Browsing by Author "Foy, Brian, committee member"

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Open Access
A new synopsis of the mosquitoes (Diptera: Culicidae) of Colorado
(Colorado State University. Libraries, 2016) Rose, Dominic Anthony, author; Kondratieff, Boris, advisor; Weissmann, Michael, advisor; Savage, Harry, committee member; Foy, Brian, committee member
Harmston and Lawson (1967) provided the most recent comprehensive review of the mosquitoes of Colorado, reporting 42 species in six genera. Over the last 48 years, a number of important studies that included mosquitoes of Colorado have been completed enhancing the knowledge of this medically important group of flies. To date, the number of Colorado mosquitoes has increased to 54 species in seven genera. Additionally, mosquito vectored pathogens of humans and animals have shifted in Colorado from primarily Western equine encephalitis virus and St. Louis encephalitis virus to primarily West Nile virus. The objective of the current project is to provide an up-to-date synopsis of the species and genera known to occur in Colorado including distributions at a county scale. The study also provides up-to-date illustrated keys to the adult females, fourth instar larvae, and a summary of the important taxonomic characteristics that allow separation of species for both adult females and fourth instar larvae. The species summary includes relevant biological notes and comments on each species such as its phenology, overwintering stage, larval habitat, host preference, medical importance and unique behaviors.
Open Access
Cationic liposome-DNA complex-based immunotherapeutic and immunization strategies for control of la Crosse virus and Leishmania major infections
(Colorado State University. Libraries, 2011) Arthun, Erik Norden, author; Dow, Steven, advisor; Foy, Brian, committee member; Lappin, Michael, committee member; Callan, Robert, committee member
To view the abstract, please see the full text of the document.
Open Access
Characterization of changes in metabolic pathways during dengue virus serotype 2 infection of the Aedes aegypti mosquito vector to identify control points for interrupting virus transmission
(Colorado State University. Libraries, 2018) Chotiwan, Nunya, author; Perera, Rushika, advisor; Blair, Carol, committee member; Foy, Brian, committee member; Huang, Claire, committee member; Di Pietro, Santiago, committee member
Dengue viruses (DENV) are mosquito-borne viruses that cause a wide range of acute symptoms from mild fever to lethal dengue shock syndrome in humans. DENV are transmitted primarily by Aedes aegypti (Ae. aegypti). These mosquitoes are widely distributed throughout tropical and subtropical areas around the world. Increasing globalization, urbanization and global warming are factors that enhance the spread of these vectors placing over 2.5 billion people at risk of contracting these viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate into several tissues in the mosquito vector. During DENV infection of its human and mosquito hosts, a visible rearrangement of lipid membrane architecture and alterations of the metabolic repertoire is induced. These events occur to facilitate efficient viral replication and virus assembly within the cell and to circumvent antiviral responses from the host. Interference with these virus-induced processes can be detrimental to virus replication and can prevent viral transmission. In this dissertation, we present the first insight into the metabolic environment induced during DENV serotype 2 (DENV2) replication in Ae. aegypti. Using untargeted high-resolution liquid chromatography-mass spectrometry, we explored the temporal metabolic perturbations that occur following dengue virus infection of the midgut, the primary site of the virus infection in the mosquito vector. Temporal changes of metabolites across early-, mid- and late-infection time points were identified. A marked increase in the /content of glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels and the accumulation of medium-chain acyl-carnitines suggested a diversion of resources during infection from energy storage to synthetic pathways and energy production. From the observations above, two active pathways, sphingolipid and de novo fatty acid synthesis pathways, were further validated to identify metabolic control hubs. Using inhibitor screening of the sphingolipid pathway, we determined that sphingolipid Δ-4 desaturase (DEGS), the enzyme that converts dihydroceramide to ceramide was important for DENV2 infection in cultured Ae. aegypti cells (Aag2). Long, double-stranded RNA-mediated knockdown of DEGS expression led to the imbalance of ceramide to dihydroceramide ratios and affected DENV2 infection in cell culture. However, the inhibitory effect to DENV2 replication was not observed during DEGS-knockdown in mosquito vectors. De novo fatty acid biosynthesis is the pathway that synthesizes the first lipid molecules, fatty acids, required in synthesizing complex lipid molecules, such as glycerophospholipids, glycerolipids and sphingolipids. As a result, this pathway serves as a bottle neck for