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- Creators: School of Life Sciences
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Description
Vaccinia virus (VV) is a prototype virus of the Orthopox viruses. The large dsDNA virus composed of 200kbp genome contains approximately 200 genes and replicates entirely in the cytosol. Since its use as a live vaccine against smallpox that leads to the successful eradication of smallpox, Vaccinia has been intensely studied as a vaccine vector since the large genome allows for the insertion of multiple genes. It is also studied as a molecular tool for gene therapy and gene functional study. Despite its success as a live vaccine, the vaccination causes some mild to serious bur rare adverse events in vaccinees such as generalized Vaccinia and encepharitis. Therefore, identification of virulence genes and removal of these genes to create a safer vaccine remain an important tasks. In this study, the author seeks to elucidate the possible relationship between immune evading proteins E3 and B19. VV did not allow double deletions of E3 and B19, indicating the existence of a relationship between the two genes.
ContributorsBarclay, Shizuka (Author) / Jacobs, Bertram (Thesis director) / Ugarova, Tatiana (Committee member) / Kibler, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Among wild rodent populations, vertical transmission is believed to constitute the primary route of infection for Lymphocytic Choriomeningitis Virus (LCMV), a non-lytic arenavirus with both acute and chronic forms. When carrier mice infected at birth with the acute Armstrong strain reproduce, they generate congenital carrier offspring containing a quasispecies of LCMV that includes Armstrong as well as its chronic Clone-13 variant. This study examined the genetic trends in the vertical transmission of LCMV from mothers infected perinatally with Clone-13. Viral isolates obtained from the serum of congenital carrier offspring were partially sequenced to reveal residue 260 in the glycoprotein-encoding region of their S segment, the site of a major amino acid change differentiating the chronic and acute strains. It was found that the phenylalanine-to-leucine mutation associated with Clone-13 was present in 100% of the isolates, strongly indicating that the offspring of Clone-13 carriers contain exclusively the chronic variant. This research has broad implications for the epidemiology of the virus, and, given the predominance of Armstrong in the wild, suggests that there must be a biological cost associated with Clone-13 infection in non-carriers.
ContributorsFrear, Cody Christian (Author) / Blattman, Joseph (Thesis director) / Hogue, Brenda (Committee member) / Holechek, Susan (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Viral infections are a significant cause of disease in humans. While some viral diseases have been eliminated, many more continue to infect millions. Viral infections are challenging to treat because viruses use host cell machinery to replicate, so it is difficult to develop drugs that can target viruses. Normally, the host’s immune system is capable of destroying the virus, but during chronic infections it becomes exhausted and T cells lose their effector functions necessary for the clearance of the virus. IL-2 can help relieve this exhaustion, but causes toxicity to the body. In mice infected with chronic LCMV, IL-2 administration causes death due to pulmonary hemorrhage. CD4 deficient mice were infected with chronic LCMV and then dosed with IL-2 and survived, but mice that were deficient for CD8 T cells died, indicating that toxicity was mediated by CD8 T cells. CD8 T cells can kill infected host cells directly by producing perforin, or can produce cytokines like IFN-γ and TNF to further activate the immune system and mediate killing. Mice that were deficient in perforin died after IL-2 administration, as well as mice that were deficient in IFN-γ. Mice deficient in TNF, however, survived, indicating that TNF was mediating the toxicity in response to IL-2. There are two different receptors for TNF, p55 and p75. p55 is known as TNFR1 and has been implicated in apoptosis of virally infected cells. P75 is known as TNFR2 and is associated more with inflammation in response to infection. My hypothesis was that if TNFR2 was knocked out, infected mice would survive IL-2 dosing. When single knockouts of TNFR1 and 2 were used in an experiment however, it was found that either receptor is capable of mediating toxicity, as both experimental groups failed to survive. This is relevant to current IL-2 therapies because there is no way to eliminate a single receptor in order to reduce toxicity. Further studies exploring the anti-viral capabilities of IFN-γ are suggested.
