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Browsing by Author "Wilusz, Jeff, committee member"

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    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 member
    Zika 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.
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    Flavivirus control of lipid metabolism: implications for virion formation, function and pathogenesis
    (Colorado State University. Libraries, 2018) Gullberg, Rebekah, author; Perera, Rushika, advisor; Crick, Dean, committee member; Di Pietro, Santiago, committee member; Geiss, Brian, committee member; Wilusz, Jeff, committee member
    Dengue viruses (DENV) are the most aggressive arthropod-born viruses worldwide with no currently available antivirals. There is a clear need to understand host viral interactions that can be exploited for therapeutic options. DENV are members of the Flaviviridae family with a positive sense single-stranded RNA genome surrounded by a virally encoded capsid protein, a host cell derived lipid envelope and an icosahedral shell of virally encoded glycoproteins. Its genome is replicated in virally–induced invaginations in the endoplasmic reticulum of the host cell that consistently develop in a time-dependent manner. These invaginations display a highly curved architecture and seem to increase the membrane contact sites within the ER and its vicinity. Functionally, these membranes condense the replication machinery, provide a scaffold to coordinate replication, and hide the viral double stranded RNA intermediate from the host cellular defenses. It has been shown that fatty acid synthesis is increased during infection to provide substrates for this membrane expansion. To identify further changes to cellular metabolism, we have profiled the metabolome of DENV serotype 2 (DENV2) infected Human Hepatoma cells (Huh7) cells at key time-points in virus replication. We have found time-dependent changes in cellular essential fatty acid metabolism. Furthermore, we have interrogated a library of siRNAs directed at the unsaturated fatty acid biosynthesis pathway to determine key enzymes involved in viral replication. We have identified that stearoyl Co-A desaturase 1 (SCD1), the rate-limiting enzyme responsible for converting stearic to oleic acid, is critical for viral replication, maturation and infectious particle formation. Finally, we have profiled the serum metabolome of acute-phase patients with dengue diseases, chikungunya virus infection, or an unknown febrile illness to identify metabolic changes with potential use as prognostic biomarkers. Hypothesis: Since dengue viruses are enveloped viruses, lipid metabolites in the human host are a critical resource hijacked by these viruses for their replicative advantage. Important metabolites will be altered during infection in a time dependent manner and can be quantified and correlated directly to their role in viral genome replication and infectious particle assembly and release. These metabolic changes could also be identified in human bio-fluids and could function as early biomarkers of disease manifestation.
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    Functional analysis of three Arabidopsis SR proteins (SCL33, SC35, SCL30A) in plant development and splicing
    (Colorado State University. Libraries, 2012) Thomas, Julie, author; Reddy, A. S. N., advisor; Bedinger, Pat, committee member; Pilon, Marinus, committee member; Wilusz, Jeff, committee member
    To view the abstract, please see the full text of the document.
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    High throughput characterization of bunyavirus diversity, ecology, and reassortment potential
    (Colorado State University. Libraries, 2023) Kapuscinski, Marylee, author; Stenglein, Mark, advisor; Kading, Rebekah, committee member; Wilusz, Jeff, committee member; Ebel, Greg, committee member; Nachappa, Punya, committee member
    Bunyavirales is an important group of viral pathogens with significant economic impacts. The Bunyavirales order contains the largest number of RNA viruses and can cause disease in plants, animals, and humans [1]. Notable plant pathogens include Tomato Spotted Wilt virus [2] which results in significant agricultural losses. Notable animal pathogens include Rift Valley fever virus [3] and Schmallenberg [4] virus resulting in significant livestock loss. Notable human pathogens include Crimean-Congo hemorrhagic fever virus [5], hantavirus [6], and La Crosse virus [7]. The effects of bunyavirus infections are felt worldwide because bunyaviruses are distributed globally. The emergence of novel bunyaviruses continues to threaten agricultural and livestock industries as well as human health. Where or when a novel bunyavirus might emerge is unknown. Predicting emergence is difficult for three reasons. First, because bunyaviruses are RNA viruses [8], the high error-rate of the RNA-dependent RNA polymerase results in a large genetic diversity within a population of viruses [9]. Secondly, many important bunyaviruses are arthropod-borne [10], resulting in an intricate lifecycle between an invertebrate and vertebrate host. This results in constantly changing genetic diversity due to different selective pressures from different host types [11]. It also results in an ever-expanding geographic range as vector range expands due to climate change and vertebrate host range changes due to urbanization, industrialization, and deforestation [12–16]. Third, bunyaviruses have segmented