the control of lipid metabolism. In this study, we annotated and characterized the expression of seven Ae. aegypti fatty acid synthase (AaFAS) genes in the different stages of mosquito development and upon exposure to different diets. We found that AaFAS1 shares the highest amino acid similarity to human fatty acid synthase (FAS) and is the dominant AaFAS that expressed in female mosquitoes. Knockdown expression of AaFAS1 expression showed a reduction in DENV2 replication in the Aag2 cells and in the midgut of Ae. aegypti mosquitoes during early infection. However, the correlation between viral infection and levels of AaFAS1 expression was difficult to elucidate. The work in this dissertation has highlighted metabolic pathways that are induced by DENV2 infection and the metabolic control points within these pathways that are critical for DENV2 infection in Ae. aegypti. Successful perturbation of metabolic homeostasis can potentially limit virus replication in the vector, presenting a novel avenue to block the transmission of DENV2 from the mosquitoes to humans.
Open Access
Evolution of mutations associated with pyrethroid resistance and the reversal of resistance in Aedes aegypti
(Colorado State University. Libraries, 2019) Vera Maloof, Farah Zamira, author; Black, William, advisor; McAllister, Janet, committee member; Foy, Brian, committee member; Bjostad, Louis, committee member; McGrew, Ashley, committee member
Worldwide vector control has been relying heavily on pyrethroid insecticides to reduce Aedes aegypti Linnaeus populations. Pyrethroids are relatively inexpensive, have low vertebrate toxicity, and have been efficient in reducing mosquito populations. Constant use of pyrethroid insecticides, however, has driven mosquito populations to develop resistance over time. In this dissertation, we have tracked the evolution of three mutations in the voltage gated sodium channel (vgsc) that are associated with pyrethroid resistance Aedes aegypti populations in Mexican. These are 410, 1,016 and 1,534, corresponding to the position of amino acid substitutions in the vgsc. A valine at locus 410 (V410) confers susceptibility, while leucine (L410) confers resistance. A valine at locus 1,016 (V1,016) confers susceptibility, while isoleucine (I1,016) confers resistance. A phenylalanine at locus 1,534 (F1,534) confers susceptibility, while cysteine (C1,534) confers resistance. We performed a linkage disequilibrium analysis of the three mutations in Mexican collections from 2000–2016. In the first study, a linkage disequilibrium analysis was performed on I1,016 and C1,534 in Ae. aegypti collected in Mexico from 2000–2012, to test, in natural populations, for statistical associations between segment six (S6) in domains II and III of the vgsc. We estimated the frequency of the four di-locus haplotypes in 1,016 and 1,534: V1,016/F1,534 (susceptible), V1,016/C1,534, I1,016/F1,534, and I1,016/C1,534 (resistant). The susceptible V1,016/F1,534 di-locus haplotype went from near fixation to extinction, and the resistant I1,016/C1,534 di-locus haplotype increased in all collections from a frequency near zero, to frequencies ranging from 0.5–0.9. The V1,016/C1,534 di-locus haplotype frequency increased in all collections until 2008. After this year, the frequencies in two collections began to decrease, likely due to the fact that the I1,016/C1,534 di-locus haplotype frequency increased in all collections. However, the I1,016/F1,534 di-locus haplotype was rarely detected; for instance, it reached a frequency of only 0.09 in one collection and subsequently declined. Pyrethroid resistance in the vgsc gene appears to require the sequential evolution of two mutations. The I1,016/F1,534 di-locus haplotype appears to have low fitness, suggesting that I1,016 was unlikely to have evolved independently. Instead the C1,534 mutation evolved first but conferred only a low level of resistance. I1,016 in S6 of domain II then arose from the V1,016/C1,534 haplotype and was rapidly selected because double mutations confer higher pyrethroid resistance. This pattern suggests that knowledge of the frequencies of mutations in both S6 in domains II and III are important to predict the potential of a population to evolve kdr. Susceptible populations with high V1,016/C1,534 frequencies are at high risk for kdr evolution, whereas susceptible populations without either mutation are less likely to evolve high levels of kdr, at least over a 10 year period. In the second chapter we describe a novel replacement V410L that was initially detected in a pyrethroid resistant insectary strain from Brazilian Ae. aegypti populations. We screened V410L in 25 Ae. aegypti historical collections from Mexico. The first heterozygote appeared in 2002, and frequencies have increased in the last 16 years, along with I1,016 and C1,534. L410 showed a strong association between 1,534 and 1,016 mutations. Individuals with the triple homozygote resistant genotype had higher survival after pyrethroid exposure, 96% of the alive individuals had the triple homozygote resistant genotype after permethrin and 76% after deltamethrin treatment. The purpose of