Department of Microbiology, Immunology, and Pathology
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These digital collections include theses, dissertations, faculty publications, and datasets from the Department of Microbiology, Immunology, and Pathology. Due to departmental name changes, materials from the following historical department are also included here: Microbiology.
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Browsing Department of Microbiology, Immunology, and Pathology by Subject "Aedes aegypti"
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Item Open Access Aedes aegypti and dengue virus investigation of anatomic, genomic, and molecular determinants of vector competence(Colorado State University. Libraries, 2009) Bernhardt, Scott Arthur, author; Blair, Carol D., advisor; Black, William C., IV, advisorDengue (DENV) causes one of the most rapidly expanding diseases in the tropics. Vector competence (VC) in Aedes aegypti for DENV-2 is a quantitative trait and has been shown to be highly variable. Questions remain as to whether variation in VC continues to exist after the primary field observation. What genetic factors contribute to VC and do these factors evolve from arbovirus exposure remain unclear.Item 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 memberDengue 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.Item Open Access Effects of immunological targeting of two mosquito antigens and oral ingestion of anthelmintic drugs on the yellow fever mosquito, Aedes aegypti (Diptera: culicidae)(Colorado State University. Libraries, 2011) Deus, Kelsey Marie, author; Foy, Brian D., advisor; Avery, Anne, committee member; Black, William C., IV, committee member; Bowen, Richard A., committee memberAedes aegypti is one of the most important mosquito vectors of human arboviruses, including dengue viruses, chikungunya virus, and yellow fever virus. Human infection with these viruses constitutes an enormous global disease burden. Current control methods rely heavily on the use of insecticides, which are rapidly losing their utility due to the spread of insecticide resistance. Anti-vector vaccines and anthelmintic drugs with insecticidal properties have been proposed as novel means to decrease pathogen transmission by reducing the daily probability of mosquito survival. The aims of this dissertation research were to: evaluate the Ae. Aegypti mosquito lysosomal aspartic protease and the glutamate-gated chloride anion channel as potential mosquitocidal antigens, evaluate drugs frequently used in mass drug administration campaigns for their ability to induce a mosquitocidal effect when imbibed in a blood meal, to assess the variation in susceptibility of Ae. Aegypti strains to orally imbibed ivermectin, and finally to determine if resistance to ivermectin could be selected for in a genetically diverse laboratory strain of Ae. Aegypti . Despite the utilization of several immunization regimens, a specific mosquitocidal immune response against the Ae. Aegypti mosquito lysosomal aspartic protease could not be verified. In vitro experiments in which high titer glutamate-gated chloride anion channel serum was fed to mosquitoes failed to elicit a mosquitocidal response, suggesting that it is an unlikely mosquitocidal antigen. In vitro blood feeding experiments with several anthelmintic drugs revealed that high concentrations of macrocyclic lactones (including ivermectin, selamectin and moxidectin) were effective in reducing adult mosquito survival and that sublethal concentrations resulted in reduced fecundity and egg hatch rate. When imbibed in a blood meal, diethylcarbamazine, albendazole-sulfoxide and pyrantel pamoate, which are all currently used in human mass drug administration campaigns for controlling parasitic pathogens in humans, had no effect on adult mosquito survival. Significant differences in susceptibility to ivermectin, according to mosquito strain, were observed, with three permethrin-resistant strains of Ae. Aegypti being the most refractory to ivermectin, suggesting a possible permethrin-induced cross resistance mechanism to ivermectin. After subjecting a genetically diverse laboratory strain of Ae. Aegypti to three successive rounds of selection with orally imbibed ivermectin, no resistance to the drug was apparent. Although mass drug administration is unlikely to have any impact on the transmission of Ae. Aegypti vectored pathogens, Ae. Aegypti may prove to be a useful model for studying the effects of ivermectin in the mosquito, including studying potential resistance and cross-resistance mechanisms to anthelmintic drugs.Item 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 memberWorldwide 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.Item Open Access Exploration of the arthropod virome, its biological impacts on host health, and its potential implementation in biocontrol(Colorado State University. Libraries, 2020) Cross, Shaun T., author; Stenglein, Mark D., advisor; Wilusz, Jeffrey, committee member; Foy, Brian D., committee member; Metcalf, Jessica L., committee memberWith the advent of next generation sequencing, viruses are being discovered at an unprecedented rate. The