genomes which allows them to reassort and produce viral progeny with an altered vector-host range, pathogenesis, and virulence [1,17,18]. Therefore, this body of work aims to increase our ability to understand bunyavirus emergence and reassortment potential as a way to aid in outbreak preparedness and early response systems. To do this, we've combined traditional surveillance data with modern bioinformatics to expand our knowledge of bunyavirus genetic diversity, ecology, and reassortment potential. Using whole-genome sequencing, we've characterized the genomes of 99 bunyaviruses, some of which have never been sequenced before. This aids in our understanding of the genetic diversity, co-infection dynamics, and reassortment potential. Next, we used existing metadata from orthobunyavirus sequences to determine reassortment potential given a shared geographic and vector-host range. Finally, we've developed a novel molecular assay to evaluate reassortment potential based on replication and transcription compatibility. Together, we've combined the strengths of viral surveillance and modern bioinformatics to demonstrate the benefit of combining both. We've developed systems that will help to delineate the mechanisms that either promote or inhibit reassortment potential, ultimately aiding in early response systems for outbreak preparedness.
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    Reframing viral infections as acute metabolic disorders: dengue viruses and their dependency on host metabolic pathways
    (Colorado State University. Libraries, 2022) St. Clair, Laura A., author; Perera, Rushika, advisor; Belisle, John, committee member; Nachappa, Punya, committee member; Wilusz, Jeff, committee member; Zabel, Mark, committee member
    Dengue viruses (DENVs) are the etiological agent of the world's most aggressive arthropod-borne disease. At present, there are no available antivirals against DENVs. This fact underscores a dire need to examine host-virus interactions to identify and develop novel therapeutic approaches. As obligate intracellular parasites, DENVs are reliant upon and hijack several host metabolic pathways both to fulfill their replicative needs, and to evade the host immune response. We and others have previously established that infection with DENVs causes significant perturbation to host lipid metabolism, including elevations in sphingolipids in both the human and mosquito host. In addition, we and others previously discovered that the DENV NS1 protein increases sialidase activity in both in vitro and in vivo models leading to increased endothelial hyperpermeability and vascular leakage which are hallmarks of severe dengue. To further clarify and characterize these previous works, we have performed siRNA-mediated loss of function studies using human hepatoma cells (Huh7 cells) on several metabolic pathways altered during DENV2 infection. First, we examined the role of acyl-CoA thioesterases, enzymes responsible for controlling the intracellular balance of activated fatty acids and free fatty acids, on the DENV2 lifecycle. In these analyses, we determined that the cytosolic ACOT1 enzyme had an inhibitory effect on DENV2 replication and release, while mitochondrial ACOT (ACOTs 2 and 7) functionality was critical for viral replication and release. Moreover, we identified several enzymes within the ACOT family whose expression was dependent on ACOT2 and ACOT7 expression. These results highlighted complex relationships between ACOTs and DENVs, as well as identified yet unknown functional interdependence between ACOT enzymes. Next, we expanded our previous understanding of the relationship between DENVs and the human sialidase enzymes (NEU1-4). While previously studies linked upregulation of these enzymes with DENV2 pathology, we provide the first evidence showing that NEU1-4 functionality is vital for DENV2 genome replication and viral egress. Moreover, our analyses also revealed previously unknown functionality of NEU4 or its downstream products as transcriptional regulators for NEU1-3. Finally, we provide the first profile of the effect of loss of function of enzymes within the entire sphingolipid metabolic pathway (as identified through KEGG pathway database) on the DENV2 life cycle. In this study, we identified that enzymes involved the sphingomyelinase and salvage pathways of ceramide synthesis as opposed to de novo ceramide synthesis were critical to DENV2 release from Huh7 cells. In addition, we determined that enzymes involved in the synthesis and degradation of glycosphingolipids were vital for DENV2 release. An especially intriguing result within this arm of sphingolipid metabolism was that the two enzymes which hydrolyze GluCer had differential effects on DENV2 replication and release. GBA1 (lysosomal) had an antiviral effect on DENV2, while GBA2 (non-lysosomal) was required for DENV2 replication and release. This prompted us to profile the changes that occur to glycosphingolipids (GSLs) during infection, and we uncovered several species of GSLs that are elevated during infection. Moreover, we identified that Ambroxol HCl, a pharmaceutical GBA1 chaperone/GBA2 inhibitor, was able to abrogate these elevations in GSLs. Combined, our results allowed us to propose a novel function for GBA2 as a GluCer recycling enzyme during DENV2 infection. In conclusion, together, the work in this dissertation highlights critical metabolic nodes that impact virus replication and provides new directions for investigating viral infections as acute metabolic diseases.