ContributorsJarvis, Jordan Alisa (Author) / Blattman, Joseph (Thesis director) / Denzler, Karen (Committee member) / McAfee, Megan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Vaccinia virus is a cytoplasmic, double-stranded DNA orthopoxvirus. Unlike mammalian cells, vaccinia virus produces double-stranded RNA (dsRNA) during its viral life cycle. The protein kinase R, PKR, is one of the principal host defense mechanisms against orthopoxvirus infection. PKR can bind double-stranded RNA and phosphorylate eukaryotic translation initiation factor, eIF2α, shutting down protein synthesis and halting the viral life cycle. To combat host defenses, vaccinia virus encodes E3, a potent inhibitor of the cellular anti-viral eIF2α kinase, PKR. The E3 protein contains a C-terminal dsRNA-binding motif that sequesters dsRNA and inhibits PKR activation. We demonstrate that E3 also interacts with PKR by co-immunoprecipitation. This interaction is independent of the presence of dsRNA and dsRNA-binding by E3, indicating that the interaction is not due to dsRNA-bridging.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
ContributorsFoster, Clayton (Co-author) / Alattar, Hamed (Co-author) / Jacobs, Bertram (Thesis director) / Blattman, Joseph (Committee member) / McFadden, Grant (Committee member) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Stress granules are cytoplasmic foci that form in response to various types of cellular stress, including viral infection. They contain mRNA, translation initiation factors, the small ribosomal subunit, RNA binding proteins, and other unique components depending on the type of stress the cell is under. Stress granules are thought to store these components until the stress as passed at which time the mRNA resumes translation. They also have an active role in the cell's antiviral response and are required for efficient induction of the interferon pathway. There are many viruses that induce or interfere with stress granules, including poliovirus. Poliovirus is a positive sense RNA virus that is part of the Picornaviridae family. Stress granules in poliovirus infected cells differ from stress granules in cells undergoing other types of stress because they contain the RNA binding protein Sam68, their formation is dependent on RNA export by the Crm1 pathway, and they are induced by poliovirus cleavage of eIF4G and PABP. It was found previously that Sam68 is found in the stress granules of poliovirus infected HeLa cells but not in oxidative stress of heat shock induced stress granules. My research shows that this finding is true in other cell lines and thus represents a biologically significant finding. The Crm1 pathway exports snRNAs and some mRNAs, rRNAs, and proteins. To determine which of these classes of RNA is necessary for stress granule formation in poliovirus infected cells but not in cells undergoing other types of stress, plasmids with modified PHAX protein were used to isolate the snRNA export pathway. More work needs to be done to determine the impact of snRNA export on stress granule formation. This research could eventually help us better understand the cell's anti-viral response and have implications for how we treat viral infections.
ContributorsErickson, Caroline Rose (Author) / Hogue, Brenda (Thesis director) / Gustin, Kurt (Committee member) / School of Life Sciences (Contributor) / Department of Management and Entrepreneurship (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Laboratory animals represent an invaluable, yet controversial, resource in the field of biomedical research. Animal research has been behind many influential discoveries in the field of emerging therapeutics. They provide the link between the theory of the lab bench and the functional application of medicine to influence human health. The use of animals in research is a consideration which must be heavily weighed, and the implementation must be carried out at a very high standard in order to retain research integrity and responsibility. We are in the process of conducting an experiment using laboratory mice to demonstrate cancer treatment using vaccinia (VACV) mutants as a possible oncolytic therapy for certain strains of melanoma. VACV is a double-stranded DNA poxvirus with a large and easily altered genome. This virus contains many genes dedicated to immune evasion, but has shown sensitivity to cell death by necroptosis in mouse studies (5). We have identified the absence of the kinase RIP3 which is vital in the necroptosis pathway as a potential target for oncolytic therapy using VACV mutants in specific strains of melanoma. Multiple groups of SCID Beige mice were inoculated with different melanoma cell lines and observed for tumor growth. Upon reaching 1 cm3 in volume, tumors were injected with either VACV- Δ83N, VACV- Δ54N, or PBS, and observed for regression. It was hypothesized that melanoma tumors that are RIP3-/- such as the MDA5 cell line will show regression, but melanoma tumors that are RIP3-positive and capable of necroptosis, such as the 2427 cell line, will resist viral replication and continue to proliferate. Our results so far tentatively support this hypothesis, but the data collection is ongoing. Strict and specific protocols with regard to the ethical and responsible use of mice have been implemented and upheld throughout the experiment. Animals are closely monitored, and if their quality of life becomes too poor to justify their continued use in the experiment, they are humanely euthanized, even at the expense of valuable data. The importance of commitment to a high ethical standard is pervasive throughout our work. Animals represent an invaluable contribution to research, and it is important to maintain high standards and transparency with regard to their use. Education and engagement in critical discussions about the use and care of animals in the laboratory contribute to the overall merit and legitimacy of biomedical research in the public and professional eye as a whole, and give legitimacy to the continued use of animals as models to advance science and health.
