Section 1: Construction and screening of a genomic library for the mosquito Aedes aegypti (Diptera, culicidae). A genomic library has been constructed for this important vector of arboviral disease. Total genomic DNA and various classes of RNA from Ae. aegypti were used to screen this library. The results obtained indicate that this species has a short period interspersion pattern of repeated sequences. Transcription of these repeats could not be detected using total cytoplasmic RNA, hnRNA or mRNA as hybridisation probes. Section 2: Sequence organisation of ribosomal DNA in Aedes aegypti. The Aedes aegypti genomic library was used to isolate clones containing the intact ribosomal DNA (rDNA) repeat of this species. This has been restriction mapped and the transcribed regions have been identified. The rDNA repeat is 9.0 Kb in length and is present as approximately 500 head-to-tail tandemly repeated copies. A low level of intraspecies polymorphism of Ae. aegypti rDNA is evident. Two restriction polymorphisms have been identified within the rDNA repeat. Section 3: Analysis of ribosomal DNA variation within Ae. aegypti'and between closely related species. Four variant rDNA clones have been isolated. One of these' may contain the end of a tandem array of ribosomal genes. Another variant contains a duplication of rDNA within the internal transcribed spacer region of the ribosomal repeat. Sequence analysis of this clone has identified regions at the 3' end of the 18S rRNA gene of Ae. aegypti which show very strong homology with the corresponding regions in other species. Some repeated sequences have been identified downstream of the 18S rRNA gene in this clone. Preliminary analysis of the two other rDNA variants indicates that one contains a duplication or insertion of DNA in the 28S rRNA coding region and one contains non-transcribed spacer homologous sequences which are not associated with rRNA coding regions. Section 4: DNA probes for species identification of mosquitoes in the Anopheles gambiae complex. DNA sequences have been isolated which distinguish four of the morphologically identical members of the An. gambiae species complex. Two sequence classes were obtained. Class 1 homologues are highly reiterated in the genomes of An. arabiensis and An. merus, present in low copy number in An. melas and were not detected in An. gambiae s. s. Class 1 sequences are male specific in An. arabiensis. Class 2 homologues are highly reiterated in the genomes of An. merus and An. melas and present in low to middle copy number in An. gambiae s. s, and An. arabiensis. Sex specificity of Class 2 homologues does not occur in the species tested (An. gambiae s. s. and An. arabiensis). Hybridisation of these species specific DNA sequences to mosquitoes squashed directly onto nitrocellulose provides a simplified method of species identification.

Flaviviruses are a single-stranded, positive sense, RNA virus that affect around 400 million people annually. Flaviviruses are transmitted by arthropod vectors, the most common vector being the mosquito. Currently, many mosquito control strategies are in use, these control strategies are diverse in both efficiency and cost. However, developing new vector control strategies is becoming increasingly important, due to climate changing affecting vector population distribution and the current limitations conventional control strategies face. Although many different control strategies exist, there is limited research pertaining to reducing viral infection in the vector. Reducing the transmission capabilities of vectors could help relieve the disease burden felt around the world. Aripo virus (ARPV), an insect-specific flavivirus, has ushered in opportunities to discover a novel approach to arbovirus control. The exclusionary effects of ARPV were explored as a means to eventually understanding superinfection exclusion (SIE) and utilizing it as a calculated defense against mosquito-borne disease. Aripo virus was evaluated for its SIE potential in vitro and experiments were performed to explore the possible mechanisms underlying SIE. Aripo virus showed significant exclusion against the flaviviruses tested, as well as an alphavirus. Additionally, West Nile virus was unable to adapt and overcome SIE barriers over 9 serial passages. Lastly, ARPV was superinfected with chimeric viruses to asses replication kinetics, and possible exclusionary bias was seen with non-structural genes. These data show ARPV is capable of reducing viral titer, as well as possible leads into understanding the underlying mechanism of SIE, a critical step in utilizing SIE as a strategy to combat vector-borne disease. / Master of Science in Life Sciences / Mosquitoes all around the globe spread disease causing pathogens. Infection with viral pathogens such as West Nile virus, Zika virus, and Japanese encephalitis virus can cause lifelong health effects or even death. Risk for increased spread of mosquito-vectored disease is on the rise. The continued effects of climate change, increasing temperatures away from the equator, and increased encroachment into natural areas for urban development, is opening the door for new infectious diseases spread by mosquitoes. There are many ways to curb the effects that these viral diseases can have on humans, including vaccines or mosquito repellents, and even eliminating mosquito populations is helping to reduce the effects of these important diseases. Sadly, no one method can solve the problem. The methods of dealing with mosquito transmitted disease are likely to be most effective when done in combination. The more countermeasures that attempt to stop the spread of disease, the greater dent in reducing the spread of disease. In this dissertation, we dive into the realm of stopping disease transmission before humans become a factor. If we can limit mosquito infections, then the disease has no way of reaching humans. An approach to controlling mosquito infections could lie within viruses themselves. Aripo virus, an insect-specific virus, is interesting in that it does not affect vertebrates, only mosquitoes. When Aripo virus infects mosquito cells at the same time as deadly viruses, the deadly viruses do not accumulate in the mosquito cells nearly as much. Within mosquito cells, Aripo virus was able to reduce the levels of West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Zika virus, and Mayaro virus. If the underlying concept of the method of Aripo virus induced exclusion of deadly viruses could be further understood, it could be used to reduce the spread of mosquito-related viral diseases.

