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Browsing by Author "Kading, Rebekah, committee member"

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    A tale of two viruses: the ability of New World mammals to host Old World viruses
    (Colorado State University. Libraries, 2022) Lewis, Juliette, author; Schountz, Tony, advisor; Kading, Rebekah, committee member; Vilander, Allison, committee member; Stoner, Kathryn, committee member
    To view the abstract, please see the full text of the document.
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    Assessment of anopheles vectorial capacity metrics and malaria transmission factors within the Rimdamal II Study
    (Colorado State University. Libraries, 2021) Gray, Lyndsey Irene, author; Foy, Brian, advisor; Kading, Rebekah, committee member; Mueller, Rachel, committee member; Stenglein, Mark, committee member
    To view the abstract, please see the full text of the document.
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    Assessment of mosquito and animal model factors in Aedes-borne arbovirus transmission and disease
    (Colorado State University. Libraries, 2021) Miller, Megan Rae, author; Foy, Brian, advisor; Wilusz, Jeffrey, committee member; Kading, Rebekah, committee member; Montgomery, Tai, committee member; Magunda, Forgivemore, committee member
    To view the abstract, please see the full text of the document.
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    Explorations in West Nile virus ecology and evolution
    (Colorado State University. Libraries, 2021) Byas, Alexandria D., author; Ebel, Gregory D., advisor; Bowen, Richard, committee member; Kading, Rebekah, committee member; Sloan, Daniel, committee member
    West Nile virus (WNV) continues to be a major cause of human arboviral neuroinvasive disease. Susceptible non-human vertebrates are particularly diverse, ranging from commonly affected birds and horses to less commonly affected species such as alligators. The literature review in Chapter 1 summarizes the pathology caused by West Nile virus during natural infections of humans and non-human animals. While the most well-known findings in human infection involve the central nervous system, WNV can also cause significant lesions in the heart, kidneys and eyes. Time has also revealed chronic neurologic sequelae related to prior human WNV infection. Similarly, neurologic disease is a prominent manifestation of WNV infection in most non-human non-host animals. However, in some avian species, which serve as the vertebrate host for WNV maintenance in nature, severe systemic disease can occur, with neurologic, cardiac, intestinal and renal injury leading to death. The pathology seen in experimental animal models of WNV infection and knowledge gains on viral pathogenesis derived from these animal models are also briefly discussed. A gap in the current literature exists regarding the relationship between the neurotropic nature of WNV in vertebrates, virus propagation and transmission in nature. This and other knowledge gaps, and future directions for research into WNV pathology, are addressed. In Chapter 2, experimental evolution work is described. For arboviruses, the vertebrate and invertebrate hosts in which they circulate shape viral evolution and can lead to the emergence of new genotypes. Previous work in mosquitoes and birds has identified species-specific effects on viral populations when species were assessed in isolation. We united mosquito and bird species to perform experimental evolution studies which paired Culex (Cx.) pipiens with American crows, Cx. quinquefasciatus with American crows and Cx. quinquefasciatus with American robins. Crow and Cx. pipiens transmission cycles were the most successful and robin and Cx. quinquefasciatus transmission cycles were the least successful at reaching three complete rounds of bird-to-mosquito transmission. These findings suggest that crows may be more important to WNV maintenance in nature over robins. The greater success of crow cycles when paired with Cx. pipiens in comparison to crows paired with Cx. quinquefasciatus may also suggest fitness losses associated with Cx. quinquefasciatus. In multiple rounds of transmission, infection rates (WNV-positive mosquito midgut) and transmission-capability (WNV-positive mosquito saliva) decreased with each subsequent round of transmission, suggesting that pairings in isolation experience fitness losses. Competitive fitness assays of transmission cycles exhibited cyclical increases and decreases in fitness as virus moved through crows and mosquitoes, respectively. That the stronger competitive fitness tended to occur with samples from the avian host while virus from mosquitoes tended to have decreased fitness may be consistent with genetic restriction and strong purifying selection in birds and genetic expansion and weak purifying selection in mosquitoes. Sequencing is needed to assess whether differences in transmission cycle success and competitive fitness can be attributed to genetic changes. In Chapter 3, the avian single cell viral environment is assessed. Error-prone replication of RNA viruses generates the viral diversity required for adaptation to rapidly changing environments. This is crucial for arboviruses whose viral populations