ContributorsBergamaschi, Julia (Author) / Kibler, Karen (Thesis director) / Jacobs, Bertram (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Yellow-bellied marmots (Marmota flavivent) are semi-fossorial ground-dwelling sciurid rodents native to the western United States. They are facultatively social and live in colonies that may contain over 50 individuals. Marmot populations are well studied in terms of their diet, life cycle, distribution, and behavior, however, knowledge about viruses associated with marmots is very limited. In this study we aim to identify DNA viruses by non-invasive sampling of their feces. Viral DNA was extracted from fecal material of 35 individual marmots collected in Colorado and subsequently submitted to rolling circle amplification for circular molecule enrichment. Using a viral metagenomics approach which included high-throughput sequencing and verification of viral genomes using PCR, cloning and sequencing, a diverse group of single-stranded (ss) DNA viruses were identified. Diverse ssDNA viruses were identified that belong to two established families, Genomoviridae (n=7) and Anelloviridae (n=1) and several others that belong to unclassified circular replication associated encoding single-stranded (CRESS) DNA virus groups (n=19). There were also circular DNA molecules extracted (n=4) that appear to encode one viral-like gene and are composed of <1545 nt. The viruses that belonged to the family Genomoviridae clustered with those in the Gemycircularvirus genus. The genomoviruses were extracted from 6 samples. These clustered with gemycircularvirus extracted from arachnids and feces. The anellovirus, extracted from one sample, identified here has a genome sequence that is most similar to those from other rodent species, lagomorphs, and mosquitos. The CRESS viruses identified here were extracted from 9 samples and are novel and cluster with others identified from avian species. This study gives a snapshot of viruses associated with marmots based on fecal sampling.
ContributorsKhalifeh, Anthony (Author) / Varsani, Arvind (Thesis director) / Kraberger, Simona (Committee member) / Dolby, Greer (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Following the journey through the sewerage system, wastewater is subject to a series of purification procedures, prior to water reuse and disposal of the resultant sewage sludge. Biosolids, also known as treated sewage sludge, deemed fit for application on land, is a nutrient-rich, semisolid byproduct of biological wastewater treatment. Technological progression in metagenomics has allowed for large-scale analysis of complex viral communities in a number of samples, including wastewater. Members of the Microviridae family are non-enveloped, ssDNA bacteriophages, and are known to infect enterobacteria. Members of the Genomoviridae family similarly are non-enveloped, ssDNA viruses, but are presumed to infect fungi rather than eubacteria. As these two families of viruses are not relatively documented and their diversity poorly classified, this study aimed to analyze the presence of genomoviruses and the diversity of microviruses in nine samples representative of wastewater in Arizona and other regions of the United States. Using a metagenomic approach, the nucleic acids of genomoviruses and microviruses were isolated, assembled into complete genomes, and characterized through visual analysis: a heat chart showing percent coverage for genomoviruses and a circular phylogenetic tree showing diversity of microviruses. The heat map results for the genomoviruses showed a large presence of 99 novel sequences in all nine wastewater samples. Additionally, the 535 novel microviruses displayed great diversity in the cladogram, both in terms of sub-family and isolation source. Further research should be conducted in order to classify the taxonomy of microviruses and the diversity of genomoviruses. Finally, this study suggests future exploration of the viral host, prior to entering the wastewater system.