Ross River virus (RRV) is an Alphavirus distributed throughout Australia. It is transmitted by mosquitoes and is known to cause moderate to severe disease symptoms in humans. Along with other alphaviruses such as Sindbis virus and Chikungunya virus, RRV is known to cause arthritic symptoms, characterised by muscle and joint inflammation. Several investigations have established the role of macrophage cells and pro-inflammatory host factors in the development of RRV-induced disease. In this study, we attempted to determine differences between RRV passaged in mammalian and mosquito cells. There is strong evidence that arthropod-borne viruses are able to display enhanced infectivity when passaged into arthropod cell line. We showed that mosquito cell-derived RRV (mos-RRV) was able to replicate to higher titres than mammalian cell-derived RRV. We also showed that mos-RRV failed to induce Type I IFN-associated antiviral responses. The second aim of this study was to investigate the role of TNF-ᬠa pro-inflammatory cytokine implicated in arthritic diseases, in the development of RRV disease. We treated RRV-infected C57BL/6J mice with a commercially available TNF-ᠩnhibitor drug and monitored disease signs. We found that the TNF-ᠩnhibitor does not ameliorate RRV disease (RRVD) symptoms, and that it does not prevent muscle and joint inflammation. We analysed histological sections of muscle and joint tissue of Enbrel-treated and untreated, RRV-infected cells. We also determined and compared host cytokine expression profiles. Finally, we sought to determine the requirement for natural killer (NK) cells in RRV disease. NK cells have been detected in the synovium of RRV-infected patients since early studies, but their role in disease pathogenesis remains unclear. Using a NK-dysfunctional mouse (C57BL/6J-Lystbg), we showed that mice lacking a functional NK system are more susceptible to RRV disease than wildtype, C57BL/6J mice. We monitored disease symptoms following RRV infection and assessed muscle and joint inflammation in Lystbg and C57BL/6J mice. This thesis examines mechanisms of viral infection and immune evasion employed by RRV, as well as into the role of host cells and cytokines in RRVD pathogenesis disease mechanisms. We showed that a functional NK cell system is required for the regulation of RRV-induced muscle and joint inflammation. Our characterisation of the use of a commercial TNF-ᠩnhibitor in RRV-induced disease in mice may provide information on the role of TNF-ᠩn viral arthritis, and may help towards developing safe and effective treatment.