exist as mutant swarms maintained between both mosquito and vertebrate hosts. By infecting cells and birds with barcoded WNV stock and sequencing single cells, we demonstrated that the richness and frequency of rare variants in crows far exceeded that found in robins. Moreover, those rare occurring variants were maintained by crows more than they were by robins. We further demonstrated that bird viremia functions as a determinant of multiplicity of infection in peripheral blood mononuclear cells (PBMCs), a significant site of viral replication. We found that increased viremia leads to increased polyinfections of individual PBMCs with maintenance of defective genomes and less prevalent variants, specifically in crows, presumably through complementation. When two pairings of variably-fit viruses were used to co-infect American robins and American crows, we observed increases in replication for one of the less fit viruses when viremia was higher. The ability of the low fitness virus to better replicate at higher viremia is likely a result of polyinfections and complementation at the cellular level. Our findings suggest that weak purifying selection in highly susceptible crows is attributable to higher viremia, polyinfections and complementation while viral divergence and fewer variants rising to fixation in robins is a result of overall lower levels of viremia and fewer polyinfections. In Chapter 4, the potential contributions of American alligators to natural WNV ecology are examined. West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle. We performed a diverse array of experiments which utilize novel techniques and technologies to characterize the mechanisms of WNV evolution. We also identified a potential non-avian WNV amplifier host in alligators. This work represents a significant contribution to the West Nile virus literature by working with the unique species which contribute to virus propagation and assessing their effects on viral evolution and ecology.
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    High throughput characterization of bunyavirus diversity, ecology, and reassortment potential
    (Colorado State University. Libraries, 2023) Kapuscinski, Marylee, author; Stenglein, Mark, advisor; Kading, Rebekah, committee member; Wilusz, Jeff, committee member; Ebel, Greg, committee member; Nachappa, Punya, committee member
    Bunyavirales is an important group of viral pathogens with significant economic impacts. The Bunyavirales order contains the largest number of RNA viruses and can cause disease in plants, animals, and humans [1]. Notable plant pathogens include Tomato Spotted Wilt virus [2] which results in significant agricultural losses. Notable animal pathogens include Rift Valley fever virus [3] and Schmallenberg [4] virus resulting in significant livestock loss. Notable human pathogens include Crimean-Congo hemorrhagic fever virus [5], hantavirus [6], and La Crosse virus [7]. The effects of bunyavirus infections are felt worldwide because bunyaviruses are distributed globally. The emergence of novel bunyaviruses continues to threaten agricultural and livestock industries as well as human health. Where or when a novel bunyavirus might emerge is unknown. Predicting emergence is difficult for three reasons. First, because bunyaviruses are RNA viruses [8], the high error-rate of the RNA-dependent RNA polymerase results in a large genetic diversity within a population of viruses [9]. Secondly, many important bunyaviruses are arthropod-borne [10], resulting in an intricate lifecycle between an invertebrate and vertebrate host. This results in constantly changing genetic diversity due to different selective pressures from different host types [11]. It also results in an ever-expanding geographic range as vector range expands due to climate change and vertebrate host range changes due to urbanization, industrialization, and deforestation [12–16]. Third, bunyaviruses have segmented genomes which allows them to reassort and produce viral progeny with an altered vector-host range, pathogenesis, and virulence [1,17,18]. Therefore, this body of work aims to increase our ability to understand bunyavirus emergence and reassortment potential as a way to aid in outbreak preparedness and early response systems. To do this, we've combined traditional surveillance data with modern bioinformatics to expand our knowledge of bunyavirus genetic diversity, ecology, and reassortment potential. Using whole-genome sequencing, we've characterized the genomes of 99 bunyaviruses, some of which have never been sequenced before. This aids in our understanding of the genetic diversity, co-infection dynamics, and reassortment potential. Next, we used existing metadata from orthobunyavirus sequences to determine reassortment potential given a shared geographic and vector-host range. Finally, we've developed a novel molecular assay to evaluate reassortment potential based on replication and transcription compatibility. Together, we've combined the strengths of viral surveillance and modern bioinformatics to demonstrate the benefit of combining both. We've developed systems that will help to delineate the mechanisms that either promote or inhibit reassortment potential, ultimately aiding in early response systems for outbreak preparedness.
