best fitting model with an adjusted R2 of 0.71 revealed a strong association between WNV infection risk and a subset of environmental risk factors including road density, stream density, and land surface temperature. This research also postulates that understanding the underlying place characteristics and population composition for the occurrence of WNV infection is important for mitigating future outbreaks. While many spatial and aspatial models have attempted to predict the risk of WNV transmission, efforts to link these factors within a GIS framework are limited. One of the major challenges for such integration is the high dimensionality and large volumes typically associated with such models and data. This research uses a spatially explicit, multivariate geovisualization framework to integrate an environmental model of mosquito habitat with human risk factors derived from socio-economic and demographic variables. Our results show that such an integrated approach facilitates the exploratory analysis of complex data and supports reasoning about the underlying spatial processes that result in differential risks for WNV. This research provides different tools and techniques for predicting the WNV epidemic and provides more insights into targeting specific areas for controlling WNV outbreaks.

Thesis (Dr. P.H.)--University of Texas School of Public Health, 2008. Thesis (Dr. P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0973. Adviser: Kristy O. Murray. Includes bibliographical references.

Le virus West Nile,WNV est responsable de milliers de cas de morbidité et de mortalité chez les oiseaux, les chevaux et l’homme. Le WNV se transmet par des moustiques du genre Culex. Les méthodes entomologiques ne permettent pas l’évaluation individuelle directe du contact hôte/vecteur. 5 protéines salivaires de Culex ont été sélectionnées, produites, et évaluées comme des candidats antigéniques de l'exposition aux piqûres de Culex. Des sérums humains du sud de France exposés à des densités de Culex distinctes et des sérums de chevaux exposés à l'infection par le WNV ont été testés. Une protéine 30kD est reconnue par les chevaux exposés à Culex. Cependant, pas de différence de réponse d’anticorps n’a été observée entre les animaux faiblement et fortement exposés. Concernant les processus physiopathologiques de la maladie causée par le WNV, la cinétique des profils d'expression de protéines de l’hôte dans le cerveau de souris infectées par le WNV, a été étudiée sur des échantillons prélevés avant et après l’apparition des signes cliniques, en utilisant 2D-DIGE et iTRAQ. 148 protéines différentiellement exprimées. Les voies de signalisation altérées au cours de l'infection précoce et tardive ont été identifiées. Les profils protéiques de LCR de patients atteints de WNND et des individus témoins ont été comparés, en utilisant l’approche iTRAQ. 47 protéines ont été trouvées différemment exprimées chez les patients WNND. Un candidat potentiel biomarqueur, la Defensine-alpha1, a été évalué par ELISA sur des échantillons humains de LCR/sérum. Les biomarqueurs putatifs identifiés dans cette étude peuvent être un outil précieux d’évaluation de la mesure de la gravité du WNV. / West Nile Virus,WNV is responsible for thousands of cases of morbidity and mortality in birds, horses and humans. WNV is transmitted mainly by mosquitoes by Culex species, to avian hosts. Entomological methods did not give direct individual evaluation of the host/vector contact. 5 salivary proteins from the Culex genus were selected for a production under recombinant forms for further evaluation as potential antigenic candidates of exposure to Culex bites. Sera from individuals living in south of France exposed to distinct Culex density and sera from horses exposed to WNV infection were tested. The recombinant protein30 kDa was recognized only by horses exposed to Culex. However, no difference of antibody response between low and high exposed to Culex. Concerning the pathophysiological processes of WNV disease, a kinetics host brain protein expression profiles of WNV-infected mice using samples collected prior and after clinical signs apparition was performed using proteomic approaches 2D-DIGE and iTRAQ. 148 distinct proteins was found altered following WNV infections. The functional signaling networks in samples collected during early and late infection have been identified. Un examination of CSF protein profiles between patients with neuroinvasive disease (WNND) and control individuals was performed using iTRAQ approach. 47 proteins were found differentially expressed in WNND patients compared to controls. A potential biomarker candidates, defensin-alpha1 was assessed by ELISA using other human paired CSF/serum samples. The putative biomarker identified in this study may potentially be a valuable tool in the assessment of the extent of WNV severity.

Doctor of Philosophy / Department of Anatomy and Physiology / Elizabeth G. Davis / Activation of innate immunity through Toll-like receptor (TLR) signaling can also enhance antigen-specific adaptive immunity. TLR9 is an endosomal receptor for unmethylated bacterial and viral cytosine-phosphate-guanine DNA (CpG-DNA). West Nile virus (WNV) infection may result in meningitis and encephalitis in humans and horses, especially aged and immunocompromised individuals. Using flow cytometric analyses and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated equine cell-mediated immunity (CMI) to an inactivated West Nile virus vaccine in healthy yearling and adult horses. We also studied the potential of enhancing equine adaptive immunity to viruses and other pathogens by activation of innate immunity though TLR9 signaling pathway. We found vaccination with inactivated WNV vaccine induced strong WNV-specific T helper type 1 (Th1) and Th2 CMI with a Th1 bias, also effectively induced WNV-specific CTLs in yearling horses. In adult horses, the pre-existing Th1 CMI bias against WNV was enhanced following booster vaccination with inactivated WNV vaccine. Molecular characterization and flow cytometric analysis of TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in neutrophils (PMNs), CD4[superscript]+ and CD8[superscript]+ T cells and other leukocytes. Conservation of equine TLR9 and a high expression profile among leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity. Unmethylated CpG-DNA can significantly activate equine PMNs. It also induces expression of interferon (IFN)-[Alpha], IFN-[Beta], IFN-[Gamma], and interleukin (IL)-12p35 in PBMCs, as well as IFN-[alpha] and IFN-[gamma] in monocyte-derived DCs. Enhanced expression of IFNs in immune cells by CpG-DNA is not only crucial for host viral clearance, but also important in mediating host immune responses due to IFNs' anti-inflammatory effects. Compared to the relatively weaker activation of equine innate immunity by inactivated WNV, the tested CpG-DNA species showed potential as vaccine adjuvants for enhancement of CTLs and Th1 CMI against intracellular pathogens, characterized by significant induction of type I IFNs and Th1-specific cytokines such as IL-12p35 and IFN-γ. These data provide a basis for further investigation of these CpG-DNA species as potentially effective vaccine adjuvants in horses.

West Nile virus (WNV) is a mosquito-borne virus that infects many bird species. Examination of American crows and house sparrows from the Winnipeg region confirmed that WNV levels were at least 1000 times higher in crows than sparrows. No species differences were observed in the level of transcripts encoding a putative WNV receptor, β3 integrin. Differences in mosquito vector competence can be due to differences in the ability of WNV to enter mosquito cells. Using RNAi techniques, the role of two clathrin coat adaptor proteins in facilitating WNV infections in mosquito cells was examined, and the findings suggest that these proteins may act as resistance factors in Aedes aegypti, and as susceptibility factors in Culex quinquefasciatus. These findings will contribute to our understanding of the molecular basis of vector competence in different mosquitoes, and may help us determine whether other species could serve as potential vectors of this health-threatening virus. / October 2015