collection of these viruses, known as the virome, and their impact on the host is relatively understudied compared to the bacterial microbiome. The underlying goal of this thesis work was to better understand how the virome interacts with the host, and this has been accomplished in two ways. First, we biologically characterized predominant virus constituents in arthropod viromes, namely arthropod-infecting partitiviruses. In a subsequent study we measured how one of these partitiviruses, galbut virus, impacted the fitness of Drosophila melanogaster. Second, we searched for evidence that these viruses are active in their interactions with fellow microbial constituents within the host. Specifically, we addressed how the virome may change disease vectors' competence in harboring and transmitting pathogens with a focus on Ixodes scapularis ticks. Partitiviruses are segmented, multipartite dsRNA viruses that until recently were only known to infect fungi, plants, and protozoans. Metagenomic surveys have revealed that partitivirus-like sequences are also commonly associated with arthropods. One arthropod-associated partitivirus, galbut virus, is common in wild populations of D. melanogaster. To begin to understand the processes that underlie this virus's high global prevalence, we established colonies of wild-caught infected flies. Infection remained at stably high levels over three years, with between 63-100% of individual flies infected. Galbut virus infects fly cells and replicates in tissues throughout infected adults, including reproductive tissues and the gut epithelium. We detected no evidence of horizontal transmission via ingestion but vertical transmission from either infected females or infected males was ~100% efficient. Vertical transmission of a related partitivirus, verdadero virus, that we discovered in a laboratory colony of Aedes aegypti mosquitoes was similarly efficient. This suggests that efficient biparental vertical transmission may be a feature of at least a subset of insect-infecting partitiviruses. To study the impact of galbut virus infection free from the confounding effect of other viruses, we generated an inbred line of flies with galbut virus as the only detectable virus infection. We were able to transmit the infection experimentally via microinjection of homogenate from these galbut-only flies. This sets the stage for experiments to understand the biological impact and possible utility of partitiviruses infecting model organisms and disease vectors. Using the galbut virus and D. melanogaster system, we set forth to answer: what are the biological effects, if any, of galbut virus infection on D. melanogaster fitness? Using multiple lines of flies from the Drosophila Genetic Reference Panel (DGRP) that differed only in their galbut virus infection status, a variety of fitness measurements were performed across both sexes. Galbut virus minimally impacted lifespan and had no effects on fecundity, but infection did significantly impact developmental speeds of flies. When challenged with various viral, bacterial, and fungal pathogens, some galbut virus infected flies had altered sensitivity to these pathogens. These susceptibility changes varied by both genetic background and sex. Galbut virus overall has minimal influences on host transcriptional responses, consistent with minimal phenotypic impacts of galbut virus infection. Major constituents of the microbiome were not perturbed by galbut virus infection. All fitness measurements alterations attributable to galbut virus were small, but they were dependent by strain and sex, highlighting the importance of these variables in phenotype outcomes. However, these altered measurements in galbut virus infected flies were dwarfed in comparison to those measurements attributable solely by fly strain and sex. These findings further support a trend of predominately cryptic phenotypes of partitivirus infections. To understand how the virome interacts with other microbial constituents, we specifically searched for polymicrobial interactions within the field of vector-borne diseases. I. scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence. Follow-up analyses on a second population of I. scapularis ticks derived from New York, USA revealed that these potential functional relationships may be population-specific. When evaluating South Bay virus, blacklegged tick phlebovirus-1, and B. burgdorferi in these individual ticks, no correlative or cooccurrence associations were observed. The lack of concordance between populations suggests that interactions between microbial constituents may be fluid, and change based upon location and populations. To characterize the biology of tick-associated viruses, an attempt to isolate South Bay virus was performed. Despite using mammalian and tick cell lines, we were unsuccessful in isolating South Bay virus through in vitro cell culture. The lack of success accents the challenge for understanding the biology of these arthropod-specific viruses. Further additional attempts to acquire infectious South Bay virus, such as creating a reverse genetics system, are warranted for its biological characterization.Item Open Access Mutagenesis