Browsing by Author "Ebel, Greg, committee member"
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Item Open Access Characterizing the genetic evolution of endemic bluetongue virus strains(Colorado State University. Libraries, 2019) Kopanke, Jennifer H., author; Mayo, Christie, advisor; Callan, Rob, committee member; Ebel, Greg, committee member; VandeWoude, Sue, committee memberBluetongue virus is an arthropod-borne virus that can cause severe disease in susceptible animals. Transmitted by biting midges in the genus Culicoides, the bluetongue virus particle (genus Orbivirus, family Reoviridae) is composed of ten segments of double-stranded RNA enclosed by a bi-layered, icosahedral capsid. While both wild and domestic ruminants are capable of becoming infected with bluetongue virus, sheep are most likely to develop severe disease characterized by systemic vasculitis, edema, and coagulopathy. Due to its relatively unusual genome structure, bluetongue virus (BTV) is able to evolve via several key mechanisms, including via the accumulation of mutations over time, or more rapidly via reassortment of genome segments. Adding to this genetic complexity, bluetongue virus must maintain fitness in two very disparate hosts: the insect vector and the ruminant. While host-switching is widely accepted as an important aspect of bluetongue virus evolution, the specific features of viral adaptation in each host are poorly characterized. Limited field studies and experimental work from other labs have alluded to the presence of these phenomena at work in the evolutionary trajectory of bluetongue virus, but our overall understanding of the factors that drive or constrain this virus's genetic diversification remains incomplete. In recent years, bluetongue virus has caused significant disease outbreaks among ruminants in enzootic regions, such as the U.S., as well as in areas where bluetongue virus was previously considered exotic, such as northern Europe. Various dynamics including vector range expansion, movement of animals, virus evolution through reassortment and mutation, and environmental factors all may have an integral role in the occurrence of these outbreaks. Not only do bluetongue epizootics carry sometimes profound animal health consequences, but they are also associated with significant economic impacts due to production declines, costly efforts to contain disease spread, and trade restrictions. Collectively, our currently limited understanding of bluetongue virus ecology and evolution dramatically hinders our ability to predict and prevent the occurrence of epizootics associated with orbiviruses. As whole genome sequencing approaches have become increasingly available and affordable, these tools provide a uniquely valuable platform for interrogating underlying viral genetic factors associated with bluetongue disease incursions and outbreaks. Coupling applied fieldwork, in vitro, and in vivo studies with sequencing tools and bioinformatics, the work described in this dissertation seeks to address specific knowledge gaps surrounding bluetongue evolution in North America. In particular, we first queried how an alternating-host transmission cycle affects bluetongue's genetic diversity using an in vitro system, where we leveraged whole genome sequencing and measures of population genetics to understand the role of viral mutation during BTV evolution. We found low rates of overall mutation, leading us to consider whether reassortment is a relatively greater contributor to bluetongue's genetic diversity. Once again using an in vitro platform, we investigated reassortment frequency and segment-specific trends between two enzootic bluetongue virus strains. Our work demonstrated that global shifts in segment frequencies emerged across serial passages, possibly representing preferred reassortant segment combinations. However, most viral segments persisted – even if at very low levels – within the overall population from passage to passage. To better characterize these trends, and to understand whether environmental factors such as temperature might affect their occurrence, we introduced these same viruses into Culicoides sonorensis midges – the predominant North American vector of bluetongue virus – and tracked virogenesis and reassortment across time at three different temperatures. Correlating with other studies, we found that higher temperatures were associated with more rapid virogenesis. However, we were surprised to find that one of the two virus strains replicated poorly in midges orally infected with biologically relevant titers, highlighting potential vector-based barriers to reassortment. Finally, we used whole genome sequencing to characterize circulating strains of bluetongue virus present in Colorado ruminants in 2015 and 2018. We found that numerous strains of