Browsing by Author "Stenglein, Mark, committee member"
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Item Open Access 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 memberTo view the abstract, please see the full text of the document.Item Open Access Exploring the hemp virome and assessing hemp germplasm for resistance to emerging pathogens(Colorado State University. Libraries, 2024) Hackenberg, Laine, author; Nachappa, Punya, advisor; Roberts, Robyn, committee member; Stenglein, Mark, committee memberHemp (Cannabis sativa L.), commonly grown for its seeds, fiber, and non-psychoactive cannabinoids, has been experiencing a resurgence in the United States with its recent legalization. While farmers across the nation have readily adopted this crop, resources for pest management are still lacking, particularly regarding the diversity and distribution of pathogens. As production increases and the crop diversifies, the emergence and spread of these pathogens are certain. To circumvent loss due to disease, research is needed understand the threats and to identify sustainable management options. The goal of this study is to describe the diversity and distribution of viruses/viroids infecting hemp in Colorado and to determine if there is genetic resistance to pathogens in hemp. The objectives of this study are to 1) characterize the virome of different hemp cultivars throughout the growing season across different locations and 2) screen a panel of genetically unique genotypes of hemp for resistance to emerging viruses/viroids of hemp. Throughout 2021 and 2022, the hemp virome was examined in four major hemp producing regions of Colorado. In total, nine fields were sampled, and each field was visited during three phenological stages (early vegetative, late vegetative, and mature flowering) in order to characterize the virome throughout the growing season. Leaf tissue samples were collected from two cultivars of hemp from each field site. These tissue samples were submitted for High Throughput Sequencing (HTS) and upon bioinformatic analysis, candidate virus/viroid sequences were validated. Across both years, a total of seven viruses were identified: Alfalfa mosaic virus (AMV), Beet curly top virus (BCTV), Cannabis cryptic virus (CanCV), Cannabis sativa mitovirus (CasaMV1), Grapevine line pattern virus (GLPV), Opuntia umbra-like virus (OULV), and Tomato bushy stunt virus (TBSV). All viruses identified had >97% nucleotide identity to the nearest GenBank accession. Between individual cultivars isolated from the same field, both similar and unique viromes were observed. Viral diversity and incidence increased as the growing season progressed for both years. The three viruses that were most commonly found across all regions were CasaMV1, GLPV, and BCTV. Dominating the virome in viral load were CasaMV1 and GLPV. Given the prevalence of BCTV in the virome, in addition to its prevalence in hemp across the western United States, 13 genotypes of hemp were screened for resistance to this pathogen. These genotypes of hemp are genetically diverse, which will aide in the discovery of candidate genes involved with resistance. BCTV is the causal agent of curly top disease which can have drastic symptomology in hemp plants, causing malformed growth, stunted plants, and crop loss up to 100%. Varying BCTV copy number was observed across the hemp genotypes. Additionally, percent disease index (PDI) was analyzed to determine the frequency of infection of individual genotypes. Two of the genotypes were observed to have a lower PDI than the others, 4587 and 4710. Hop latent viroid (HLVd) has been emerging as a threat to the cannabis industry. It has been described across North America but is believed to be worldwide due to its global distribution in hops. HLVd has been documented to cause drastic reduction in cannabinoid content in mature inflorescences and therefore has the potential for substantial economic losses. Although not identified within the 2021 or 2022 virome, HLVd was determined to be an important threat facing hemp production therefore it was included in the screening. Similarly to BCTV, a panel of 14 genetically unique genotypes of hemp were analyzed for resistance to HLVd. Resistance was identified in a single genotype, 517, which had a lower frequency of infection than the others. However, no varying viroidal loads were observed between genotypes. Throughout this study, viruses associated with hemp were described as well as the identification of genetic resistance to emerging pathogens. This work will help to further integrated pest management strategies and promote sustainable agriculture.Item Open Access Extrinsic incubation temperature impacts on Zika virus evolution and vector competence during systemic Aedes infection(Colorado State University. Libraries, 2020) Murrieta, Reyes David A., author; Ebel, Gregory, advisor; Olson, Kenneth, committee member; Stenglein, Mark, committee member; Sloan, Dan, committee memberArthropod-borne viruses (arboviruses) are distinctive in that they are required to constantly replicate in different hosts and in a wide range of temperatures for their perpetuation in nature. Vertebrate hosts tend to maintain temperatures of approximately 37°C - 40°C, but arthropods hosts are poikilotherms and subject to ambient temperatures which can have a daily temperature fluctuation of > 10°C. Invertebrate host genus, species, and strain in combination with arbovirus strain and preparation methods are known to have large impacts on vector competence and vectorial capacity. Seemingly small differences in host geographic isolation, virus strain, and preparation methods can have significant impacts on