insecticide resistance management strategies is to minimize the selection for resistance to any one type of insecticide, or to help regain susceptibility in insect populations in which resistance has already arisen. A key component of resistance management assumes that there will be a negative fitness associated with resistance alleles, so that when insecticides are removed, resistance alleles will decline in frequency. In the third chapter we tested for the loss of pyrethroid resistance from eight field populations of Ae. aegypti, (six field collections from or near the city of Merida, and two collections from Tapachula and Acapulco in southern Mexico) to assess variation in the rate of loss of pyrethroid resistance. Collections were maintained for up to eight generations after pyrethroids were discontinued. We recorded changes in the frequencies of two kdr mutations, I1,016 and C1,534, and the analysis of resistance ratios (RR) with permethrin (pyrethroid type 1) and deltamethrin (pyrethroid type 2). In generations F3, F6, and F8, we also evaluated fecundity to test for parallel changes in a fitness trait during the eight generations. This was analyzed because a negative association between resistance and fecundity had previously been described in two studies [1, 2]. We demonstrate that the frequency of the Ae. aegypti pyrethroid resistance alleles I1,016 and C1,534 decline when pyrethroid pressure is removed in the laboratory; however, the pattern of decline is strain dependent. In agreement with earlier studies, fecundity was negatively associated with the frequency of resistance alleles.
Open Access
Geospatial analyses of childhood malaria following repeated village-wide Ivermectin administrations: secondary analyses for the RIMDAMAL pilot study
(Colorado State University. Libraries, 2017) Barnett, Chelcie A., author; Magzamen, Sheryl, advisor; Foy, Brian, committee member; Hahn, Micah, committee member
Malaria has long been a major public health concern, with historic roots dating back thousands of years. This febrile disease is caused by a parasite that is transmitted among vertebrates by mosquitoes. Over the past century, global eradication programs have focused on minimizing populations of the insect vectors, and administering treatments to people infected, especially young children and pregnant women, as they are the most vulnerable to suffering severe complications. Overall, these programs have decreased the geographic distribution and global disease burden; however, malaria remains a major problem in regions where these efforts have been unsuccessful. In 2015, there were an estimated 214 million cases throughout the world, resulting in approximately 438,000 deaths; however, over 3 billion people are living at risk of becoming infected with malaria. Widespread use of the few available effective insecticides and anti-malarial drugs has conferred resistance in both parasitic and mosquito species, decreasing the effectiveness of current interventions. As anti-malarial resistance and insecticide resistance spread, the need for novel malaria interventions becomes more urgent. One novel approach to combatting malaria was pilot-tested by researchers in the Department of Microbiology, Immunology and Pathology at Colorado State University. The Repeated Ivermectin Mass Drug Administration to control Malaria, or the RIMDAMAL study, evaluated the safety and effectiveness of repeated village-wide administrations of an anti-parasitic drug to prevent malaria in children 5 years old. The RIMDAMAL study was a randomized trial carried out in Burkina Faso, a small tropical country in West Africa. Ivermectin (IVM) is a common anti-parasitic used around the world to prevent and treat parasitic diseases. Recent evidence has demonstrated that IVM is toxic to malaria-transmitting mosquitoes, and can inhibit the propagation of some life stages of malaria parasites. Initial analyses of the RIMDAMAL data found significantly fewer childhood malaria cases in intervention villages that received repeated IVM administrations, compared to control villages. This study is a geospatial analysis of the RIMDAMAL data to provide further insight as to how this intervention could be implemented. There were two study aims for this research: 1) identify significant clustering of high and low childhood malaria incidence within each study village; and 2) identify significant clustering of high and low childhood malaria incidence throughout the entire study region. In total, eight villages were enrolled in the study, four of which served as controls, while the other four received the intervention. Residents of each village live in concessions, or compounds of extended family. Geospatial coordinates were collected for each concession within a study village, along with data on the participants within each concession. Using this data, incidence density of malaria among children 5 years old or younger was calculated at the concession level. Concessions were mapped, and spatial clustering of incidence density values was evaluated using the Getis-Ord Gi* (G-I-star) spatial autocorrelation statistic. To evaluate within village