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    The role of retroviral cyclin in the development of walleye dermal sarcoma
    (Colorado State University. Libraries, 2015) Birkenheuer, Claire, author; Rovnak, Joel, advisor; Quackenbush, Sandra L., advisor; Nyborg, Jennifer, committee member; VandeWoude, Susan, committee member; Wilusz, Jeff, committee member
    The retroviral cyclin (RV-cyclin) is an accessory protein encoded by walleye dermal sarcoma virus (WDSV). This virus causes the formation of walleye dermal sarcoma, and requires the tumor tissue to replicate. RV-cyclin is one of only two proteins expressed by the virus during tumor development before production of the WDSV virion, and the mechanism by which RV-cyclin causes tumor formation was explored and is presented here. RV-cyclin interacts with TAF9 and CDK8, which are cellular proteins that regulate RNA Pol II transcription. RV-cyclin's influence on transcription was explored by analyzing transcript levels of CCND1, CDKN2D, FOS, EGR1, and JUN. All of these genes are important oncogenes in human cancers, and were hypothesized to contribute to development of walleye dermal sarcoma (WDS). Quantitative reverse transcription PCR analysis of these genes in HeLa and HCT116 cell lines expressing wt or mutant RV-cyclin, or over-expressing cyclin C was carried out. These analyses showed that wt RV-cyclin enhances expression of all genes tested and that the interactions with TAF9 and with CDK8 contribute to this enhancement. Western blot analysis of phosphorylated Elk1 and SRF, two transcription factors that initiate RNA Pol II transcription, revealed that RV-cyclin's mechanism of gene activation is downstream of transcription factor phosphorylation. Messenger RNA (mRNA) decay assays demonstrated that RV-cyclin does not alter the rate of mRNA decay, framing RV-cyclin's mechanism of activation to the level of RNA Pol II transcription. Nuclear run-on and chromatin immunoprecipitation analysis of EGR1 with an RNA Pol II antibody support a role for RV-cyclin in transcription elongation and re-initiation. ChIP analysis of the mutant RV-cyclin cells and cells over-expressing cyclin C demonstrated that RV-cyclin requires both a functional CDK8 and TAF9 interaction for efficient activation and extension of the EGR1 serum response. CDK8 ChIPs demonstrated that the RV-cyclin enhances CDK8 occupancy at the EGR1 gene locus, and HA-ChIPs show that CDK8 and RV-cyclin occupancy correlate with each other on the locus. At this point, it was hypothesized that RV-cyclin functions to enhance CDK8 kinase activity in addition to bringing more CDK8 to select oncogenic loci in vivo. For this reason kinase assays using recombinant, baculovirus-produced CDK8/RV-cyclin or CDK8/cyclin C constructs were attempted. CDK8 bound to RV-cyclin was able to autophosphorylate to similar levels as CDK8 bound to cyclin C. Other CDK8 substrates remain to be tested. RV-cyclin's function in transcription requires activation of the MAPK pathway, suggesting RV-cyclin needs an outside signal like serum stimulation for efficient WDS formation. This signal likely comes from the presence of the Orf b protein, the other WDSV accessory protein expressed during tumor development. The protein Orf b causes phosphorylation and activation of the p90RSK complex. This event likely causes aberrant phosphorylation of SRF and Elk-1 giving RV-cyclin the activated transcription factors required for its function on specific proto-oncogenes. Taken together, the Orf b protein and RV-cyclin illustrate a previously un-described, trans-acting mechanism of retroviral-induced oncogenesis.