vector competence studies. The role of temperature on the ability of an arthropod vector to acquire, maintain, and transmit a pathogen has been investigated for numerous arboviruses. Changing the extrinsic incubation temperature between distinct constant temperatures has been shown to alter arbovirus vector competence, extrinsic incubation period, and mosquito survival, in which moderate temperatures of 28°C-32°C are optimal and temperatures higher and lower have deleterious effects. The mean and range of daily temperature fluctuations (diurnal temperature) have likewise been shown to influence arbovirus perpetuation and vector competence, in which large daily temperature fluctuations negatively affect mosquito development, survival, and vector competence. However, little is known as to how temperature alters arbovirus genetic diversity during systemic mosquito infection or how differences in arbovirus hosts and viral strains impact arbovirus genetic diversity in relationship to temperature. Therefore in the study completed in chapter two, we characterized the impact that constant temperatures of 25°C, 28°C, 32°C, and 35°C, and the diurnal fluctuation from 25°C to 35°C during extrinsic incubation periods have on the Puerto Rican isolate of Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti (Poza Rica) and Aedes albopictus (Florida) mosquitoes. To characterize the impact that temperature has on ZIKV population diversity in different host species and viral isolates, in the study completed in chapter three, we used a Tapachula, Mexico Aedes aegypti line and a Chiapas, Mexico ZIKV isolate to assess ZIKV population dynamics during 20°C, 24°C, 28°C, 32°C, 34°C, and 36°C constant extrinsic incubation temperatures. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species, while transmission peaked at 10 days post-infection for Aedes aegypti and 14 days post-infection in Aedes albopictus. The diurnal temperature group is not predicted by the constant temperature distribution. Instead, when using the mean daily temperature of the diurnal group as a predicter, its VC lies between the moderate (28°C and 32°C) and extreme (25°C and 35°C) temperature group VCs. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the ZIKV selective environment during infection. During mosquito infection, constant temperatures more often elicited positive selection whereas diurnal temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology within mosquitoes and has distinct effects on the selective environment within mosquitoes. Additionally, the selective pressures induced by temperature are consistent across host species and viral strain and have similar impacts on shaping the viral population structure. However, input viral populations are still a driving factor of diversity and expansion during systemic mosquito infection. While our findings and those of others suggest that vector competence is impacted unimodally regardless of temperature, this is only applicable for constant temperatures. Future work assessing daily temperature fluctuation range and mean are needed to have a clear understanding of the role extrinsic incubation temperature plays on vector competence.Item Open Access From Retroviridae to Flaviviridae: adventures in molecular virology(Colorado State University. Libraries, 2021) Butler, Molly, author; Rovnak, Joel, advisor; Quackenbush, Sandra, committee member; Stenglein, Mark, committee member; Moreno, Julie, committee memberThe work presented here encompasses two avenues of investigation: the first, regarding the identification of a novel retrovirus in Gunnison's prairie dogs, and the second regarding the role of cyclin-dependent kinases 8 and 19 (CDK8 and CDK19) as transcriptional regulators during infection with dengue virus serotype 2 (DENV2) and during the innate immune response. Part I: During the course of research and wildlife disease surveillance efforts, we identified three cases of thymic lymphoma in free-ranging Gunnison's prairie dogs (Cynomys gunnisoni). As Gunnison's prairie dogs are keystone species, that is, critical for the maintenance of their ecosystems, we investigated the potential for an association between the observed thymic lymphomas and retroviral infection. We identified a novel retroviral sequence which exhibits genetic organization consistent with a type D betaretrovirus and which was highly associated with thymic lymphoma in Gunnison's prairie dogs. The proposed name of this virus is Gunnison's prairie dog retrovirus (GPDRV). Part II: CDK8 and CDK19 are transcriptional regulators which are critical for modulating gene expression changes during induced states such as hypoxia and starvation. We investigated the role of CDK8 and CDK19 in two distinct but related induced states: infection with DENV2 and the type I interferon response. We found that in the context of DENV2 infection, CDK8/19 regulate metabolic gene expression changes, the result of which is a reshaping of the host cell metabolic environment which is ultimately beneficial to viral replication. Therefore, chemical inhibition or reduced expression of CDK8 or CDK19 significantly restricted viral replication. Both within the context of DENV2 infection and with non-viral stimulation of innate immunity, we identified a role for CDK8 and CDK19 as regulators of the type I interferon response. CDK8 and CDK19 have distinct and overlapping functions as regulators of IFN-β expression dependent on the nature of the stimulus. This work not only furthers our understanding of host transcriptional regulation during DENV2 infection and within innate immunity, but also the diverse and complex functions of CDK8 and CDK19 as key modulators of cellular