clustering, each of the eight study villages were analyzed individually, and between village clustering was evaluated by analyzing the entire study region. Within each village, several "hot spots," or statistically significant clusters of high malaria incidence density values were recognized during analyses with max clustering, at the 95% confidence level. Statistically significant clusters of low incidence density were identified in one study village during the analysis with max clustering. The proportion of concessions identified as significant clusters varied by village, ranging from 12% to 91.3%. There seems to be no trend in clustering patterns seen within each village; some villages had randomly distributed hot or cold spots, while others appeared more clustered. The spatial clustering patterns in the whole study region are more telling. Max clustering occurs in a bimodal pattern with two peaks; at 2,100 meters and 10,000 meters. The clustering patterns that occur indicate regions of similar malaria incidence. The proximity and locations of these villages may imply the RIMDAMAL protocol has regional impacts. Additional research is needed to evaluate how to most effectively implement this intervention to protect against malaria.
Open Access
Illustrating the post-introduction ecology of Rift Valley fever virus in the United States of America
(Colorado State University. Libraries, 2021) Hartman, Daniel A., author; Kading, Rebekah C., advisor; Ebel, Gregory, committee member; Foy, Brian, committee member; Webb, Colleen T., committee member
Rapid urbanization, increasing international travel, and our changing climate are modifying the existing interspecies interactions at the interfaces between human, wildlife, and livestock interfaces, increasing the potential for outbreaks and transboundary disease introductions. It is more important than ever to maintain proactive research programs that integrate data across disciplines to maintain a working knowledge the potential transmission cycles of high-threat pathogens in novel environments. For vector-borne pathogens, entomological parameters, as well as interactions with the pathogen and host are highly informative, while representing avenues for control prior to an introduction. The work of this dissertation seeks to inform the potential transmission cycles of Rift Valley fever virus (RVFV) in the United States. Currently restricted to Africa and the Arabian Peninsula, RVFV infects domestic ruminants and humans with substantial degrees of morbidity and mortality. Throughout its current range, transmission involves a diversity of vectors, which are capable of transmitting the virus horizontally between vertebrates and vertically to mosquito progeny. The ecology of RVFV presents a great deal of complexity, with many unknown factors such as the roles of wildlife hosts, and relative contributions of vectors to transmission. To gain some insight into the potential ecology of RVFV in the United States, we first performed extensive sampling of mosquitoes at feedlots in northern Colorado to explore the potential for these operations to act as amplification foci after an introduction. We discovered that the most competent mosquito in Colorado that has been tested to date is highly abundant, and feeds readily on cattle, making these operations high risk for an epizootic. In this previous study we also identified blood-feeding on deer for some mosquitoes (Ae. vexans, Ae. melanimon, Ae. dorsalis) as well as domestic ruminants. We then set out to determine whether Ae. melanimon is capable of transmitting RVFV biologically, as there were no data to date for this species. We conducted infection experiments with these three Aedes species and others to determine the efficiencies with which they can transmit RVFV horizontally and vertically. We found substantial evidence for horizontal transmission and susceptibility of ovaries to infection, a prerequisite for vertical transmission, in all species but for Aedes increpitus. For these data we also developed a model to estimate the infection susceptibilities and barriers in mosquito organs in a functional manner. Finally, we sought to investigate the potential for transmission of RVFV in white-tailed deer by describing the community of mosquitoes in a riparian woodland habitat. We revealed some interesting patterns in the abundances of some mosquito species which stood in contrast to those observed at the feedlots. Several mosquito species exhibited the capacity to feed on white-tailed deer, including Ae. increpitus and Ae. vexans, both previously shown the ability to transmit RVFV by bite. By scoring the digestive stage of the blood meals in mosquitoes that were later identified to vertebrate source species, we uncovered an interesting pattern suggestive of interrupted feeding on eastern cottontail rabbits, in contrast to blood meals taken from white-tailed deer, from which mosquitoes fed to repletion. The implications of interrupted feeding for transmission by mosquitoes is unclear, but highlights the important factor of behavioral interactions between mosquito vectors and hosts which is often overlooked.