of the dengue virus envelope glycoprotein gene can significantly alter virus infectivity phenotypes in cultured cells and live mosquitoes(Colorado State University. Libraries, 2011) Erb, Steven Michael, author; Blair, Carol D., advisor; Roehrig, John T., committee member; Olson, Kenneth E., committee member; Chen, Chaoping, committee memberThe dengue virus (DENV) envelope (E) glycoprotein is the primary determinant for initiation of host cell infection. To date, studies investigating the contribution of DENV genetics to mosquito infection are limited. A infectious clone cDNA of DENV type 2 strain 16681 (30P-NBX) provided the ability to introduce site-specific amino acid (AA) mutations into the E protein. The results of the studies herein analyze the effects that AA mutations in the E protein have on infectivity of cultured cells and live mosquitoes. The ability of 30P-NBX to infect Aedes aegypti RexD strain mosquitoes after oral infectious blood-meal was investigated and showed that both 30P-NBX and the parent virus 16681 have low, but equivalent midgut infection rates (MIRs). Mosquito midgut infection with 30P-NBX is not affected by the virus titer in the blood-meal as long as titers are above 6 log 10 pfu/ml or 7 log 10 TCID 50 /ml. Additionally, multiple experimental repetitions with at least 20 mosquitoes per infectious blood-feed were required to obtain an accurate average MIR for 30P-NBX. Serial passage of 30P-NBX in RexD mosquito midguts identified a single AA mutation at position 122 in domain II of the E protein from lysine to glutamic acid that correlated with increased MIRs. Introduction of this AA mutation into the infectious clone (mutant virus K122E) reproduced the results from the serial passage experiment. Compared to 30P-NBX, K122E was not only shown to infect a higher proportion of mosquitoes as early as day 2 post blood-feed, but also to produce a disseminated infection in a higher proportion of mosquitoes by day 6 post blood-feed. Also, K122E consistently produced a midgut infection that spread throughout the entire tissue while 30P-NBX stayed restricted by comparison. Virus attachment to midgut cells was compared and showed that 30P-NBX and K122E could attach with equal efficiencies via our midgut-virus attachment assay. Additionally, incorporation of a single AA mutation into the infectious clone at E protein AA 120 from arginine to threonine significantly enhanced mosquito midgut infection compared to 30P-NBX. This is the first time that mosquito infection determinants have been identified in the DENV E protein. Amino acid mutations were engineered into the E protein on the lateral ridge of domain III, the fusion peptide at the distal end of domain II, and the molecular hinge region between domains I and II. Mutant virus phenotypes were analyzed in cell culture and live mosquitoes. In contrast to previous suggestions, domain III mutant virus phenotypes showed that the FG loop structure (previously suggested as a mosquito-specific infection determinant) and not the specific AA sequence is important for infection of mammalian cells and live mosquitoes, while the structure and sequence of the FG loop is dispensable for infection of cultured C6/36 cells. Additionally, mutations that remove positively charged residues from the A strand in DIII significantly attenuate infection of mosquitoes after oral infectious blood-meal and completely abrogate infection in mammalian cells. The results of this study suggest that there may be multiple structures in the E protein that are contributing to virus-receptor interactions. Viruses with mutations in the fusion peptide and hinge region of the E protein were intrathoracically (IT) inoculated into mosquitoes and showed variable infectivity phenotypes. All of the mutants except for one virus from both the fusion peptide and hinge region viruses attenuated infection of mosquito tissues outside the midgut. Importantly, considering that almost all of these viruses were able to replicate as efficiently as wild type in C6/36 cells, the IT inoculation results provide evidence that C6/36 cells are not a complete surrogate for DENV replication in mosquitoes.Item 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 memberThe 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.Item Open Access Small RNAs in Aedes aegypti: one giant step for virus control in mosquitoes(Colorado State University. Libraries, 2022) Williams, Adeline E., author; Olson, Ken, advisor; Antolin, Mike, committee member; Calvo, Eric, committee member; Franz, Alexander, committee member; Wilusz, Jeffrey, committee memberAedes aegypti mosquitoes are key vectors of medically relevant arthropod-borne (arbo) viruses such as Zika (ZIKV), dengue (DENV1-4), and yellow fever (YFV). When Ae. aegypti become infected with arboviruses, RNA interference (RNAi) is a critical antiviral immune mechanism that is a key determinant for successful virus transmission. The major antiviral pathway is the RNAi small-interfering RNA (siRNA) pathway, although evidence shows that the Piwi-interacting RNA (piRNA) pathway also acts as an important RNAi mechanism for controlling persistently infective viruses. The overarching goals of