stress responses.Item Open Access Influence of management practices on virulence factors, antimicrobial resistance genes and heavy metal resistance genes in broiler chicken production(Colorado State University. Libraries, 2023) Woyda, Reed Richard, author; Abdo, Zaid, advisor; Oladeinde, Adelumola, committee member; Daniels, Josh, committee member; Sloan, Dan, committee member; Stenglein, Mark, committee memberThe main bacterial species associated with food-borne illness in humans are Escherichia coli, Salmonella species and Campylobacter species. The ability of a bacterial strain to survive the food-production pipeline and to mount an infection and cause disease in humans is dependent on an array of genetic factors. The presence of specific virulence factors will influence the severity of disease while antimicrobial resistance genes affect the choice and efficacy of treatment. Management practices in poultry production aim at reducing the incidence of poultry and human bacterial pathogens and, in general, at maintaining a healthy flock and a healthy global population. However, the influence of management practices, in a post-antibiotic era, on pathogenic bacterial species, and in particular the selective pressures imposed on genetic factors such as antimicrobial and metal resistance and virulence factors, are understudied. In Chapter 2, we provide a robust bacterial genomic analysis pipeline which is used for subsequent analysis in the following chapters. Chapter 3 provides an understanding of the current antimicrobial resistance and virulence factors present in chicken production and human clinical settings. This work found these host sources harbored different antimicrobial resistance genes and virulence factors that can classify them into phylogroups and host origin. In Chapter 4, through characterization of Campylobacter species isolated from broiler litter, we determined the reused litter environment selected for Campylobacter species lacking virulence factors aiding in colonization of chicken and human hosts. In Chapter 5, we determined the practice of adding copper sulfate to drinking water, commonly used for growth promotion or sanitization, may have selected for, and provided a reservoir for, Salmonella strains harboring plasmid-borne copper resistance genes. Overall, this work provides a computational pipeline for the high-throughput analysis of bacterial genomes and provides insights into selective pressures imposed on pathogenic bacterial species by modern-day management practices.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 Low temperature effects on the transcriptome of Yersinia pestis and its transmissibility by Oropsylla montana fleas(Colorado State University. Libraries, 2016) Williams, Shanna K., author; Borlee, Brad, advisor; Bearden, Scott, advisor; Gage, Ken, committee member; Stenglein, Mark, committee member; Archibeque, Shawn, committee memberYersinia pestis, the causative agent of plague, is primarily a rodent-associated, flea-borne zoonosis. Transmission to humans is mediated most commonly by the flea vector, Oropsylla montana, and occurs predominantly in the Southwestern United States. In these studies, we hypothesized that Y. pestis-infected O. montana fleas held at temperatures as low as 6ºC could serve as reservoirs of the plague bacillus during the winter months in temperate regions with endemic plague foci. With few exceptions, previous studies showed O. montana to be an inefficient vector at transmitting Y. pestis at 22-23°C particularly when such fleas were fed on susceptible hosts more than a few days after ingesting an infectious blood meal. We examined whether holding fleas at sub-ambient temperatures (for purposes of these studies, ambient temperature is defined as 23°C) affected the transmissibility of Y. pestis by this vector. Colony-reared O. montana fleas were given an infectious blood meal containing a virulent Y. pestis strain (CO96-3188), and potentially infected fleas were maintained at different temperatures (6ºC, 10°C, 15°C, or 23ºC). Transmission efficiencies were tested by allowing groups of ~15 infectious fleas to feed on each of seven naïve CD-1 mice on days 1-4, 7, 10, 14, 17, and 21, 28, 35, and 42 post infection (p.i.). Fleas held at 6ºC, 10°C and 15°C were able to effectively transmit at every time point p.i. The percentage of transmission to naïve mice by fleas maintained at low temperatures was higher than for fleas maintained at 23ºC and indicates that O. montana fleas efficiently transmit Y. pestis at low temperatures. Moreover, bacterial loads of flea cohorts maintained at temperatures of 6ºC, 10ºC and 15ºC were statistically higher than fleas maintained at 23ºC. In addition, whole transcriptomes of Y. pestis bacteria grown at 6ºC, 10°C, 15°C and 23ºC were analyzed to assess differential gene expression at each temperature to identify genes which may contribute to an increase in virulence or survivability of the plague pathogen at the lower temperatures when compared to ambient temperature. This is the first comprehensive study to demonstrate efficient transmission of Y. pestis by O. montana fleas maintained at temperatures as low as 6ºC. Our findings further contribute to the understanding of plague ecology in temperate climates by providing support for the hypothesis that Y. pestis is able to overwinter within the flea gut and potentially cause infection during the following transmission season. The findings also might hold implications for explaining the focality of plague in tropical regions where plague occurs in cooler environments, primarily located at higher elevations.Item Open Access Modeling the evolution of SIV progenitor viruses towards