Open Access
Macrophage immunometabolism during flavivirus infection
(Colorado State University. Libraries, 2022) Donkoh, Jasmine, author; Rovnak, Joel, advisor; Quackenbush, Sandra, committee member; Foy, Brian, committee member; Perera, Rushika, committee member; Chen, Chaoping, committee member
Dengue virus (DENV) and Zika virus (ZIKV) are mosquito borne flaviviruses that are transmitted by the Aedes spp. mosquito and have caused outbreaks in Africa, Asia, the south Pacific, and the Americas. Infection with DENV can cause severe illness, such as dengue hemorrhagic fever and dengue shock syndrome, while infection with ZIKV can result in congenital abnormalities, such as microcephaly, and spontaneous abortions. Although disease outcome for these viruses is markedly different, both DENV and ZIKV both target monocytes and macrophage for pathogenesis. Macrophage are among the first cells to be infected by DENV and ZIKV and are disseminated throughout the body. While macrophage are an important cell in flavivirus pathogenesis, the mechanisms by which viruses modulate macrophage function are not fully understood. In this dissertation, I present data that attempts to explain the interaction between macrophage and flaviviruses, as well as investigate the mechanisms in which DENV and ZIKV control macrophage gene expression and metabolism. The most widely used macrophage cell line, THP-1 cells, are cultured as immature monocytes. To become naïve macrophage, these cells are treated with phorbol 12-myristate- 13 acetate (PMA). Once THP-1 monocytes are differentiated into naïve macrophage, they can be polarized into different macrophage subsets. Even though THP-1 macrophage are widely used, the protocols in which to differentiate and polarize cells are not consistent. In chapter 2, we optimize methods to differentiate and polarize THP-1 cells. We measure gene expression and cellular metabolism during differentiation and polarization to characterize macrophage phenotype. These data, coupled with published literature, show that this model is a reliable system to study macrophage biology and flavivirus-macrophage interactions. We use the methods developed in this aim throughout the dissertation. Macrophage metabolism and phenotype determine immune function. Inflammatory (M1) macrophage are inflammatory and mount a strong anti-viral response, while anti-inflammatory (M2) macrophage dampen anti-viral responses. Viruses can alter macrophage phenotype for efficient replication and immune evasion. In chapter 3 we elucidated the role of macrophage polarization on DENV replication, showing that M1 macrophage have suppressed DENV replication while M2 macrophage support replication. In addition, we characterized the impact of DENV infection on M1 and M2 gene expression and metabolism. DENV infection resulted in an upregulation of inflammatory and anti-inflammatory genes in both M1 and M2 macrophage. Infection resulted in similar metabolic profiles in M1 and M2 cells, suggesting that DENV infection reprograms cellular metabolism in a way that is favorable for replication, regardless of macrophage phenotype. The key difference between M1 and M2 cells was the upregulation of interferon genes, where M1 mounted a strong interferon response, M2 mounted a subdued response. The difference in the interferon response could explain the difference in DENV replication observed in the two phenotypes. These data add to the ongoing literature on immunometabolism and its impact on viral pathogenesis. Cyclin dependent kinase 8 (CDK8) and CDK19 are transcriptional cofactors that regulate expression of inflammatory and anti-inflammatory genes. In addition, inhibition of CDK8/19 during DENV infection leads to decreased replication, as well as metabolic shifts in Huh7 cells, a liver cell line. In chapter 4, we investigate the role of CDK8/19 on viral replication and inflammatory/ anti- inflammatory gene expression. We found that inhibition of CDK8/19 kinase activity increased DENV replication and anti-inflammatory gene interleukin 10 (IL-10) expression. In contrast, inhibition of kinase activity decreased expression of inflammatory genes C-X-C motif chemokine ligand 10 (CXCL10). Furthermore, I found distinct mechanisms for each kinase through analysis of DENV-infected CDK8 and CDK19 knockdown cells. Knockdown of CDK8 mimics chemical inhibition of CDK8/19, while knockdown of CDK19 did not change expression in CXCL10 or IL-10. These data indicate that CDK8 and CDK19 regulate the transcription of different genes during DENV