Browsing by Author "VandeWoude, Sue, advisor"
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Item Open Access Cytokines, antibodies and plasma viremia of cats infected with feline immunodeficiency virus(Colorado State University. Libraries, 2013) Wood, Britta Ann, author; VandeWoude, Sue, advisor; Avery, Paul, committee member; Zabel, Mark, committee member; Hussey, Gisela, committee memberFeline immunodeficiency viruses (FIVs) are naturally occurring lentiviruses (family Retroviridae) of felid species, including domestic and wild cats. Studies on FIVs are beneficial for understanding the host immune response associated with disease progression (e.g., domestic cat FIV) or the viral kinetics and molecular ecology associated with naturally occurring infections in wildlife (e.g., bobcat and mountain lion FIVs). Here we describe the development and validation of the following microsphere immunoassays (MIAs) for evaluating the cytokine and antibody response of domestic cats: i) the quantification of cytokines (interferon gamma (IFNγ), interleukin (IL)-10, and IL-12/IL-23) in cell culture supernatant, and ii) the quantification of these cytokines in plasma; iii) the quantification of total IgG and IgA in plasma, and iv) the detection of IgG and IgA antibodies to feline CD134 (the primary cell receptor for FIV), and FIV capsid (CA) and surface (SU) proteins in plasma. These assays were used to evaluate temporal cytokine and antibody responses of domestic cats experimentally infected with various FIV strains. To analyze viral RNA loads associated with naturally occurring FIV infections in bobcats or mountain lions, we are adapting existing quantitative PCR assays for use with plasma samples. The eight assays described here are/will be beneficial for addressing questions related to lentiviral immune response and viral kinetics.Item Open Access Determining the cancer risks presented by space radiation: genomic mapping in outbred mice reveals overlap in genetic susceptibility for HZE ion and γ-ray induced tumors(Colorado State University. Libraries, 2016) Edmondson, Elijah F., author; VandeWoude, Sue, advisor; Weil, Michael, advisor; Thamm, Douglas, committee member; Olver, Christine, committee member; Kamstock, Debra, committee memberTo view the abstract, please see the full text of the document.Item Open Access Evaluation of parathyroid hormone and zoledronic acid in promoting bone healing after stereotactic radiation therapy for local control of osteosarcoma in an orthotopic rat model(Colorado State University. Libraries, 2014) Curtis, Ryan C., author; VandeWoude, Sue, advisor; Donahue, Seth, advisor; Custis, Jamie, committee member; Ehrhart, Nicole, committee member; Ehrhart, EJ, committee memberClinical studies using definitive-intent stereotactic radiation therapy (SRT) for the local treatment of canine osteosarcoma (OSA) have achieved similar median survival times in patients as the current standard of care (amputation and adjuvant chemotherapy). Despite this, there remains an unacceptably high risk of pathologic fracture following radiation treatment. Zoledronic acid (ZA) and parathyroid hormone (PTH) are therapeutic candidates for decreasing this fracture risk post-irradiation. Due to differing mechanisms, we hypothesized that the combined treatment with ZA and PTH would significantly improve bone healing more than ZA or PTH treatment alone. Using an orthotopic model of canine osteosarcoma in athymic rats, we evaluated bone healing following clinically-relevant doses of radiation therapy (12Gy x 3 fractions, 36 Gy total). Groups included 36 Gy SRT only, 36 Gy SRT plus ZA , 36 Gy SRT plus ZA and PTH, 36 Gy SRT plus PTH, and 36 Gy SRT plus localized PTH treatment. Our study showed significant increases in bone volume and polar moments of inertia within the region of interest (distal femoral metaphysis) 8 weeks after radiation in the combined (ZA/PTH) treatment group as compared to radiation treatment alone. Histomorphometric analysis revealed evidence of active mineralization at study endpoint as well as successful tumor-cell kill across all treatment groups. This work provides further evidence for the expanding potential indications for ZA and PTH therapy, including post-irradiated bone disease due to canine osteosarcoma.Item Open Access Extrinsic and intrinsic drivers of feline immunodeficiency virus evolution in the mountain lion(Colorado State University. Libraries, 2018) Malmberg, Jennifer L., author; VandeWoude, Sue, advisor; Crooks, Kevin, committee member; Quackenbush, Sandra, committee member; Perera, Rushika, committee memberViruses are among the most rapidly evolving entities in biology and are so intricately associated with their obligate hosts that the boundary between host and pathogen, and thus the study of one versus the other, is blurred by intimate interactions at scales ranging from proteins to populations. Viral genetic variation