HIV-1 and HIV-2 in a humanized mouse surrogate model(Colorado State University. Libraries, 2020) Curlin, James Zachary, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Stenglein, Mark, committee member; Wiese, Claudia, committee memberHuman Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2), the causative agents of Acquired Immunodeficiency Syndrome (AIDS) first emerged in humans over the past century. Despite significant advances in treatment options, the pandemics continue with millions of new infections every year. Both HIV-1 and HIV-2 likely emerged through the cross-species transmission of primate lentiviruses originating from nonhuman primates (NHPs) including chimpanzees (SIVcpz), gorillas (SIVgor), and sooty mangabeys (SIVsm). SIVsm shares a remarkable degree of homology with HIV-2, while SIVcpz and SIVgor are most closely related to HIV-1. Nonhuman primates infected with these lentiviruses frequently come into contact with humans due to the prevalence of bushmeat hunting practices in various African countries. Other lentiviruses such as SIVmac239 represent independent instances of primate lentiviruses crossing into novel host species. The repeated exposure of primate lentiviruses to a human immune environment allowed the accumulation of adaptive genetic changes uniquely suited to overcoming the evolutionary pressures of a new host. Host-restriction factors such as tetherin, SAMHD1, APOBEC3G and SERINC3/5 exert species-specific antiviral activity and must be overcome for a virus to adapt to a new host cell. These evolutionary pressures could be a guiding force in the direction that these viruses adapt. In order to recapitulate these genomic cross-species adaptations, we used humanized mice engrafted with human hematopoietic stem cells (hu-HSC mice). These mice produce a full spectrum of human immune cells such as B cells, T cells, macrophages, monocytes, and dendritic cells, and are susceptible to HIV infection. Representative progenitor viruses of both HIV-1 (SIVcpzEK505, SIVcpzMB897, and SIVcpzLB715) and HIV-2 (SIVsmE041) as well as other viruses of interest, namely, SIVmac239, SIVhu and SIVB670 lineages were intraperitoneally injected into hu-HSC mice. Following successful infections, the derivative viruses were subsequently passaged serially through multiple generations to simulate the repeated exposures that originally produced HIV-1 and HIV-2. Viral adaptation was assessed primarily through three different criteria. Plasma viral RNA levels were measured on a weekly basis using qRT-PCR to determine changes in viral replication kinetics over time. We found that the plasma viral loads of the viruses tested varied during serial passages, and mostly increased over time in many cases. Human CD4+ T cell engraftment decline as assessed by flow cytometry biweekly acts as a measure of AIDS progression in cases of human infection. CD4+ T cell levels declined over time with increasing rapidity upon further passaging in many cases. Additionally, viral RNA collected from the infected mice at multiple timepoints in each generation was used to generate overlapping amplicons spanning the length of the viral genome in order to be used with Illumina-based deep sequencing. Numerous nonsynonymous mutations arose in the first generation of passaging and were maintained across multiple sequential passages. While the mutations occurred throughout the viral genome, the bulk of the mutations were found in env and nef. Many of these mutations were present in known CD4+ binding sites, motifs involved in protein interactions, and other areas involved in host-restriction factor antagonism. While these results are revealing, further inquiry is needed to determine the true functionality of these genetic changes. These data showcase the value of using humanized mice to model lentiviral evolution and provide important insights into understanding the origin of HIVs.Item Open Access Modern considerations for the use of naive Bayes in the supervised classification of genetic sequence data(Colorado State University. Libraries, 2021) Lakin, Steven M., author; Abdo, Zaid, advisor; Rajopadhye, Sanjay, committee member; Stenglein, Mark, committee member; Stewart, Jane, committee memberGenetic sequence classification is the task of assigning a known genetic label to an unknown genetic sequence. Often, this is the first step in genetic sequence analysis and is critical to understanding data produced by molecular techniques like high throughput sequencing. Here, we explore an algorithm called naive Bayes that was historically successful in classifying 16S ribosomal gene sequences for microbiome analysis. We extend the naive Bayes classifier to perform the task of general sequence classification by leveraging advancements in computational parallelism and the statistical distributions that underlie naive Bayes. In Chapter 2, we show that our implementation of naive Bayes, called WarpNL, performs within a margin of error of modern classifiers like Kraken2 and local alignment. We discuss five crucial aspects of genetic sequence classification and show how these areas affect classifier performance: the query data, the reference sequence database, the feature encoding method, the classification algorithm, and access to computational resources. In Chapter 3, we cover the critical computational advancements introduced in WarpNL that make it efficient in a modern computing framework. This includes efficient feature encoding, introduction of a log-odds ratio for comparison of naive Bayes posterior estimates, description of schema for parallel and distributed naive Bayes architectures, and use of machine learning classifiers to perform outgroup