Browsing by Author "Ebel, Gregory D., advisor"
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Item Open Access A circuitous journey of virus characterization and surveillance in North and Central America(Colorado State University. Libraries, 2023) McMinn, Rebekah J., author; Ebel, Gregory D., advisor; Quackenbush, Sandra, committee member; Brault, Aaron, committee member; Neuwald, Jennifer, committee memberThe burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV, Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of human disease. In this dissertation we evaluated the recent emergence of POWV from a culmination of field (chapter 2), in vitro (chapter 3), and in-vivo (chapter 4) studies. In addition, we determined the applicability of a vector-enabled surveillance method (xenosurveillance) in Central America (chapter 5). We first used a genetic approach to evaluate the emergence of lineage II POWV, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the northeastern United States with an average infection rate of 1.4%. High-depth whole genome sequencing of eighty-four new and archival POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the northeastern United States and patterns of geographic dispersal within and between regions. Bayesian skyline analysis demonstrated DTV population expansion over the last fifty years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus. We then assessed in vitro phenotypes of POWV on human neuronal cells using 16 genetically diverse isolates obtained from a broad geographic and temporal range. We determined over a 10,000-fold range in peak viral titer and significantly decreased cell mortality for two Midwest DTV isolates, though no clear correlation between in vitro phenotype and geo-temporal characteristics could be made. We then performed whole genome sequencing of virus post neuronal cell passage to identify potential residues of interest. Again, no residues could be linked to phenotype, though several interesting residues with increased frequency post-neuronal cell culture were identified. Based on the significant in vitro diversity observed, we sought to assess pathogenesis and tick transmission phenotypes between isolates. We noted neurological disease in mice in both lineages of POWV, with potential low-virulence strains derived from coastal New York. Additionally, we observed an early neuroinvasion phenotype for a Midwest DTV isolate. The ability to infect I. scapularis ticks was determined by feeding on infected host mice (viremic) and through an artificial infection method. Surprisingly, infection rates in ticks via viremic or artificial infection remained consistent between all five isolates tested, resulting in 12-20% infection rate. Taken together, these data demonstrate potential genotype-independent ability to infect ticks and conversely, strain-dependent differences in pathogenesis. In chapter 5, we evaluated a vector-enabled surveillance method ('xenosurveillance') in rural Guatemala. Surveillance methods that permit rapid detection of circulating pathogens are desperately needed. Xenosurveillance is a novel surveillance approach that takes advantage of mosquito feeding behavior to identify blood-borne pathogens that may be circulating in human and animal hosts. This approach circumvents invasive blood sampling of individuals and results in an abundant sample source derived from both humans and animals. In this study, twenty households from two villages (Los Encuentros and Chiquirines) in rural, southwest Guatemala were enrolled and underwent weekly prospective surveillance for 16 weeks. When febrile illness was reported in a household, recently blood-fed mosquitoes were collected from within dwellings and blood samples taken from each member of the household. Mosquitoes were identified to species and blood sources identified by sequencing. Shotgun metagenomic sequencing was used to identify circulating viruses. Culex pipiens (60.9%) and Aedes aegypti (18.6%) were the most abundant mosquitoes collected. Bloodmeal sources were most commonly human (32.6%) and chicken (31.6%), with various other mammal and avian hosts detected. Several mosquito-specific viruses were detected, including Culex orthophasma virus. Human pathogens were not detected. While more intensive sampling may be needed to detect human pathogens, sampling mosquitoes that feed on humans and domestic animals may prove valuable for monitoring pathogens with zoonotic potential.Item Open Access Dynamics of West Nile virus evolution during infection of wild birds, mosquitoes, and the human brain: unraveling the compelexities of selection, drift, and fitness(Colorado State University. Libraries, 2016) Grubaugh, Nathan D., author; Ebel, Gregory D., advisor; Black, William C., committee member; Foy, Brian D., committee member; Brault, Aaron C., committee member; Sloan, Daniel B., committee memberTo view the abstract, please see the full text of the document.Item Open Access 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 memberWest 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.Item Embargo 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 memberVector 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.Item Open Access Profiling the antiviral small RNA response in mosquitoes to arbovirus infection: intra- and interspecies comparisons and relationship to vector competence(Colorado State University. Libraries, 2014) Prasad, Abhishek N., author; Ebel, Gregory D., advisor; Olson, Kenneth E., committee member; Blair, Carol D., committee member; Brault, Aaron C., committee member; Hess, Ann M., committee memberTo view the abstract, please see the full text of the document.