bluetongue virus were present among sentinel animals, and that many isolates contained signatures of reassortment. Collectively, our findings demonstrate that reassortment among virus strains is a prominent feature of bluetongue viral evolution. Importantly, there appear to be preferred segment combinations that arise following coinfection, but vector-virus interactions seem to play a central role in modulating the ultimate emergence of reassortant viruses. These studies and others promise to improve our understanding of bluetongue's evolution and ecology, ultimately contributing to the development of better predictive models and management strategies to reduce future impacts of bluetongue epizootics.Item Open Access Development and efficacy testing of broad alphavirus vaccines and antivirals and characterization of alphavirus neuroinvasion(Colorado State University. Libraries, 2016) Rico, Amber, author; Olson, Ken, advisor; Ebel, Greg, committee member; Powers, Ann, committee member; Bowen, Richard, committee member; Chen, Chaoping, committee memberAlphaviruses are mosquito-borne pathogens that cause worldwide disease and death in humans and animals. Several alphaviruses are select agents and are a legitimate biosafety and bioweapon concern. Additionally, several alphaviruses are emerging infectious diseases. Climate change and urbanization have expanded mosquito populations and increased human-mosquito interactions within this decade and will continue into future decades. As mosquito populations expand, naïve human populations are exposed to arthropod-borne viruses, including alphaviruses, and vector-borne diseases have surged. The increasing prevalence of arthropod-borne disease has highlighted the global need to develop measures that prevent or treat arthropod-borne disease infection. Currently, vaccines to prevent alphavirus infection are limited to investigational new drug status and no therapeutics are available to treat alphavirus disease. This dissertation will describe projects aimed at preventing or treating alphavirus infection and characterizing the process of alphavirus neuroinvasion. To address the concern of potential outbreaks of an intentional or natural nature, alphavirus vaccines based on the ectodomain of alphavirus E1 were designed and tested. Cationic liposomes complexed with nucleic acid adjuvants and alphavirus E1 protein (lipid-antigen-nucleic acid complexes; LANACs) provided the best platform for alphavirus E1 vaccination. Interestingly, western equine encephalitis virus (WEEV) E1 (LANAC WEEV E1) protected against both mouse WEEV and eastern equine encephalitis virus (EEEV) challenge but not Venezuelan equine encephalitis virus (VEEV); whereas, VEEV E1 (LANAC VEEV E1) protected against both VEEV and EEEV challenge but not WEEV. LANAC VEEV E1 + WEEV E1 vaccination protected mice against EEEV, VEEV, and WEEV challenge. Mice immunized with LANACs (LANAC WEEV E1, LANAC VEEV E1 or LANAC VEEV E1 + WEEV E1) mounted strong humoral immune responses, but were lacking neutralizing antibody. Hamsters immunized with LANAC WEEV E1 failed to mount humoral immune responses and were not protected from challenge. Antibody derived from E1 vaccination binds infected cells and purified E1, but not intact virions. E1 antibody is non-neutralizing yet protective against CHIIKV, EEEV, SINV, VEEV, and WEEV in vitro. In vivo we have demonstrated that antibody is protective against all three new world alphaviruses (NWAs0. Antibody affects late stages of the viral life cycle and likely inhibits virus release or cell death. Following a screen, conducted by our collaborators, of FDA approved and ex-US approved compounds for effectiveness against viral encephalitic and hemorrhagic fever viruses, we tested compounds for anti-alphavirus activity to develop therapeutics to treat alphavirus infection. Yield reduction assays identified four compounds that inhibited virus replication by two to four logs. These compounds were further tested in vitro for mechanism of action. Compounds P-75802, P-75803, P-75805, and P-75811 affected early stages of replication. Compound P-75802 was tested in vivo and was found to possess limited antiviral activity. The process of alphavirus neuroinvasion, particularly following peripheral inoculation, is poorly defined. In the studies described here, encephalitic alphavirus neuroinvasion following EEEV, VEEV, and WEEV intranasal and subcutaneous inoculation were described relying on bioluminescent in vivo and ex vivo imaging, CLARITY imaging, and histopathology. We found that neuroinvasion occurs through two routes dependent on inoculation method. The cranial nerves, particularly the olfactory tract nerve, were involved in neuroinvasion following intranasal inoculation. Neuroinvasion from the periphery, footpad inoculation, occurred via a route