sequence classification. Finally in Chapter 4, we explore a variant of the Dirichlet multinomial distribution that underlies the naive Bayes likelihood, called the beta-Liouville multinomial. We show that the beta-Liouville multinomial can be used to enhance classifier performance, and we provide mathematical proofs regarding its convergence during maximum likelihood estimation. Overall, this work explores the naive Bayes algorithm in a modern context and shows that it is competitive for genetic sequence classification.Item Unknown Of bats and bugs: characterizing arboviral transmission at the human-wildlife interface(Colorado State University. Libraries, 2021) Fagre, Anna C., author; Kading, Rebekah C., advisor; Bowen, Richard, committee member; Schountz, Tony, committee member; Stenglein, Mark, committee member; Stoner, Kathryn, committee memberTo view the abstract, please see the full text of the document.Item Unknown SARS-CoV-2 evolution and within-host variation in nonhuman animals(Colorado State University. Libraries, 2024) Bashor, Laura, author; VandeWoude, Sue, advisor; Stenglein, Mark, committee member; Bosco-Lauth, Angela, committee member; Sloan, Dan, committee member; Gagne, Roderick B., committee memberThe COVID-19 pandemic originated following spillover of SARS-CoV-2 from non-human animals into humans. Despite concentrated efforts before and after the pandemic, current research is constrained by the impracticality of witnessing initial host shift events and transmission dynamics that shape infectious disease emergence. SARS-CoV-2 transmission from humans to a range of domestic and wild species has been well documented; furthermore, spillback into humans from white-tailed deer, mink, hamsters, domestic cats, and lions has also been reported. SARS-CoV-2, like other RNA viruses, has the ability to adapt rapidly following host shifts. These cross-species transmission events can accelerate novel variant emergence through selection for genetic variation that improves virus fitness in a novel host environment. To evaluate the possibility that cross-species transmission accelerates SARS-CoV-2 evolution and variant emergence, we employed next-generation sequencing of viral genomes recovered from experimentally and naturally infected animals to characterize within-host virus populations. We demonstrated the use of experimental exposure studies as a controlled system to test hypotheses surrounding SARS-CoV-2 adaptation in cats (Felis catus), dogs (Canis lupus familiaris), hamsters (Mesocricetus auratus), ferrets (Mustela putorius furo), deer mice (Peromyscus maniculatus), bushy-tailed woodrats (Neotoma cinerea), Brazilian free-tailed bats (Tadarida brasiliensis), striped skunks (Mephitis mephitis), red foxes (Vulpes vulpes) and mule deer (Odocoileus hemionus). We also evaluated publicly available sequencing data from infected felids, and investigated within-host dynamics in natural infections of Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) in a zoo environment. Our initial work investigated SARS-CoV-2 evolution across three passages in Vero cells and experimentally infected cats (n = 6), dogs (n = 3), hamsters (n = 3), and a ferret (n = 1). We observed the rapid selection and fixation of five SARS-CoV-2 mutations in Vero cells, followed by their reversion in dogs, cats and hamsters 1-3 days post-infection. We noted 14 emergent variants across the SARS-CoV-2 genome, including increased variation in the SARS-CoV-2 spike protein. Emergent variants included mutations not detected in the original virus stocks used for inoculation, and several defining mutations of variant lineages of concern in humans. Finally, we noted increased signs of adaptation in dogs, which did not shed infectious virus, including six nonsynonymous mutations in the SARS-CoV-2 open-reading frames (ORFs) encoding proteins for virus replication. In particular, this work underscored the potential for accelerated viral evolution in cell culture systems used commonly in virological research. This work has been published and represents Chapter 2 of this dissertation. Our next study built upon this work by investigating SARS-CoV-2 evolution in three experimental cohorts of domestic cats (n=23) infected through direct inoculation and cat-to-cat contact transmission. We observed high numbers of within-host variants in SARS-CoV-2 genomes recovered from cats compared to what is documented in humans, over half of which were nonsynonymous changes. The number of variants detected was positively correlated with the experimental dose of virus inoculum, and fewer variants were observed in contact cats. Similar to the previous study, mutations occurring at the same positions as defining VOC mutations, and signatures of positive selection in the viral spike (S) gene were observed. Our concurrent analysis of publicly available SARS-CoV-2 sequences showed no evidence for independent evolutionary trajectories associated with natural infections of domestic cats or other felids, and confirmed susceptibility of felids to the breadth of variants circulating in human populations. This work has also been published and represents Chapter 3 of this dissertation. We subsequently investigated SARS-CoV-2 evolution in longitudinal samples collected from Amur tigers (n=2), African lions (n=11), and spotted hyenas (n=4) infected during an outbreak at the Denver Zoo. Longitudinal nasal swabs were collected from infected individuals over an approximately three-month sampling period. We determined that the outbreak was caused by a single introduction of the Delta sublineage AY.20, which was a rare variant circulating in human populations at the time. We inferred a transmission