ContributorsSchreck, Joshua Reuben (Author) / Varsani, Arvind (Thesis director) / Rolf, Halden (Committee member) / Misra, Rajeev (Committee member) / School of Film, Dance and Theatre (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Arachnids belong to the phylum Arthropoda, the largest phylum in the animal kingdom. Ticks are blood-feeding arachnids that vector numerous pathogens of significant medical and veterinary importance, while scorpions have become a common concern in urban desert cities due to the high level of toxicity in their venom. To date, viruses associated with arachnids have been under sampled and understudied. Here viral metagenomics was used to explore the diversity of viruses present in ticks and scorpions. American dog ticks (Dermacentor variabilis) and blacklegged ticks (Ixodes scapularis) were collected in Pennsylvania while one hairy scorpion (Hadrurus arizonensis) and four bark scorpions (Centruroides sculpturatus) were collected in Phoenix. Novel viral genomes described here belong to the families Polyomaviridae, Anelloviridae, Genomoviridae, and a newly proposed family, Arthropolviridae.
Polyomaviruses are non-enveloped viruses with a small, circular double-stranded DNA (dsDNA) genomes that have been identified in a variety of mammals, birds and fish and are known to cause various diseases. Arthropolviridae is a proposed family of circular, large tumor antigen encoding dsDNA viruses that have a unidirectional genome organization. Genomoviruses and anelloviruses are ssDNA viruses that have circular genomes ranging in size from 2–2.4 kb and 2.1–3.8 kb, respectively. Genomoviruses are ubiquitous in the environment, having been identified in a wide range of animal, plant and environmental samples, while anelloviruses have been associated with a plethora of animals.
Here, 16 novel viruses are reported that span four viral families. Eight novel polyomaviruses were recovered from bark scorpions, three arthropolviruses were recovered from dog ticks and one arthropolvirus from a hairy scorpion. Viruses belonging to the families Polyomaviridae and Arthropolviridae are highly divergent. This is the first more extensive study of these viruses in arachnids. Three genomoviruses were recovered from both dog and deer ticks and one anellovirus was recovered from deer ticks, which are the first records of these viruses being recovered from ticks. This work highlights the diversity of dsDNA and ssDNA viruses in the arachnid population and emphasizes the importance of performing viral surveys on these populations.
Polyomaviruses are non-enveloped viruses with a small, circular double-stranded DNA (dsDNA) genomes that have been identified in a variety of mammals, birds and fish and are known to cause various diseases. Arthropolviridae is a proposed family of circular, large tumor antigen encoding dsDNA viruses that have a unidirectional genome organization. Genomoviruses and anelloviruses are ssDNA viruses that have circular genomes ranging in size from 2–2.4 kb and 2.1–3.8 kb, respectively. Genomoviruses are ubiquitous in the environment, having been identified in a wide range of animal, plant and environmental samples, while anelloviruses have been associated with a plethora of animals.
Here, 16 novel viruses are reported that span four viral families. Eight novel polyomaviruses were recovered from bark scorpions, three arthropolviruses were recovered from dog ticks and one arthropolvirus from a hairy scorpion. Viruses belonging to the families Polyomaviridae and Arthropolviridae are highly divergent. This is the first more extensive study of these viruses in arachnids. Three genomoviruses were recovered from both dog and deer ticks and one anellovirus was recovered from deer ticks, which are the first records of these viruses being recovered from ticks. This work highlights the diversity of dsDNA and ssDNA viruses in the arachnid population and emphasizes the importance of performing viral surveys on these populations.
ContributorsSchmidlin, Kara (Author) / Varsani, Arvind (Thesis advisor) / Van Doorslaer, Koenraad (Committee member) / Stenglein, Mark (Committee member) / Arizona State University (Publisher)
Created2019