independent of the olfactory tract. Neuroinvasion occurred in areas where the blood brain barrier is naturally absent including the hypothalamus, anterioventral third ventricle region (AV3V), area postrema, and the pineal body.Item Open Access Development of electrochemical assays and biosensors for detection of Zika virus(Colorado State University. Libraries, 2019) Filer, Jessica, author; Geiss, Brian, advisor; Chen, Tom, advisor; Henry, Charles, committee member; Wilusz, Jeff, committee member; Ebel, Greg, committee memberZika virus (ZIKV) emerged as a significant public health concern after the 2015-2016 outbreak in South and Central America. Severe neurological complications and birth defects in adults and children respectively underscore the need for quick and accurate diagnosis so that proper medical observation and intervention can be done. Electrochemical assays and biosensors are attractive as alternative diagnostic tools due to their sensitivity and ease of miniaturization. This dissertation describes three novel electrochemical assays and biosensors to detect ZIKV specific nucleic acid, antibodies, and virus particles. A nuclease protection ELISA (NP-ELISA) was developed for nucleic acid detection by enzymatic readout. The assay was validated using synthetic complementary oligos for absorbance, chemiluminescence, and electrochemical enzymatic readout. Two horseradish peroxidase substrates, 3,3',5,5'-Tetramethylbenzidine (TMB) and hydroquinone, were characterized electrochemically and compared for electrochemical assay use. Electrochemical TMB readout demonstrated better sensitivity compared to all tested detection modalities with a limit of detection of 3.72×103 molecules mL-1, which compares well to the amount of ZIKV RNA in clinical samples and to other approved assays like the CDC's Trioplex assay. For serological analysis, a capacitive microwire biosensor was developed and validated using immunized mouse sera to detect a ZIKV antibody response. Measurements were taken through a wide serial dilution range of 1:1018 to 1:103 and two dilutions (1:1012 and 1:106) were used for analysis for optimal sensitivity. A statistically significant immune response was detected four days after immunization at a 1:1012 dilution and was specific for ZIKV when compared with Chikungunya virus (CHIKV). These results indicate that serological analysis can be performed four days earlier with the wire sensor compared to ELISAs using ultra-dilute samples. The sensor also was used to differentiate between IgG and IgM antibodies and compared well with ELISA results. Lastly, an impedance array sensor was designed and validated for detection of ZIKV particles. The array allows for simultaneous handling of many electrodes, which increases throughput compared to other biosensor designs. The sensor demonstrated good sensitivity with an LOD of 22.4 focus forming units (FFU) which compares well to other reported sensors. In addition, it was optimized for specificity and tested using Sindbis virus (SINV) as a negative control. These novel platforms comprise new advancements in biosensor technology by simplifying existing assays, increasing sensitivity, and providing a new platform for handheld measurements.Item Embargo High throughput characterization of bunyavirus diversity, ecology, and reassortment potential(Colorado State University. Libraries, 2023) Kapuscinski, Marylee, author; Stenglein, Mark, advisor; Kading, Rebekah, committee member; Wilusz, Jeff, committee member; Ebel, Greg, committee member; Nachappa, Punya, committee memberBunyavirales is an important group of viral pathogens with significant economic impacts. The Bunyavirales order contains the largest number of RNA viruses and can cause disease in plants, animals, and humans [1]. Notable plant pathogens include Tomato Spotted Wilt virus [2] which results in significant agricultural losses. Notable animal pathogens include Rift Valley fever virus [3] and Schmallenberg [4] virus resulting in significant livestock loss. Notable human pathogens include Crimean-Congo hemorrhagic fever virus [5], hantavirus [6], and La Crosse virus [7]. The effects of bunyavirus infections are felt worldwide because bunyaviruses are distributed globally. The emergence of novel bunyaviruses continues to threaten agricultural and livestock industries as well as human health. Where or when a novel bunyavirus might emerge is unknown. Predicting emergence is difficult for three reasons. First, because bunyaviruses are RNA viruses [8], the high error-rate of the RNA-dependent RNA polymerase results in a large genetic diversity within a population of viruses [9]. Secondly, many important bunyaviruses are arthropod-borne [10], resulting in an intricate lifecycle between an invertebrate and vertebrate host. This results in constantly changing genetic