chain from tigers to lions to hyenas, which was consistent with the appearance of clinical signs in infected animals. We observed expansion and diversification of within-host virus populations, and signatures of both purifying and positive selection. The strongest signs of positive selection were evident in the viral nucleocapsid (N) gene, and in viruses recovered from hyenas. Four candidate species-specific adaptive mutations, two of which are in the N gene, were identified in lions and hyenas (N A254V) and hyenas alone (ORF1ab E1724D, S T274I, and N P326). This work is presented in Chapter 4 of this dissertation. In Chapter 5, we evaluated a large dataset of peridomestic wildlife species experimentally infected with two SARS-CoV-2 variants, WA01 and Delta. Study species included deer mice (n=3), bushy-tailed woodrats (n=3), Brazilian free-tailed bats (n=4), striped skunks (n=5), red foxes (n=9), and mule deer (n=6). Distinct dynamics were observed in within-host virus populations recovered from WA01- and Delta- infected animals. This included increased within-host variation, relative effective population size, and genomic signatures of positive selection in WA01 animals. In contrast to our first study in domestic dogs, Brazilian free-tailed bats, which also did not shed infectious virus, did not show increased signs of adaptation. We also observed a potential host barrier to infection in skunks and one fox, followed by the emergence of potential de novo mutations. Six novel mutations were also detected in contact-exposed mule deer. Our findings suggest that mule deer populations, similar to what has been documented in closely related white-tailed deer, should be investigated for accelerated SARS-CoV-2 evolution. Collectively, our work reveals the unique dynamics of SARS-CoV-2 evolution and transmission in both naturally- and experimentally- infected felids. We observed rapid viral adaptation both in vitro and in vivo, highlighting advantages and limitations of experimental animal infections for studies of viral evolution. In each study, we used publicly available data to contextualize our experimental data and identify broader patterns. Furthermore, we identified specific SARS-CoV-2 mutations and genomic regions under selective pressures across a range of animal species, setting the groundwork for future mechanistic studies. Our findings underscore the importance of a One Health approach to understanding SARS-CoV-2 evolution, and the need for surveillance in animal populations.Item Open Access The effects of genome expansion on transposable element diversity in salamanders(Colorado State University. Libraries, 2021) Haley, Ava, author; Mueller, Rachel, advisor; Sloan, Daniel, committee member; Stenglein, Mark, committee memberTransposable elements (TEs) are repetitive sequences of DNA that replicate and proliferate throughout genomes. Taken together, all the TEs in a genome form a diverse community of sequences, which can be studied to draw conclusions about genome evolution. TE diversity can be measured using ecological models for species distribution that consider richness and evenness of communities. It is currently not well studied how genome expansion impacts the diversity of transposable elements. However, there are a few models that predict TE diversity decreasing as genomes expand due to varying mechanisms such as selection against ectopic recombination and competition between TEs and silencing machinery. Salamanders are known to have some of the largest vertebrate genomes. Salamanders of the genus Plethodon in particular have very large genomes consisting of high levels of TEs, with sizes ranging from 30 to 70 Gigabases (Gb). Here, I use Oxford Nanopore sequencing to generate low-coverage genomic sequences for four species of Plethodon that encompass two independent genome expansion events, one in the eastern clade and one in the western clade: Plethodon glutinosus (41.4 Gb), P. cinereus (30.5 Gb), P. idahoensis (71.7 Gb), and P. vehiculum (50.5 Gb). I classified the TEs in these datasets using RepeatMasker and DnaPipeTE and found ~51 superfamilies which accounted for 27-32% of the genomes. For each genome I calculated the Simpson's and Shannon's diversity indices to quantify diversity, taking into account both TE richness and evenness. In all cases, the values for Simpson's index were within 0.75 and 0.79, and for Shannon's index all species were within 1.88 and 1.99. We conclude that once genomes reach large sizes, they maintain high levels of TE diversity at the superfamily level, in contrast to observations made by previous studies done on smaller genomes.Item Open Access The plastid caseinolytic protease complex as a model for cytonuclear coevolution(Colorado State University. Libraries, 2021) Williams, Alissa Marie, author; Sloan, Daniel, advisor; Bedinger, Patricia, committee member; Mueller, Rachel, committee member; Pilon, Marinus, committee member; Stenglein, Mark, committee memberCoevolution, or evolution in response to reciprocal selective pressures, is important to biological function and the persistence of populations. Competition or mutualisms between organisms can drive coevolution, as can predatory or parasitic relationships. However, coevolution also occurs within cells, as coevolution can result from the interactions between proteins within complexes as well as between the multiple genomes within eukaryotic cells. In protein complexes, subunits must bind tightly and specifically to one another. Changes in one protein subunit are often correlated with changes in the other subunits to preserve the functionality of the complex. Thus, in many protein complexes, correlated rates of evolution are