this work were twofold: (1) to determine the potency of the Ae. aegypti siRNA pathway against Zika virus and (2) to understand molecular mechanisms underlying piRNA-mediated antiviral immunity and its implications on mosquito vector competence. To achieve these goals, we (1) engineered transgenic Ae. aegypti mosquitoes that synthetically triggered the endogenous siRNA pathway against ZIKV and then quantified virus resistance in these mosquitoes, (2) sequenced small RNAs (sRNAs) of the mosquito virome that may impact vector competence and virus persistence, and (3) characterized structural features of Piwi4, an antiviral protein, involved in sRNA binding and subcellular localization to gain insights on its role in the piRNA and siRNA pathways. A major challenge in the fight against arboviruses is the lack of effective vaccines and limited therapeutic options. Vector control remains the primary method of preventing disease, and integrated vector management (IVM), including the genetic control of mosquitoes, is imperative to prevent emerging arboviral diseases. To this end, we designed an antiviral effector gene – a ZIKV-specific double stranded (ds) RNA –that synthetically triggered the mosquito's siRNA pathway after a bloodmeal in transgenic Ae. aegypti. Small RNA analyses in transgenic midguts revealed ZIKV-specific 21 nucleotide (nt) siRNAs 24 hours after a non-infectious bloodmeal. Nearly complete (90%) inhibition of ZIKV replication was found 7-to-14 days post-infection (dpi); furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that the siRNA pathway can be synthetically exploited to generate ZIKV-resistant Ae. aegypti mosquitoes. In the context of gene drive, antiviral effectors expressed in transgenic Ae. aegypti will be an invaluable tool for a population replacement vector control approach. piRNA-mediated antiviral immunity involves an endogenous viral element (EVE) – viral derived cDNA (vDNA) integrated into host genomes – as well as infection with a cognate virus, which together trigger piRNA amplification and lead to virus silencing. EVEs are from viruses that infected a population in previous generations, and most are derived from insect-specific viruses (ISVs) that persistently infect Ae. aegypti. We hypothesized that ISVs and ISV-derived piRNA populations, like EVEs, have geographic structure and impact vector competence to arboviruses. To test this hypothesis, we sequenced sRNAs from geographically distinct Ae. aegypti and characterized virus-derived sRNAs (vsRNAs). Overall, the distribution of total sRNAs was highly variable. Small RNAs derived from ISVs were diverse and dependent on geographic origin. We next infected Ae. aegypti from Poza Rica, Mexico with DENV2 and analyzed changes in the sRNA virome. DENV2 intrathoracic inoculation resulted in DENV2-specific siRNAs and piRNAs. We also found increased loads of sRNAs against the ISVs verdadero (Partitiviridae: unclassified), Aedes anphevirus (Xinmoviridae: Anphevirus), and chaq-like virus (Partitiviridae: unclassified) after DENV2 infection compared to ISV-derived sRNAs in controls. Overall, our study highlights the diversity of infective ISVs and the complexity of the sRNA virome across Ae. aegypti populations, which likely has consequences on sRNA crosstalk, virus replication, and vector competence. To gain insights on how Piwis, piRNA-binding proteins, are involved in virus control, we characterized structural features of an antiviral Piwi, Piwi4, involved in RNA binding and subcellular localization. We found that Piwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3'-terminal ends of piRNAs, bound to mature (3'-terminal 2'-O-methylated) and 3'-terminal unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 μM and KD of 5.0 ± 2.2 μM, respectively) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins had significant impacts on binding behaviors, likely by impacting protein secondary structure. We also found that Piwi4 was both cytoplasmic and nuclear in mosquito tissues, and we identified a Piwi4 nuclear localization signal in the N-terminal region of the protein. These studies provide insights on the dynamic role of Piwi4 in RNAi and pave the way for future studies aimed at understanding Piwi4 interactions with diverse RNA populations.Item Open Access The role of midgut serine proteases in Aedes aegypti vector competence(Colorado State University. Libraries, 2007) Brackney, Douglas Eugene, author; Olson, Kenneth E., advisorNumerous gut-associated viruses utilize host proteolytic enzymes to facilitate enhancement of infection. Similarly, arboviruses infecting the invertebrate host (vector) through the alimentary tract may exploit serine proteases in the midgut to enhance vector infection. Recent genetic and biochemical experiments have demonstrated that dengue virus type 2 (DENV-2) may require proteolytic processing by midgut trypsins to efficiently infect Aedes aegypti. These results suggest midgut serine proteases may influence A. aegypti vector competence. The requirement of serine proteases in DENV-2 infection of the vector provides unique targets for development of novel control strategies through approaches such as transmission blocking vaccines.