Aedes mosquitoes are responsible for the transmission of several arboviruses throughout tropical and subtropical regions of the world. In the Americas, Aedes aegypti is the most competent vector of dengue, chikungunya, Zika, and yellow fever. With up to 400 million cases globally each year, dengue fever is the fastest growing vector-borne disease in the world, and it has become an increasingly significant risk to global health. In the Western Hemisphere, dengue, and more recently chikungunya and Zika, exists throughout much of Latin America and the Caribbean. Both diseases are spread via Aedes mosquitoes and both constitute major health risks in Costa Rica. A dengue outbreak in Costa Rica in 2013 was the largest epidemic in the country since the return of the disease in 1993. Moreover, between 2014 and 2015 there was a 54.9% increase in dengue cases in Costa Rica, further demonstrating the relevancy of research on mosquito-borne diseases. Given this context, this study employs qualitative methods to critically investigate measures to combat Aedes mosquito-borne diseases in Costa Rica. Data were collected using household interviews (n = 80); semi-structured key informant interviews with public health officials, researchers, nonprofit organizations, and community leaders (n = 22); and a focus group discussion in each of two study areas. The results provide broad and place-specific information about mosquito control efforts and other actions deployed in Costa Rica to mitigate mosquito-borne diseases. The data reveal differences in perceptions and disease incidence among household interviewees as well as where households source information about mosquitoes and mosquito-borne diseases. Varying degrees of knowledge on mosquitoes and mosquito-borne diseases were uncovered across the study sites, as were differing attitudes among the general population regarding mosquito control techniques (e.g., fumigation, education campaigns, and household visits by public health officials). Additionally, data indicated a gap in knowledge regarding the ability of the public to report mosquito-related problems to health authorities. Households also exhibited discrepancies in knowledge pertaining to chikungunya transmission and details about the vector. / Master of Science

The state of Florida is the third most populous state in the United States of America, with six (6) of its metropolitan areas dubbed as the fastest growing in the entire country. A mosquito bite may mean the transmission of a virus or disease which might be fatal. Hence, there is a need for the state to control mosquitoes through the various Departments of Mosquito Control in each of its sixty-seven (67) counties. Six locally acquired mosquito-borne viruses which affect humans and animals in the state of Florida were considered. This thesis used statistical methods to examine data for rainfall, population estimate, as well as, the data on six (6) arboviruses, over the course of thirteen (13) years, namely 2002 to 2014. The first hypothesis that was tested, was that greater precipitation increased the likelihood of a greater number of arbovirus cases. It was important to also examine the relationship that this growing human population had with mosquito-borne diseases, and so the second hypothesis that was tested, was that, an increase in the human population would increase the likelihood of a greater number of arbovirus cases. Subsequently, an analysis was done for eleven (11) of Florida*s 67 counties with the greatest cumulative occurrence of human and animal arbovirus cases combined. Of the eleven counties, seven exhibited a weak associated between the size of the human population and the spread of animal and human arbovirus cases; three exhibited a somewhat moderate association; and one – Osceola County – had a strong negative association. This indicated that, as the size of the human population increased in Osceola County, the combined number of human and animal arbovirus cases decreased, which refuted the second hypothesis of this thesis. A linear regression model for the data for Osceola County was derived and that model was used to simulate what will occur in future years with the use of population projection data. In each simulated year, the number of combined human and arbovirus cases was negative. This prediction meant that, as the projected population increased from year to year, then the number of cases should be zero in each year. The reliability of these predictions are questionable, since Osceola County does not exist in a vacuum and it cannot be isolated from the surrounding counties which may be experiencing an outbreak of arboviruses.

Mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) are major threats to global public health resulting in millions of infections and hundreds of thousands of deaths annually. The presence of these viruses and their increasing emergence/spread continues to escalate. Notably, Usutu virus (USUV; Genus: Flavivirus; Family: Flaviviridae) is one such pathogen currently causing mass die-offs of avian hosts throughout Europe. USUV is categorized in the Japanese Encephalitis virus (JEV) antigenic complex and thus shares many antigenic and pathologic characteristics with fellow members, such as JEV and WNV. Respective to human infections, USUV cases are generally asymptomatic; nonetheless, acute cases have been reported. These acute cases typically cause mild symptoms, such as fevers and rashes; however, more severe cases can result in neurologic diseases, such as encephalitis and/or meningoencephalitis. In addition to these pathologic similarities, USUV shares several ecological and geographical traits with WNV, a pathogen responsible for several outbreaks during its spread from Africa, to Europe, and eventually the United States. Currently, WNV is considered endemic in areas across the United States due to its transmission via Culex spp.; mosquitoes that are ubiquitous in the United States. These parallels suggest the possible emergence of USUV into the United States and therefore, it is imperative to broaden our knowledge of USUV and assess its potential to become a major global health concern. The overall goal of this thesis was to characterize USUV and evaluate its emergence potential in the United States by: (1) developing infectious clones of recent European and African USUV isolates as tools for characterization and analysis of USUV and (2) assessing the transmission potential of several species of North American mosquitoes. In Aim 1, we show that the aforementioned infectious clones infect and replicate similarly to their parental strains in vitro in both vertebrate and invertebrate models, as well as in transiently immunocompromised CD-1 and IFNAR-/- murine models, and thus serve as useful tools for future molecular studies focusing on USUV. Furthermore, in Aim 2, we describe the ability of field-caught (Southwest Virginia, USA) Culex spp. and Aedes spp. mosquitoes to become infected with a recent European isolate of USUV; although, we report an overall limited potential for these species to transmit this virus. Altogether, these studies form a foundation for understanding the potential emergence of USUV in the United States as well as provide necessary tools needed to aid future research on USUV emergence, transmission, and pathogenesis. / Master of Science / Usutu virus (USUV) is an emerging mosquito-borne virus that was first isolated from a mosquito in 1959 in South Africa, and since then, has become a major problem throughout Africa and Europe causing acute to severe infection in dozens of patients. Additionally, this virus is causing massive die-offs in Eurasian blackbird populations. This is particularly problematic because birds play a critical role in ecosystems as they act as forms of pest control, pollinators, and seed dispersers. Depletion of these species could lead to an imbalance and, eventually, collapse of our natural ecosystem. Additionally, there is a growing concern of USUV making its way into the United States, following a similar track of emergence to WNV's introduction in New York in 1999 and its subsequent spread throughout the states. WNV's introduction to the United States was detrimental to native bird populations and humans, and has caused tens of thousands of infections and thousands of deaths since this introduction. Research has shown USUV causes similar disease symptoms to WNV. The self-limiting illness from these viruses typically includes fever and rashes but some infections can result in more severe cases causing inflammation of the brain and surrounding areas. Like many other prominent mosquito-borne viruses, there is no specific treatment or vaccine for WNV or USUV. Because USUV is so closely related to WNV, and their similar characteristics may point towards similar emergence in the United States, it is essential to garner more information on USUV. The overall goal of this thesis was to establish a reliable tool(s) for further characterization of USUV and demonstrate the potential for USUV emergence in the United States. We first developed molecular tools, known as viral clones, that are valuable to the scientific community which allows the manipulation of USUV genetic material to perform further downstream studies. Our objective for this initial study was to create a molecular tool that would behave similarly to their natural, or "parental", virus. The results from this study suggest we have successfully produced these tools. Furthermore, we sought to determine the potential for field-caught mosquitoes from Southwest Virginia, USA to transmit a recently isolated strain of USUV. These data suggest that while these mosquitoes do have the ability to become infected with USUV, they have a limited potential to transmit this virus to animal hosts. Altogether, these studies have allowed us to expand our knowledge on USUV's potential emergence in the United States and develop powerful tools to continue this essential research.

Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease

Virginia's temperate climate is suitable for several mosquito species capable of transmitting pathogens to humans. In southwest Virginia, La Crosse encephalitis and West Nile fever are most prominent. The objective of this research, which uses the Health Belief Model (HBM) as a theoretical framework, is to assess knowledge of mosquito-borne disease in southwest Virginia, as well as perceptions and practices of mosquito prevention. Given that several cases of La Crosse encephalitis have been reported in Wise and Tazewell counties, they were selected as study sites to conduct surveys. Five demographic and socioeconomic variables (gender, age, income, education level and length of time one has lived in the county) were used as predictor variables in logistic regression analyses. Gender, age, and length of residence time in the county were found to be statistically significant predictors of specific health-related behaviors. Within the framework of the HBM, barriers to removing standing water around the home and wearing insect repellent were highlighted. Knowledge of mosquito-borne diseases within the area was generally low, with only one individual correctly identifying La Crosse encephalitis as a threat in the region. Higher numbers (6%) were aware of West Nile virus, while 4% reported malaria in the region, demonstrating a disconnect between actual and perceived risk. These results can enhance existing public health programs by increasing knowledge, addressing public uncertainty about insect repellent safety, and addressing ways to make recommended practices more effective with the knowledge of how different aspects are perceived by varying groups within the community. / Master of Science

No description available.

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