is both ecologically and molecularly determined, and thus viruses serve as measurably evolving populations that provide a window into adaptations and behaviors of their vertebrate hosts. Of all viral families, the biology of retroviruses is coupled especially tightly to that of the host due to permanent integration of viral DNA into eukaryotic chromosomes, producing an inherently dynamic infection that persists for life. Feline immunodeficiency virus (FIV) is among the oldest of viruses in the Lentivirus genus and puma (Puma concolor) are the most extensively ranging New World terrestrial mammal. We used molecular analyses to investigate the host-pathogen interactions between pumas and FIV across geographic and temporal space, within and across populations, and among FIV subtypes. In Chapter One, we investigate cross-species transmission of FIV from bobcats to pumas and compare the outcome of spillover infections in two populations separated by vast geographic space. Our findings reveal that the puma is typically a dead-end host of bobcat FIV infection, although altered population dynamics can promote stuttering chains of infection following spillover events. In Chapter Two, we employed a novel next generation sequencing technique to investigate the impact of management interactions such as population supplementation on FIV dynamics in the endangered Florida panther. Results from this chapter show evidence for cointroduction of one subtype of FIV with translocated pumas from Texas, followed by local extinction of the previously circulating, 'less fit' subtype in the puma host. Chapter Three describes an important intrinsic driver of viral evolution through characterization of the APOBEC3 protein A3Z3 in the puma, a primary cellular restriction factor against FIV. We show evidence that at least one geographically associated genotype of puma FIV is able to evade lethal hypermutation typical of A3Z3 activity despite a deficiency in the viral counter protein Vif. The collective findings of this work explore the ancient relationship between a vastly ranging apex predator and a chronic lentiviral infection by applying both novel and conventional methodologies to a unique, naturally occurring host-pathogen system. Although our questions were specific to FIV in pumas, the methodologies described here can be applied to other systems and models to address inherent limitations of opportunistic field studies including DNA degradation and sequencing of low copy number templates from archival biological samples. Ancient viral infections have the potential to elucidate the life history of mammalian hosts, which is particularly useful in the study of elusive and broadly ranging carnivores threatened by urbanization and habitat fragmentation. Future objectives of this work will expand analyses to incorporate additional populations, such as the modern Texas puma, and more thoroughly investigate genotype variation in Vif-A3Z3 interactions. Collectively, our results will inform additional studies that seek to elucidate determinants of host-pathogen interactions in naturally-occurring systems across diverse ecosystems and broad spatiotemporal scales.Item Open Access Feline foamy virus in domestic cats: use as a vaccine vector, characterization of infection, and association with other diseases(Colorado State University. Libraries, 2019) Ledesma-Feliciano, Carmen Denise, author; VandeWoude, Sue, advisor; Quimby, Jessica, committee member; Schountz, Tony, committee member; Basaraba, Randall, committee member; Frye, Melinda, committee memberFoamy viruses (FVs) are retroviruses from the Spumaretrovirinae subfamily. FVs are globally prevalent retroviruses with a unique molecular biology. FVs establish apparently apathogenic lifelong infections. Due to this, FVs are considered attractive vectors for vaccine and gene therapy development. Feline foamy virus (FFV) infects domestic cats and has widespread and high prevalence around the world. However, FFV has also been isolated from cats suffering from concurrent disease, including renal syndromes and other retroviral co- infections such as feline immunodeficiency virus (FIV). Much remains unknown about FFV infection and in vivo experimental infections are rare in the literature. To test FFV's use as a vaccine vector and understand the interaction between viral proteins and host antiviral restriction factors, we developed an infective chimeric vaccine containing lentiviral FIV vif replacing FFV bet. FFV Bet and FIV Vif counteract feline innate APOBEC3 (feA3) restriction factors through different mechanisms. FeA3 action on retroviral genomes lead to hypermutation and degradation of viral DNA. In vitro, we show that vif can replace bet to yield replication-competent chimeric viruses. We experimentally inoculated 12 domestic cats (n=4 per group in naïve, wild-type, and chimera-inoculated groups) with the FFV-Vif chimera and