West Nile virus has become a major risk to humans since its first appearance in New York City in 1999. Physicians and state health officials are interested in new and more efficient methods for monitoring disease spread and predicting future outbreaks. This study modeled habitat suitability for mosquitoes that carry West Nile virus. Habitat characteristics were used to derive risk maps for the entire state of Mississippi. Statistical significance tests yielded objective evidence for choosing among many habitat variables. Variables that were significantly correlated with diagnosed human cases for 2002 were combined in weighted linear algebraic models using a geographic information system (GIS). Road density, slope, and summer precipitation minus evaporation (P-E) were the most significant variables. GIS-based model results were compared with results from logistic regression models. The algebraic model was preferred when validated by 2003 human cases. If adopted, GIS-based risk models can help guide mosquito control efforts.

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<p>The incidence and severity of infectious diseases increases in elderly people (>60 years of age). It is believe that the age-associated changes in the immune system, termed immunosenescence, lead to diminished effectiveness of the immune system leaving the aged susceptible to infectious pathogens and associated diseases. The limited efficacy of the currently available vaccines in elderly populations contribute immensely to the frequency of infectious diseases in the globally growing aging population. As such, the demographic shift warrants the development of effective prophylactic vaccines for the elderly.</p> <p>West Nile virus (WNV) became endemic in North America in 1999, and although it infects people of all ages, the incidence of severe neuroinvasive disease is more prevalent in the elderly. I hypothesized that the susceptibility of the elderly towards severe WNV disease is a consequence of aberrant immune function, and specifically lack of functional virus-specific CD8+ T cells. Contrary to my hypothesis, I found that the magnitude, breadth and functionality of WNV-specific CD8+ T cells were not different between the age cohorts. These results argue that advanced age does not limit the development of functional CD8+ T cell responses following primary infections with an acute virus. Furthermore, the aged members of our cohort maintained functional CD8+ T cells to cytomegalovirus (CMV) and Epstein-Barr virus (EBV), common persistent viruses.</p> <p>Collectively, my results demonstrate that development of vaccines designed to elicit CD8+ T cell immunity may be warranted for elderly individuals and perhaps live vectors should be considered for this population.</p> / Doctor of Philosophy (Medical Science)

WN virus has spread for over 60 years creating endemic and epidemic areas throughout Africa, Asia, and Europe, affecting human, bird, and equine populations. Its 1999 appearance in New York shows the ability of the virus to cross barriers and travel great distances, emerging into new territories previously free of infection. Spreading much faster than expected, WN virus has infected thousands of birds, equine, and humans throughout the conterminous United States (US). Case and serological studies performed in the Eastern hemisphere prior to 1999 offer detailed descriptions of endemic and epidemic locations in regards to geography, land cover, land use, population, climate, and weather patterns. Based on the severity of WN activity within each study area, the patterns associated with these environmental factors allow for the identification of values associated with different levels of risk. We can then model the landscape of the disease within the US and identify areas of high risk for infection. State and county public health officials can use this model as a decision-making tool to allocate funding for disease prevention and control. Dynamic factors associated with increased transmission, such as above average temperature and precipitation, can be closely monitored and measures of prevention can be implemented when necessary. In turn, detailed information from higher resolution analyses can be documented to an online GIS (Geographic Information System) that would contribute to a global collaboration on outbreaks and prevention of disease. / Master of Science