diversity due to different selective pressures from different host types [11]. It also results in an ever-expanding geographic range as vector range expands due to climate change and vertebrate host range changes due to urbanization, industrialization, and deforestation [12–16]. Third, bunyaviruses have segmented genomes which allows them to reassort and produce viral progeny with an altered vector-host range, pathogenesis, and virulence [1,17,18]. Therefore, this body of work aims to increase our ability to understand bunyavirus emergence and reassortment potential as a way to aid in outbreak preparedness and early response systems. To do this, we've combined traditional surveillance data with modern bioinformatics to expand our knowledge of bunyavirus genetic diversity, ecology, and reassortment potential. Using whole-genome sequencing, we've characterized the genomes of 99 bunyaviruses, some of which have never been sequenced before. This aids in our understanding of the genetic diversity, co-infection dynamics, and reassortment potential. Next, we used existing metadata from orthobunyavirus sequences to determine reassortment potential given a shared geographic and vector-host range. Finally, we've developed a novel molecular assay to evaluate reassortment potential based on replication and transcription compatibility. Together, we've combined the strengths of viral surveillance and modern bioinformatics to demonstrate the benefit of combining both. We've developed systems that will help to delineate the mechanisms that either promote or inhibit reassortment potential, ultimately aiding in early response systems for outbreak preparedness.Item Open Access Ivermectin-treated bird feed to control West Nile virus transmission(Colorado State University. Libraries, 2018) Nguyen, Chilinh, author; Foy, Brian, advisor; Ebel, Greg, committee member; Bowen, Richard, committee member; Huyvaert, Kathryn P., committee memberWest Nile virus is the leading cause of arboviral fever and encephalitis in the United States. The highest WNV disease incidence occurs along the Great Plains region of the United States, as the ecology and land use provide a supportive habitat for the main WNV enzootic and bridge vector of the region, Culex tarsalis. However, due to the lack of dense human population, this area often does not benefit current WNV control measures as applied by conventional mosquito control districts. Based on the ecology of WNV transmission in the Great Plains region, a strategy that targets Cx. tarsalis through its ornithophilic blood feeding behavior could disrupt WNV transmission. Given that the majority of Cx. tarsalis blood meals on the northern Colorado plains may come from doves and passerine species during the WNV transmission season, effective targeting of these or other local preferred hosts with endectocide-treated bird feed could result control of WNV transmission. This study develops and characterizes the effects of IVM-treated bird feed in birds and biting Cx. tarsalis mosquitoes in both a laboratory and field setting. In Chapter 2, the effects of IVM on Cx. tarsalis survival were examined using both in vitro membrane blood meals and direct blood feeding on IVM-treated birds. Chickens and wild Eurasian Collared Doves fed solely on IVM-treated bird feed concentrations up to 200 mg IVM/kg feed exhibited no signs of toxicity, and most Cx. tarsalis that blood fed on these birds died compared to controls. Mosquito survivorship following blood feeding correlated with IVM serum concentrations at the time of blood feeding, which dropped rapidly after the withdrawal of treated feed. These results suggested IVM-treated bird feed should be further explored as a hyper-localized control strategy for WNV transmission. Chapter 3 presents the development of a method to detect and quantify IVM in individual blood meals of Anopheles gambiae and Cx. tarsalis, which will be important in measuring the coverage of this intervention in the field, and accurately assessing IVM's mosquitocidal effects in field situations. This ability to detect IVM in mosquito blood meals was similar between blood fed Cx. tarsalis and An. gambiae, and between sampling times of 0 or 12 hours post blood feed. The quantity of IVM ingested in individual mosquitoes was also compared to the venous serum concentrations of live animals. Chapter 4 presents promising results from two separate pilot field trials of IVM-treated bird feed that were conducted during the summers of 2016 and 2017. Results from 2016 showed that wild birds frequently visit the IVM-treated feeders. In addition, there was an observable trend where "far" traps that are expected to be beyond the zone of control had more WNV-positive pools compared to "near" traps at both ELC and ARDEC South sites. Results from the 2017 study continued to be