wild- type FFV in order to compare viral replication kinetics through PCR and specific antibody development through ELISA. Inoculation with the chimeric vector resulted in the development of a specific immune response against FFV Gag and Bet and FIV Vif proteins. In addition, we show that the domestic cat can be superinfected with different strains of FFV. The chimeric virus displayed attenuated infection in vivo, as provirus was not detected in PBMC for any chimera-only inoculated animals. Thus, Bet may have additional functions other than A3 antagonism required for successful in vivo infection. Our studies further exemplify how FV vaccine vectors are an attractive tool to counteract lentiviral infections and poses the possibility to induce immunity against other lentiviral antigens. In order to further characterize wild-type infection, we also collected blood, saliva, and urine over a 6-month time-period with a necropsy and tissue collection at the end of the study. Animals were monitored daily for clinical signs of disease and temperature and weight data were collected weekly. None of the cats showed clinical signs of infection and complete blood count and chemistry were unremarkable. However, we found significant differences in blood urea nitrogen, one of the markers used to assess renal function, when comparing infected versus control animals. All animals inoculated with wild-type virus showed a persistent proviremia (in PBMCs) and viral tissue tropism was primarily lymphoid with the exception of one cat that had an expanded tissue tropism to other lymphoid tissues and oral mucosa. This animal had altered viral kinetics compared to the rest of infected animals, in addition to a negative correlation between lymphocyte count and viral load. Histopathological analysis showed evidence of microscopic pathology in the kidneys, lung, and brain of infected animals. This same cat had an increase in urine protein at the time of highest PBMC proviremia. Additionally, transmission electron microscopy showed ultrastructural changes indicative of transient renal injury in the kidneys of infected animals. We additionally found electron dense structures in the cytoplasm of tubular epithelial of as of yet unknown origin. Due to the renal changes we saw in the experimental study and pathology reported in the literature, we conducted a survey of FFV in pet cats in the USA and Australia (AU) suffering from chronic kidney disease (CKD) and compared findings to age- and sex-matched controls without CKD. We found an association between CKD and FFV in males, and males in general are also at a significantly increased risk of FFV infection. We then assessed through an FFV serosurvey whether FFV was associated with FIV and causing potentiation of FIV disease in two cohorts of naturally FIV-infected cats. One of the groups consisted of cats living in 1-2 cat households that did not experience much FIV-related morbidity and mortality, while the second group of cats housed in a large multicat household suffered from severe clinical symptoms and mortality. We hypothesized the reason for this discrepancy could be an increase in FFV/FIV co-infection rate in the group of cats with higher morbidity and mortality. We found that FFV is associated with FIV in these groups and that males are also at an increased risk for FFV infection. Finally, we conducted an in vitro co- infection study to assess potentiated infection as determined by more rapid development of cytopathic effects (CPE) and higher viral titers in the supernatant. GFox cells were inoculated with FFV and FIV in single, and dual simultaneous and staggered inoculations. A p26 ELISA was used to determine amount of FIV reactivity in the cells, while a chemiluminescent β- galactosidase assay was used to detect amount of β -gal produced in FeFAB FFV reporter cells. The in vitro assays showed increased permissivity of either virus following an initial infection of the other virus, showing these two retroviruses can accelerate and potentiate a secondary infection regardless of which virus infected initially. Overall, we have demonstrated the suitability of FFV as a vaccine vector candidate. Additionally, we have documented that FFV may cause subclinical alterations that in certain cohorts of domestic cats, may contribute to disease development in chronic cases. Finally, we showed that FFV interacts with another retrovirus and could potentially affect FIV-related disease. More studies should focus on the effects of FFV in chronic infections in addition to the effect of FFV on co-morbidities in a chronic timeline.Item Open Access Host cell antigen and T-lymphocyte subset contribution to simian immunodeficiency virus pathogenicity(Colorado State University. Libraries, 2008) Stump, Debora Shawn, author; VandeWoude, Sue, advisorThe continuity of the host cell plasma membrane and the simian immunodeficiency virus (SIV) envelope at the