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    Impact of chorionic somatomammotropin in vivo RNA interference phenotype on uteroplacental expression of the IGF axis
    (Colorado State University. Libraries, 2023) Hord, Taylor, author; Anthony, Russel V., advisor; Winger, Quinton A., advisor; Bruemmer, Jason, committee member; Kading, Rebekah, committee member
    While fetal growth is dependent on many factors, optimal placental function is a prerequisite for a normal pregnancy outcome. The majority of fetal growth restricted (FGR) pregnancies result from placental insufficiency (PI). The insulin-like growth factors (IGF1 and IGF2) not only stimulate fetal growth, but also placental development and function. Previously, we demonstrated that in vivo RNA interference (RNAi) of the placental hormone, chorionic somatomammotropin (CSH), resulted in two phenotypes. One phenotype exhibits significant placental and fetal growth restriction (PI-FGR), impaired placental nutrient transport, and significant reductions in umbilical insulin and IGF1. The other phenotype does not exhibit statistically significant changes in placental or fetal growth (non-FGR). It was our objective to further characterize these two phenotypes by determining the impact of CSH RNAi on placental (maternal caruncle and fetal cotyledon) expression of the IGF axis. The trophectoderm of hatched blastocysts (9 days of gestation, dGA) were infected with a lentivirus expressing either a non-targeting sequence (NTS RNAi) control or CSH-specific shRNA (CSH RNAi) prior to embryo transfer into synchronized recipient ewes. At ≈125 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies. Nutrient uptakes were determined and tissues were harvested at necropsy. In both CSH RNAi non-FGR and PI-FGR pregnancies, uterine blood flow was significantly reduced (P≤0.05), while umbilical blood flow (P≤0.01), both uterine and umbilical glucose and oxygen uptakes (P≤0.05), and umbilical concentrations of insulin and IGF1 (P≤0.05) were reduced in CSH RNAi PI-FGR pregnancies. Fetal cotyledon IGF1 mRNA concentration was reduced (P≤0.05) in CSH RNAi PI-FGR pregnancies, whereas neither IGF1 nor IGF2 mRNA concentrations were impacted in the maternal caruncles, and either placental tissue in the non-FGR pregnancies. Fetal cotyledon IGF1R and IGF2R mRNA concentrations were not impacted for either phenotype, yet IGF2R was increased (P≤0.01) in the maternal caruncles of CSH RNAi PI-FGR pregnancies. For the IGF binding proteins (IGFBP1, IGFBP2, IGFBP3), only IGFBP2 mRNA concentrations were impacted, with elevated IGFBP2 mRNA in both the fetal cotyledon (P≤0.01) and maternal caruncle (P=0.08) of CSH RNAi non-FGR pregnancies. These data support the importance of IGF1 in placental growth and function, but may also implicate IGFBP2 in salvaging placental growth in non-FGR pregnancies.
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    Investigating the resistance status to permethrin and temephos in Aedes aegypti (the yellow fever mosquito)
    (Colorado State University. Libraries, 2024) Arthur, Nicholas Wynne, author; Norton, Andrew, advisor; Camper, Matt, committee member; Kading, Rebekah, committee member; Saavedra-Rodriguez, Karla, committee member
    Aedes aegypti (Ae. aegypti) is the principle urban vector of several viruses of high medical significance which carry a disease burden on a global scale. Ae. aegypti is anthropophilic and lives in close association with humans. This places nearly half of the global population at risk of becoming infected with an arboviral pathogen every year. Therefore, emphasis must be placed on investigating methods for controlling this vector to combat and reduce the spread of human disease. This is especially true in areas where socioeconomic factors promote sustained transmission cycles. While vector control programs use a variety of strategies, the primary method of reducing vector populations is through insecticide use. Widespread use of insecticides has placed intense selection pressures on Ae. aegypti populations and resistance mechanisms have developed. Target site modifications and the expression of detoxifying enzymes are the most significant resistance mechanisms to date. Several single nucleotide polymorphisms resulting in amino acid changes within the voltage-gated sodium channel (VGSC) have been shown to reduce binding site sensitivity and confer resistance to pyrethroids. Specifically, mutations at the knockdown-resistant (kdr) 410, 1,016, and 1,534 sites have been associated with a reduction in pyrethroid sensitivity. I investigated the resistance status to permethrin and temephos at five locations in Hidalgo County, Texas. I determined the presence of permethrin resistance using a well-characterized susceptible colony as a reference for insecticide sensitivity. The resistant allele C1,534 reached fixation at all sites and L410 and I1,016 were found at high frequencies. The permethrin resistance was over 40-fold when compared to the reference colony. The sites were less resistant to temephos at approximately 6-fold to 12-fold, which I attributed to cessation of this insecticide in the continental United States since 2016. In the absence of selection pressures mosquito populations trend towards susceptibility, which suggests that there are potential fitness costs associated with insecticide resistance. Studying these associations is important to public health as they may support different strategies to reduce vector populations. I used two collections from Tapachula, Mexico, that were free of pyrethroid exposure since 2013, to determine the presence of two previously described fitness cost metrics: wing length and egg production. I found that the average wing length of V410L and V1,016I homozygous resistant individuals were significantly smaller compared to homozygous susceptible individuals. The interaction between wing length and genotype had no effect on egg production. Wing length had no significant effect on egg production. Most notably, L410 and I1,016 resistant alleles had no effect on egg production.