Field studies were conducted in southwestern Virginia to determine the bionomics and ovipositional preferences of Culex restuans Theobald and Culex pipiens Linnaeus using ovitraps and gravid traps. Both species have been implicated as enzootic and epizootic vectors of West Nile virus (WNV) and these studies provide information on the relative abundance of gravid mosquitoes. Ovitraps were used in the summers of 2002 and 2003 to measure the oviposition activity of Culex mosquitoes, mainly Cx. restuans and Cx. pipiens. In 2002, 1,345 egg rafts were collected from 5 traps set at different locations in the New River Valley (NRV). Cx. restuans constituted 93.2% of the catch; the remainder was Cx. pipiens (6.7%) and Cx. salinarius (<1%). In 2003, 4 ovitraps were placed at each of 6 locations in the NRV. Of 9,794 egg rafts collected, Cx. restuans comprised 92.8%, Cx. pipiens 6.5%, and Cx. salinarius <1%. Oviposition patterns were similar in both years. Cx. restuans oviposition was detected about mid-May, and raft numbers were highest in late June and the middle of July and then showed a steady decline throughout the remainder of each season. Cx. pipiens oviposition activity began later in the season and gradually increased, reaching its peak in August. Although the number of egg rafts of Cx. restuans decreased in August and September while the number of Cx. pipiens egg rafts increased, a crossover in the relative abundance of the two species never occurred. In 2003, the attractiveness of four infusions (cow manure, straw, grass, and rabbit chow) were compared in oviposition traps. For Cx. restuans, the manure infusions collected more egg rafts than the other three infusions for the first four weeks, with two of the weeks showing significance. During week 1, the manure infusions collected significantly more egg rafts than straw (P<0.01), grass (P<0.01), and rabbit (P<0.001). During week 2, manure collected more than grass (P<0.05) and rabbit (P<0.01). The straw and grass infusions yielded the most egg rafts after week 2, and only three weeks showed any significance. When traps began to collect Cx. pipiens, the majority were collected in the straw and grass infusions. During week 6, the hay infusions had significantly more egg rafts when compared to manure (P<0.001) and rabbit infusions (P<0.001) and the grass infusions had significantly more when compared to manure (P<0.05) and rabbit (P<0.01). Week 9 also showed significance when the hay infusions collected significantly more egg rafts than manure and rabbit (P<0.01 and P<0.01, respectively). The attractiveness of the cow manure and straw infusions were also compared in gravid traps. Because it is difficult to accurately discriminate between Cx. pipiens and Cx. restuans that have been collected as adults in gravid traps, these collections were combined into Culex. More Culex mosquitoes were collected in the manure infusions for the first two weeks (P<0.05). No significant differences were found between the numbers of mosquitoes captured in the traps baited with the different infusions after the second week. The shift observed in oviposition preference for both types of traps may have been due to cooler temperatures in the early part of the season. The straw infusions were aged outdoors for 3 days prior to use, and a sufficient incubation temperature to generate the bacteria producing the attractant chemicals may not have been attained. In addition, the manure lagoon had been drawn down and the consistency of the remaining manure became much drier by this time. No chemicals were ever added to the lagoon, but the draw down may have affected the attractiveness of the manure. / Master of Science

West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging mosquito-borne flaviviruses. These viruses are phylogenetically closely related and belong to the Japanese encephalitis serocomplex group. Similar to other flaviviruses, these viruses are enveloped, with genomes comprising positive-sense, single-stranded RNA approximately 11 kb in length. Upon translation, a single polyprotein is produced, consisting of three structural and seven non-structural proteins. These proteins function in virus binding to the cell membrane, entry into cells, replication, immune evasion, and the production of new virus progeny. Typically, these viruses are maintained in a sylvatic cycle involving avian hosts, such as passerine birds, and mosquitoes. However, they can accidentally spill over to humans through mosquito bites or wildlife exposure. Although humans generally remain asymptomatic and do not support sufficient viral replication for transmission, they can develop febrile disease and, in some cases, severe neuroinvasive diseases, especially among the elderly or immunocompromised individuals. Due to their co-circulation in the same geographical areas and sharing similar hosts and vectors, individuals in Italy and Germany have been detected as seropositive for WNV and USUV, while seropositivity for WNV and SLEV has been observed in the Americas. Viruses in the Japanese encephalitis virus serocomplex group exhibit significant antigenic similarity. The envelope protein alone contains 12 distinct epitopes and at least three highly conserved epitopes among the JEV serocomplex. Consequently, infection with one member of the JEV serocomplex group, such as WNV, induces WNV-specific antibodies and heterotypic antibodies that can cross-neutralize other members of the JEV serocomplex group, such as USUV and SLEV. Therefore, cross-reactive epitopes can protect against heterologous virus challenges to varying extents, depending on the accessibility of the antibodies to the epitopes. Prior infection with WNV or its envelope domain III (EDIII) or non-structural protein 1 (NS1) protected mice from lethal JEV challenges. Vaccination against WNV protected mice from lethal USUV challenges, and vice versa. Immunity to JEV or SLEV protected hamsters from lethal