found between the sequences of component subunits. This covariation is strong enough to be used as a method to predict which proteins are connected physically and/or functionally. The coevolution between multiple genomes in eukaryotic cells is known as cytonuclear coevolution. Plants, for example, have a nuclear genome and two cytoplasmic genomes found in the plastid (chloroplast) and mitochondrion. Many protein complexes within these organelles consist of subunits deriving from both the nucleus and the organelle itself. Since the nuclear genome and organelle genomes differ in modes of transmission, mutation rates, and selective pressures, partnerships between proteins originating from two cellular compartments are great models for understanding protein complex evolution. Protein complexes are frequently shaped via gene duplication. Many protein complexes contain paralogous proteins at their cores; the duplication of a self-binding protein leads to dimerization of the paralogous proteins and subsequent recruitment of additional subunits. Gene duplication after establishment of a heteromeric complex allows subunits to specialize. The plastid caseinolytic protease (Clp) complex provides a model system for studying protein complex evolution, in the context of cytonuclear interactions, gene duplication, and evolutionary rate variation. This complex is highly conserved across bacteria and consists of adaptors, chaperones, and a proteolytic core. It is present in both plastids and mitochondria because these organelles are derived from ancient bacterial endosymbionts. The Clp core contains 14 subunits; in mitochondria and most bacteria, all 14 subunits are encoded by the same gene. However, in the cyanobacterial and plastid lineage, multiple rounds of gene duplication have led to a core encoded by nine different genes in the model plant species Arabidopsis thaliana. Further, only one of these plastid Clp core subunit genes is encoded by the plastid itself—the remaining eight are encoded by the nucleus, the result of gene transfers from the organelle to the nucleus early in the history of green plants. In addition to representing multiple rounds of gene duplication, the plastid Clp core also demonstrates extreme rate variation across green plants. The plastid-encoded subunit (ClpP1) is typically highly conserved across species. However, in some species, ClpP1 is one of the most rapidly evolving genes across all three genomes. In this dissertation, I use these features of the plastid Clp complex to shed light on protein complex evolution in various contexts. After a general introduction to the field in Chapter 1, Chapter 2 focuses on the evolutionary history of ClpP1, looking at rate variation and the loss of introns, RNA editing sites, and catalytic sites across green plants. Through mass spectrometry, I determine that ClpP1 is still a functional protein in Silene noctiflora, which has one of the most divergent plastid Clp complexes known. This work also includes an evolutionary rate covariation analysis between ClpP1 and the nuclear-encoded Clp core genes. Chapter 3 provides genomic resources, including a high-quality, long-read transcriptome, for S. noctiflora, which is a species of interest for the reason outlined above. Analysis of the transcriptome revealed a triplication of one of the nuclear-encoded Clp core genes in this species. Chapter 4 discusses the recent duplication history of the nuclear-encoded Clp core genes across a broad range of flowering plants. I use these data to examine and characterize post-duplication evolutionary fates of paralogs. These analyses are extended to another plastid complex, acetyl-CoA carboxylase (ACCase). Taken together, these chapters elucidate various features of plastid Clp complex evolution as well as provide insight into the possible causes and consequences of rate variation and gene duplication in the coevolution of protein complex subunits.Item Open Access Uncovering mechanisms driving variation in mutation rates in organellar and nuclear genomes(Colorado State University. Libraries, 2024) Waneka, Gus, author; Sloan, Daniel B., advisor; Lockridge Mueller, Rachel, committee member; Argueso, Juan Lucas, committee member; Stenglein, Mark, committee memberMutations are changes to DNA sequences which drive evolution by supplying raw genetic variation for natural selection to act upon. At the same time, mutations tend to have negative fitness consequences and are the source of genetic diseases. Such costs and benefits of mutation create opposing forces of selection on mutation rate modifiers, which are alleles (typically in DNA repair genes) responsible for increases or decreases to mutation rates. Essentially all eukaryotes possess at least two genomes: the nuclear genome (nucDNA) and the endosymbiotically derived mitochondrial genome (mtDNA). Photosynthetic plants and algae additionally possess endosymbiotically derived plastid genomes (cpDNAs). Together, the mtDNA and cpDNA are referred to as organellar genomes. Chapter 1 of this dissertation provides a framework for understanding how DNA repair machinery and mutation rates have evolved in complex eukaryotic cells. Chapters 2 and 3 focus on specific repair pathways active in organellar genomes. Finally, Chapter 4 shifts focus to understand how environmental perturbations in expression level impact mutation in plant nuclear genomes. Repair of organellar genomes is conducted by nuclear-encoded genes that are translated in the cytosol and targeted to the organelles. In terms of evolutionary history, organellar repair machinery is a mosaic network of bacterial-like repair genes, which came into the cell with the organelles, and nuclear