time of budding results in the incorporation of host membrane antigens. Of these host antigens, major histocompatibility complex class II (MHCII), is abundantly represented on the virion surface. In Chapter 1, the investigation the potential of antibodies specific for MHCII to block viral infection by binding viral envelope MHCII in vitro is presented. Our results did not demonstrate viral neutralization associated with anti-MHCII antibodies but illustrate that viral infectivity is influenced by target cell membrane and immunological signaling characteristics. In Chapter 2 we investigated the utility of alloimmunization of genetically divergent rhesus macaques in eliciting immune responses specific for host cell antigens capable of limiting SIV infectivity in vivo. Our results suggest that alloimmune responses are not sufficient to protect animals from SIV challenge. We were also able to assess differences in response to pathogenic SIV infection in rhesus macaques of Chinese origin (ChRh) compared to Indian origin (InRh) in Chapter 3. ChRh in our study were better able to control viral replication and resist disease progression compared to InRh. Peripheral immunocyte kinetics were evaluated using four color flow cytometry in order to define parameters of the differential immune response. No consistent differences were evident, demonstrating that peripheral immune correlates of viral control and disease progression remain unknown. Natural SIV infection has been identified exclusively in primate species inhabiting continental Africa. Serological evidence of exogenous lentiviral infection has been noted in wild lemurs in Madagascar. In Chapter 4, we investigated evidence of a naturally occurring lentivirus, possibly related to African SIVs, in samples from a captive population of L. catta at the Indianapolis Zoo. We show confirmatory serological reactivity to diverse lentiviral antigens but failed to amplify lentiviral specific sequences using established degenerate primer sets. In total, this work represents investigations that interrogate important aspects of nonhuman primate lentiviral pathogenicity. While results were primarily negative in nature, these studies provide important new information and point to additional studies required that will continue investigations into the complex nature of lentiviral host: virus relationships.Item Open Access Novel approaches to characterizing feline-associated dermatophytic fungi(Colorado State University. Libraries, 2022) Moskaluk, Alexandra Elizabeth, author; VandeWoude, Sue, advisor; Daniels, Josh, committee member; Schissler, Jennifer, committee member; Reynolds, Melissa, committee memberDermatophytes are highly infectious fungi that cause superficial infections in keratinized tissues in humans and animals. This group of fungi is defined by their ability to digest keratin and encompasses a wide range of species. Classification of many of these species has recently changed due to genetic analysis, potentially affecting clinical diagnosis and disease management. In Chapter One, we review dermatophyte classification including name changes for medically important species, current and potential diagnostic techniques for detecting dermatophytes, and an in-depth review of Microsporum canis, a prevalent zoonotic dermatophyte. M. canis commonly causes dermatophytosis in humans and cats, and is adapting to its primary host (domestic cats) as one of its mating types (MAT1-2) appears to be going extinct. Assessment of genetic variation among M. canis isolates in the United States has not been conducted. Further, M. canis mating type and assessment of disease severity associated with genotypic characteristics have not been rigorously evaluated. In Chapter Two, M. canis was isolated from 191 domestic cats across the US and characterized genotypes by evaluation of ITS sequence, MAT locus, and microsatellite loci analysis. The genes SSU1 and SUB3, which are associated with keratin adhesion and digestion, were sequenced from a subset of isolates to evaluate potential genetic associations with virulence. Analysis of microsatellite makers revealed three M. canis genetic clusters. Both clinic location and disease severity were significant predictors of microsatellite variants. 