WNV challenges. Although human sera immune to WNV cross-neutralized USUV and SLEV in vitro during serodiagnosis, the actual mechanism of cross-protection among WNV, USUV, and SLEV remains poorly characterized. Therefore, this study aims to understand the mechanism of cross-protection. Specifically, this research investigated whether human plasma immune to WNV could cross-protect mice from encephalitis caused by SLEV or USUV. Initially, WNV-specific human convalescent plasma and mouse WNV convalescent serum (as a positive control) neutralized WNV and cross-neutralized USUV and SLEV in vitro in a neutralization test. Subsequently, immunocompetent mice were intraperitoneally injected with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum the day before being challenged with WNV, SLEV, or USUV via footpad injection. We found that human WNV convalescent plasma provided mice with strong protection against neuroinvasive encephalitis caused by WNV. Additionally, human WNV convalescent plasma reduced the viremia titers of SLEV and USUV for several days during acute infection. Human WNV convalescent serum also demonstrated a trend towards protecting mice from SLEV-induced encephalitis, as evidenced by lower SLEV titers in the brain and histopathology scores. These findings will aid in decoding the mechanisms of cross-protection among the JEV serovars, developing therapeutic strategies against WNV, SLEV, and USUV, and anticipating potential disease outcomes, especially in regions where multiple viruses of the JEV serocomplex are endemic. / Master of Science / West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging flaviviruses transmitted by mosquito bites, primarily among perching birds. However, mosquitoes can also transmit these viruses to animals and humans, especially in regions where these viruses are prevalent. The immune system, which defends against pathogens and other diseases, usually combats these viruses effectively, preventing most people from developing symptoms. The immune system has two main branches: the innate immune system, which confers immediate defense, and the adaptive immune system that includes antibodies and certain long-lasting memory cells, that can fight off infections years after the initial exposure to the same or similar disease-causing agents. Occasionally, the immune system fails to fight these viruses, particularly in the elderly or those with chronic diseases, leading to fever or severe brain inflammation called encephalitis. Currently, WNV and SLEV are circulating in the Americas, while WNV and USUV are present in European countries. Due to similar transmission methods, infection patterns, and geographical overlap, individuals might be sequentially infected with WNV and USUV in Europe, and WNV and SLEV in the Americas in their lifetime. These viruses also share common antigens, which can induce similar immune responses. Therefore, the immune response to one virus might protect against another with similar antigens. It has been reported that the immune response induced by WNV can protect against encephalitis caused by USUV or SLEV. However, it remains unclear whether this cross-protection is mediated by antibodies or a certain type of immune cells called T cells. This study investigates whether antibodies induced by WNV infection can protect against SLEV or USUV in a mouse model. Plasma, the part of blood containing antibodies, is referred to as convalescent plasma when collected after an individual has recovered from an infection or disease. Human WNV convalescent plasma was tested against SLEV and USUV using a plaque reduction neutralization test to determine the antibodies’ ability to prevent viral infection in a laboratory setting. Human WNV convalescent plasma effectively prevented SLEV and USUV from infecting cells. We then developed a mouse model that could be infected with SLEV or USUV and mimic human disease. Groups of mice were systematically transferred with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum one day before the infection with WNV, SLEV, or USUV. Disease conditions, such as weight loss, reduced movement, hunchback, fur loss, and occasional paralysis, were monitored until the infected mice were humanely euthanized. After euthanasia, the brains of the mice were collected to measure viral load and examine signs of encephalitis. We observed asymptomatic disease outcomes reflecting natural human infection. Both human and mouse WNV convalescent samples reduced viral load in the blood for a period in both SLEV and USUV-challenged groups. Mice treated with human WNV convalescent plasma showed a trend of lower SLEV in their brains. Additionally, mice treated with mouse WNV convalescent serum had lower SLEV titers in their brains compared to those treated with mouse normal serum. Overall, these findings suggest that human WNV convalescent plasma provides some crossprotection against SLEV- and USUV-induced diseases. Understanding the mechanism of crossprotection is crucial for developing therapeutics against these viruses and predicting disease outcomes in areas where multiple viruses of the Japanese encephalitis virus serocomplex are prevalent.