repair genes that have been co-opted for organellar function. In some cases, repair proteins are targeted to both the nucDNA and mtDNA (and/or cpDNA in plants) to perform similar functions. This is the case as for many base excision repair (BER) proteins, which identify and remove of chemically damaged bases. In contrast, organellar repair arsenals are thought to lack canonical mismatch-repair (MMR) and nucleotide excision repair (NER), which are both important repair pathways in nuclear genomes. Instead, the diverse eukaryotic lineages have adopted unique strategies for organellar genome maintenance. As a result, there is a tremendous diversity in mtDNA mutation rates, which show over a 4000-fold variation across eukaryotes. Interestingly, much of this variation is driven by the extremely low point mutation rates plant mtDNAs. In addition, plant organellar genomes are more recombinationally active and plant mtDNAs are structurally unstable compared to the mtDNAs of other eukaryotes. Chapter 2 of this dissertation explores the mechanistic basis of low point mutation rates and recombination-mediated repair in plant organellar genomes. We performed high-fidelity Duplex Sequencing on a panel of Arabidopsis thaliana lines lacking specific organellar genome repair genes. We report large point mutation increases in mutant lines lacking MSH1, a mutS homolog that has been proposed to induce double-stranded breaks at the site of DNA mismatches, effectively shuttling such lesions into homologous recombination (HR) pathways that play important roles in plant organellar genome replication and repair. We see smaller point mutation increases in other mutant lines lacking RADA, RECA1 and RECA3. In addition, we generated long-read Oxford Nanopore sequencing to characterize repeat-mediated recombination in several of the mutants in the panel. Our findings provide valuable insights into the mechanisms driving the fascinating patterns of organellar genome evolution in land plants. The aforementioned lack of NER in organellar genomes is surprising given the importance of NER as a 'catchall' for repair of a variety of bulky DNA lesions in nuclear genomes. Chapter 3 focuses in on the fate of photodamage (an important type of bulky DNA damage) in organellar DNA. To do so, we leverage publicly available XR-seq datasets, which were generated to quantify and map active NER excision products in nuclear genomes following UV exposure. The taxonomic scope of chapter is expanded from plants to also include fungi (the brewer's yeast Saccharomyces cerevisiae) and animals (the model fruit fly Drosophila melanogaster). We find that mtDNA-derived XR-seq reads in A. thaliana and S. cerevisiae have distinct and repeatable patterns in terms of length and internal positioning of pyrimidine dimers (known targets of photodamage formation). These data mirror established patterns of NER-derived reads originating from the nuclear genomes, raising the exciting possibility that NER-like repair pathways may exist in for repair of photodamage in organellar genomes. The focus of chapter 4 shifts to understanding how environmental changes impact mutation in plant nuclear genomes. The textbook view of mutation and adaptation is that mutations occur randomly with respect to their environment-specific fitness consequences. However, this view of random mutation is challenged as evidence increasingly establishes a correlation between increased expression and decreased mutation via the coordination of transcription and DNA repair machinery at the molecular level. As a result of this correlation, intragenomic mutation rates likely vary with changing environments given that expression levels are environmentally labile. Therefore, certain genes may be predisposed to higher or lower mutation rates depending on the environment, though the magnitude and importance of this effect remains largely untested. A technical challenge in addressing these questions is that large scale plant mutation datasets are time and resource intensive to generate. A recent plant study relied on low frequency somatic calls from Illumina based shotgun libraries to generate a large number of mutations, but others report that most of these inferred mutations are sequencing errors. To overcome these challenges, we took a novel approach to measuring somatic mutations by using Duplex Sequencing to quantify mutations in targeted regions of the A. thaliana nucDNA. We identified a set of differentially expressed genes in plants grown at different temperatures, which we then targeted for mutation detection using hybrid capture. In addition to wild type (WT) lines, we also studied mutant lines deficient in BER and MMR to test if either of these pathways are responsible for the correlation between expression and mutation in plants. We found large point mutation increases in the MMR mutants compared to WT plants, which displayed surprisingly few mutations at either temperature. Though the small number of WT mutations precluded a meaningful comparison of expression and mutation in a WT background, this result if nonetheless valuable for establishing that the true frequency of somatic mutations in plants is indeed very low suggesting that previous estimates likely conflated Illumina based sequencing artifacts with mutations. Mutation rates vary by over three orders of magnitude across the tree of life. Much of this variation is captured in mitochondrial mutation rates. The chapters of this dissertation provide valuable insights into the molecular processes that drive mutation rate variation in eukaryotic genomes. Such mechanistic understandings are critical for advancing the broader field of mutation rate evolution.