infection in macrophage. These data contribute the basic understanding of CDK8/19 regulation during viral infection. Macrophage phenotype plays a large role in ZIKV pathogenesis, where macrophage found near the placenta are an anti-inflammatory phenotype and are susceptible to infection. In chapter 5, we investigated the role of cyclin dependent kinase 8 and phenotype in Zika virus pathogenesis. We found CDK8 gene expression increase throughout infection, while CDK8 kinase inhibition decreased viral replication. Furthermore, inhibiting CDK8/19 kinase activity led to a decrease in CXCL10 and an increase in IL-10, as seen in a DENV model of infection. We also found that M2 macrophage were more susceptible to infection than M0 or M1. These data suggest that CDK8/19 kinase activity could be a pan-flavivirus mechanism to regulate host gene expression during infection.
Open Access
Population genetics and vector competence of Aedes aegypti in West Africa
(Colorado State University. Libraries, 2014) Dickson, Laura B., author; Black, William C., IV, advisor; Miller, Barry, committee member; Foy, Brian, committee member; Blair, Carol, committee member; Huyvaert, Kate, committee member
The mosquito, Ae. aegypti is the primary vector for all four serotypes of dengue (DENV 1-4) and yellow fever viruses worldwide. Dengue remains an important public health problem with an estimated 390 million cases per year and yellow fever outbreaks are still continuously reported even though a safe and effective vaccine exists. Aedes aegypti (L) is found globally in tropical and sub-tropical climates and exists as two subspecies: Ae. aegypti aegypti (Aaa) and Ae. aegypti formosus (Aaf) which have previously been defined by the presence or absence of scales on the first abdominal tergite. This classification system was developed in East Africa and is contradictory in West Africa where this mosquito is genetically diverse and exhibits great variation in susceptibility to DENV and YFV, which is a quantitative genetic trait. Understanding the population genetics, the vector competence, and the way in which genetic diversity contribute to vector competence of Ae. aegypti can improve our general understanding of mosquito/virus interactions and lead to potential ways to control these mosquitoes in nature. In this dissertation, the distribution of FST values calculated from deep sequencing data between mosquitoes from two diverse locations in Senegal, one location in Mexico, and one location in Thailand across the entire genome were compared to identify the degree of genomic divergence as well as identify genes involved in speciation between the various populations. The distribution of FST values were also compared in different gene regions and mutation types to identify which parts of the genome provide the greatest resolution of subspecies population structure. Genetic crossing experiments and deep sequencing of the sex determining locus of Ae. aegypti from Senegal and Thailand was used to demonstrate discrete genetic differences between the subspecies. The vector competence for a local sylvatic isolate of DENV-2 and two genetically diverse YFV isolates was compared between various collections of Ae. aegypti throughout Senegal to demonstrate that vector competence in these mosquitoes is dependent on the viral isolate. The genetic diversity of an important immune sensing gene, Dcr2 of the exo-siRNA pathway, was determined from mosquitoes with various vector competence phenotypes to test how the genetic diversity of this gene in individuals and populations of mosquitoes contribute to vector competence. Overall, data in this dissertation suggest 1) allopatric speciation between Ae. aegypti from Senegal and Mexico or Thailand, and sympatric speciation within Senegal based on the distributions of FST values and variations in the sex determining locus between populations, 2) vector competence of Ae. aegypti from Senegal is dependent on the flavivirus species and viral genotype, and 3) increased genetic diversity of Dcr2 in individual Ae. aegypti, but not from populations of Ae. aegypti, from Senegal is correlated with the ability of Ae. aegypti to control DENV-2 infection. These results aid in our general understanding of the role of genomic divergence in speciation, as well as our understanding of genetics, vector competence, and the genetics of vector competence of West African Ae. aegypti, which could provide insight into the way we identify subspecies and make predictions about vector competence in this region.