100% of the M. canis isolates were MAT1-1 mating gene type, indicating that MAT1-2 is very rare or extinct in the US and that asexual reproduction is the dominant form of replication. No genetic variation at SSU1 and SUB3 was observed. These findings pave the way for novel testing modalities for M. canis and provide insights about transmission and ecology of this ubiquitous and relatively uncharacterized agent. Chapter Three evaluated four dermatophytosis cases occurring in kittens collected from the study in Chapter Two that yielded fungi with colony morphology more similar to Arthroderma species than Microsporum. Morphologic and microscopic examinations were conducted, and gene segments for the ITS, β-tubulin, and translation elongation factor 1-alpha (TEF1) regions were sequenced from DNA extracted from these cultures. Sequences were aligned to other dermatophytes using maximum likelihood and neighbor-joining trees and were compared to previously described fungal species to assess nucleotide homology. We identified two previously undescribed fungal species, herein as Arthroderma lilyanum sp. nov. and Arthroderma mcgillisianum sp. nov. M. canis co-cultured in two of the four cases. Other physiologic tests supported this diagnosis. These species have significance as potential pathogens and should be considered as rule-outs for dermatophytosis in cats. The potential for infection of other species, including humans, should be considered. In Chapter Four, we investigated a critical adhesion protein (Sub3) utilized by M. canis during initial stages of infection, analyzing its production and expression under varying growth conditions. Additionally, as this protein must be expressed and produced for dermatophyte infections to occur, we developed and optimized a diagnostic antibody assay targeting this protein. While clinical samples of M. canis were found to have low Sub3 production, Sub3 levels were increased in culture when grown in baffled flasks and supplemented with either L-cysteine or cat hair. As Sub3 was also produced in cultures not supplemented with keratin or cysteine, this study demonstrated that Sub3 expression is not reliant on the present of keratin or its derivatives. These findings could help direct future metabolic studies of dermatophytes, particularly during the adherence phase of infections. Chapter Five explored two molecular approaches for developing diagnostic assays for dermatophytosis based on keratin metabolites: sulfite and S-sulfocysteine (SSC). Currently, fungal culture is still considered the "gold standard" for diagnosing dermatophytosis, however, modern molecular assays have overcome the main disadvantages of culture, allowing for tandem use with cultures. The first approach involved a starch and iodine indicator that reacts with sulfite and SSC, resulting in a visual color change. While this method had a low limit of detection, the indicator had many off-target reactions, leading to low specificity for dermatophyte metabolites. The second approach utilized tandem liquid chromatography with mass spectrometry, targeting SSC. Using the same cultures performed in Chapter Four, we were able to detect and quantify SSC from M. canis cultures grown with hair at days 15 and 18 post inoculation. These findings demonstrated that SSC is consumed/degraded by the fungi, particularly during early growth stages. Collectively, this work provides future directions for genetic and metabolic studies of dermatophytes and how to leverage unique characteristics of dermatophytes for developing novel diagnostic assays. We conclude that M. canis genetics influence clinical disease presentation and further whole genome studies could help elucidate key genetic regions involved in dermatophyte pathogenesis. Furthermore, as M. canis continues to adapt to its primary host of cats, having a rapid, accurate diagnostic assay will become even more critical, particularly in high-density populations.Item Open Access Role of endogenous retrovirus in control of feline leukemia virus infection and implications for cross species transmission(Colorado State University. Libraries, 2019) Chiu, Elliott S., author; VandeWoude, Sue, advisor; Hoover, Edward A., committee member; Ebel, Gregory D., committee member; Funk, W. Chris, committee memberEndogenous retroviruses (ERV) are markers of ancient retroviral infections, though evolutionary forces have limited the capacity for ERV replication and virulence. While they are seldom considered infectious alone, they maintain the ability to interact with their exogenous retroviral (XRV) progenitors. In Chapter One, we review the interactions that exist between ERV and XRV dyads. One such couplet includes feline leukemia virus (FeLV), a common domestic cat pathogen. In Chapter Two, we review FeLV subgroup taxonomy and the methods used from which they were originally characterized. Though the domestic cat is regarded as the natural host for the virus, recent reports have documented FeLV infections in wild felids with pathogenic consequences. Chapter Three examines the root of a contemporary FeLV outbreak in Florida panthers (Puma concolor coryi), a species that lacks endogenous FeLV. Our phylogenetic analysis of the contemporary FeLV outbreak has further implicated domestic cats (Felis catus) as the origin of FeLV infections in wild felids. Furthermore, we detected a recombinant oncogenic variant in Florida panthers that is believed to be non-horizontally transmissible. These field studies have prompted us to examine the cellular basis of infection and intrinsic resistance