promising, where birds were again visiting IVM-treated feeders and IVM could be detected in the sera of birds sampled by IVM feeders. There was also a trend of higher VI for the control sites compared to IVM sites for the 2017 season. The efficacy of IVM-treated bird feed was evaluated in two pilot trials where natural WNV transmission cycles occurred in wild birds and Cx. tarsalis, but should be followed up with field seasons with many control and IVM sites to allow for a robust analysis of IVM effects. This study introduces the novel concept of using systemic endectocides for controlling WNV transmission, and this concept could be explored for other arboviruses.Item Open Access Natural cases of salamander hybridization suggest a consistent relationship between genetic distance and reproductive isolation across tetrapods(Colorado State University. Libraries, 2019) Melander, Scott, author; Mueller, Rachel, advisor; Sloan, Dan, committee member; Ebel, Greg, committee memberHybridization between populations along the path to complete reproductive isolation can provide snapshots of speciation in action. Here, we present the first comprehensive list of natural salamander hybrids and estimate genetic distances between the parental hybridizing species using a mitochondrial and nuclear gene (MT-CYB and RAG1). Salamanders are outliers among tetrapod vertebrates in having low metabolic rates and highly variable sex chromosomes. Both of these features might be expected to impact speciation; mismatches between the mitochondrial and nuclear genomes that encode the proteins for oxidative metabolism, as well as mismatches in heteromorphic sex chromosomes, can lead to reproductive isolation. We compared the genetic distances between hybridizing parental species across four main tetrapod clades that differ in metabolic rates and sex chromosome diversity: salamanders, lizards, mammals, and birds. Our results reveal no significant differences, suggesting that variation in these traits across vertebrates does not translate into predictable patterns of genetic divergence and incompatible loci in hybrids.Item Embargo Probing the metabolic secrets of Anopheles stephensi mosquitoes to enhance cryopreservation techniques(Colorado State University. Libraries, 2024) Ramirez, Gabriela, author; Dobos, Karen, advisor; Ebel, Greg, committee member; Barfield, Jennifer, committee member; Broeckling, Corey, committee memberThe lipid profile and cryopreservation methods of Anopheles stephensi mosquitoes have significant implications for mosquito management and research. The intricate life cycle of mosquitoes is governed by lipid metabolism, involving lipogenesis, lipolysis, and fatty acid metabolism, which are critical for transitioning between life stages. Despite the importance of these processes, mosquito cryopreservation has faced challenges, mainly due to the impenetrable nature of mosquito eggs to traditional cryoprotective agents. While other insects like honeybees and fruit flies have seen some success in cryopreservation, mosquitoes have posed unique difficulties. Initial attempts to cryopreserve mosquito eggs were unsuccessful, and there remains a need for effective cryopreservation protocols that maintain the viability and normal development of mosquitoes post-thaw. This study investigates lipid metabolism across mosquito life stages and advances cryopreservation techniques. The lipid profile analysis focused on major lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelins (SM), and triacylglycerides (TGs), as well as non-bilayer lipids like diacylglycerol (DG) and lysophospholipids (LysoPC, LysoPE). For cryopreservation, a new protocol using methylformamide (MF) with trehalose was developed for first-instar larvae (L1s) older than 1.5 hours post-hatching. The hypotheses were that 1) Lipid metabolism plays a crucial role in the successful development and reproduction of mosquitoes, and 2) Inducing diapause and using suitable cryoprotectants can improve the recovery rates of cryopreserved mosquitoes. The lipid profile analysis revealed that PE is vital for protein anchoring required for embryogenesis and immune responses and that steroids like 20-hydroxyecdysone (20E) are crucial for molting and development. The cryopreservation study showed that supercooling larvae for up to 60 minutes resulted in normal development to adulthood, although longer durations inhibited adult emergence. Supercooled larvae and their offspring exhibited typical sex ratios and developmental patterns, indicating genetic and phenotypic stability. This research underscores the well-orchestrated metabolic strategies in mosquitoes. It suggests that understanding these biochemical processes is essential for effective cryopreservation, potentially paralleling natural cold survival strategies seen in other insects.