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    Linking mosquito midgut and virus population biology at the molecular and cellular level
    (Colorado State University. Libraries, 2024) Fitzmeyer, Emily Anne, author; Ebel, Gregory D., advisor; Stenglein, Mark, committee member; Kading, Rebekah, committee member; Anderson, Brooke, committee member
    Vector competence (VC) refers to the efficiency of pathogen transmission by vectors. Each step in infection of a mosquito vector constitutes a barrier to transmission that may impose bottlenecks on virus populations. West Nile virus (WNV) is maintained by multiple mosquito species with varying VC. However, the extent that bottlenecks and VC are linked is poorly understood. Similarly, quantitative analyses of mosquito-imposed bottlenecks on virus populations are limited. We used molecularly barcoded WNV to quantify tissue-associated population bottlenecks in three variably competent WNV vectors. Our results confirm strong population bottlenecks during mosquito infection that are capable of dramatically reshaping virus population structure in a nonselective manner. In addition, we found that mosquitoes with differing VC uniquely shape WNV population structure: highly competent vectors are more likely to contribute to the maintenance of rare viral genotypes. These findings have important implications for arbovirus emergence and evolution. The mosquito midgut functions as a key interface between virus and vector. However, studies of midgut physiology and associated virus infection dynamics are scarce, and in Culex tarsalis - the primary vector of West Nile virus (WNV) in the contiguous United States - nonexistent. We performed single-cell RNA sequencing on dissociated, WNV-infected Cx. tarsalis midguts. We identified populations of distinct midgut cell-types consistent with existing descriptions of insect midgut physiology and found that all midgut cell populations were permissive to WNV infection. However, we observed high levels of viral RNA suggesting enhanced replication in enteroendocrine cells and cells enriched for mitochondrial genes. In addition, we found no significant upregulation of mosquito immune genes associated with WNV infection at the whole-midgut level, rather, a significant positive correlation between immune gene expression and WNV viral RNA load at the individual cell level. These findings illuminate the midgut infection dynamics of WNV, providing insight into cell-type specific enhancement of, and immune response to, WNV infection in a primary vector.
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    Microglial innate and adaptive immune function modulates disease pathology in and environmental pesticide model of Parkinson's disease
    (Colorado State University. Libraries, 2022) Rocha, Savannah M., author; Zabel, Mark, advisor; Tjalkens, Ronald B., advisor; Bouma, Jerry, committee member; Kading, Rebekah, committee member; Moreno, Julie, committee member
    Parkinson's Disease (PD) is the world's foremost movement disorder with pathological features including loss of dopaminergic neurons (DAn) within the substantia nigra pars compacta (SNpc), chronic activation of glial cells, and the misfolding and aggregation of a-synuclein (a-syn). Compounding evidence gathered over the past two centuries suggests environmental exposures, genetics, and aging can induce complicated cell-to-cell interactions that evoke and facilitate chronic inflammatory states; but the role that individual glial cells, in particular microglia, have in the progression of disease remains unknown. Difficulties in recapitulating the three pathological hallmarks of PD underscore the need for better animal models. To address this gap in functional investigation, the studies herein provide, for the first time, an optimized environmental exposure model with the pesticide rotenone (2.5mg/kg/day) in murine, which has proven effective at mirroring DAn degeneration, gliosis and misfolded a-syn accumulation. The pathology observed was region-, time- and dose-dependent, emphasizing the importance of environmental exposure and associated PD diagnosis. The successful optimization of this exposure model has allowed for its implementation in transgenic mice, which was previously unfeasible. To determine microglial specific innate inflammatory reactions in the progression of PD, we targeted the inflammatory transcriptional regulator NF-kB by use of transgenic CX3CR1-Cre