to the virus. In Chapter Four, we interrogate the cellular basis of FeLV infections between puma (P. concolor) and domestic cat cells using in vitro approaches. We demonstrated that puma cells support greater infection and replication. Additionally, we documented enFeLV long terminal repeats (LTR) in domestic cats are negatively correlated to FeLV infection outcomes in vitro. Natural FeLV infections in both Florida panther and domestic cat tissues offered us the opportunity to examine end stage disease dynamics, which demonstrate that Florida panthers have the ability to produce more virus despite having lower proviral loads than domestic cats. The results of both in vivo and in vitro experiments prompted us to further investigate enFeLV-LTRs and their role in FeLV infection. Chapter Five took advantage of the publicly available data in the NCBI Sequence Read Archive (SRA) to evaluate enFeLV-LTR basal transcription levels. Data-mining the domestic cat transcriptome showed that lymphoid cells, which are relatively resistant to in vitro FeLV infection, transcribe more enFeLV elements than relatively susceptible cells (i.e., fibroblasts). We also identified microRNA transcripts are produced that have the potential ability to down-regulate FeLV RNA transcripts. In Chapter Six, we innovated a new methodology to characterize the enFeLV-LTR integration sites across the entire genome of 20 related and unrelated domestic cats in an attempt to uncover genes that may be influenced by LTR enhancement of gene expression. We found one LTR integration site in a limited number of cats that is within 1MB of APOBEC1, an antiviral gene, and that the most common gene found in close proximity to LTR integration sites are zinc fingers, a broad-acting class of regulatory proteins. Collectively, this groundwork provides future directions to uncover direct and indirect mechanisms of enFeLV-mediated restriction of FeLV infection. We conclude that because wild felids lack enFeLV, they may be more vulnerable to FeLV infection. As urbanization forces niche overlap and contact between wild and domestic felids, the risk of infection of these species is likely to increase, and thus it will be important to consider contacts between FeLV-infected domestic cats and wild felid populations during development of conservation action plans.Item Embargo 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 molecular ecology and evolution of puma letivirus in bobcats and mountain lions in North America(Colorado State University. Libraries, 2013) Lee, Justin S., author; VandeWoude, Sue, advisor; Crooks, Kevin R., advisor; Funk, W. Chris, committee member; Troyer, Jennifer L., committee memberHost-pathogen dynamics are influenced by ecological and evolutionary processes at all levels of biological organization. Within individuals, viruses that cause chronic infection must either avoid or escape the pressures of the host immune system. Furthermore, viruses adapted to one host environment may have low fitness when transmitted to different individuals, populations, and species. At the landscape level, the movement and distribution of directly transmitted obligate pathogens are inextricably associated with their hosts. We used molecular analyses to investigate the ecology and evolution of feline immunodeficiency virus (FIV) in bobcats and mountain lions within individuals, among populations, and between species of hosts. In Chapter One we investigated the effects of urban development on the movement of bobcats and feline immunodeficiency virus (FIV) among a fragmented landscape in southern California. Our results demonstrate that bobcat movement and gene flow are restricted across a major freeway that bisects the study area, resulting in two genetically and physically distinct subpopulations connected by a low level of migration. However, the FIV population is not similarly structured, suggesting that movements and contacts sufficient for disease transmission continue despite the low level of host migration observed. Chapter Two investigates the causes and effects of FIV evolution among bobcats and mountain lions across North America. Our results illustrate a dynamic host-pathogen relationship characterized by host-immune pressures and a rapidly evolving virus with a highly plastic genome. Finally, in Chapter Three we describe a pilot project aimed at improving the efficiency with which pathogen genetic data can be collected by combining the use of two modern technologies - targeted genome capture and next-generation sequencing. The results suggest this is a promising approach to detecting and sequencing multiple pathogens from biological samples. Collectively, the work described in this dissertation combines new and existing methodologies to generate, analyze, and interpret molecular data to answer complex questions about the ecological and evolutionary determinants of host-pathogen dynamics.