Open Access
Western equine encephalitis virus: development and application of a new world alphavirus transducing system
(Colorado State University. Libraries, 2012) Stauft, Charles Brandon, author; Olson, Kenneth, advisor; Brennan, Carol, committee member; Foy, Brian, committee member; Bowen, Richard, committee member
A recombinant western equine encephalitis virus (WEEV) was generated that expressed firefly luciferase (FLUC) as a marker of infection. In vivo imaging technology was used to visualize bioluminescence in the context of WEEV infection of outbred (CD-1) and inbred (C57/BL6) strains of mice as well as Culex tarsalis mosquitoes. Bioluminescent imaging permitted us to follow a neurovirulent strain of WEEV in the living tissue of a single animal over time. The recombinant virus also permitted detection by bioluminescence of WEEV in the mosquito vector, Culex tarsalis. In vivo imaging was used to test the hypothesis that an alphavirus transducing system could be used to predict efficacy of a cationic lipid RNA complex (CLRC) immunomodulator in the suppression of WEEV infection. Bioluminescent imaging in screening potential antivirals for activity against WEEV in vivo was confirmed to be consistent, clear, and in agreement with traditional survival curve analysis. WEEV is maintained in an enzootic cycle through transmission by Culex tarsalis to passerine bird species. Tangential transmission to equine or human hosts has been associated with severe outbreaks of disease in the past. These hosts are considered to be dead-end hosts as they may become infected during epizootics but do not generate sufficient viremia titers to infect a bloodfeeding mosquito. Understanding the determinants of transmission to the vector from the host, dissemination within the vector, and secretion in saliva of WEEV are crucial to understanding the overall cycle. The recent development of a WEEV transducing system facilitated the study of WEEV interaction with the midgut, ovary, and salivary gland tissue of C. tarsalis. The expression by a recombinant alphavirus of monomeric cherry fluorescent protein allowed an overall picture of infection, dissemination, and transmission with both enzootic (IMP181) and epidemic (McMillan) strains of WEEV. Salivary gland infection rate was hypothesized to be greater for IMP181 than McMillan. IMP181 was hypothesized to be transmitted at a higher rate compared to McMillan and present in higher viral titers in saliva. The barriers to McMillan infection of salivary glands or transmission were hypothesized to be dose dependent. Increased viral titer of injected McMillan was expected to result in a higher salivary gland infection rate, transmission rate, and amount of virus detected in the saliva. A midgut barrier to infection was circumvented by injection of each virus strain into mosquitoes. There was no significant difference in McMillan and IMP181's ability to infect salivary glands or transmit at 7 and 14 days post infection. IMP181 infection resulted in higher viral titers found in expectorated saliva. The use of chimeric recombinant WEEV also revealed WEEV sequence determinants in the structural coding regions and 3'UTR of IMP181 that enhanced virus titers in expectorated C. tarsalis saliva. The transmission rate and not the salivary gland infection rate were found to be dose dependent after intrathoracic injection with both strains of WEEV.