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Description
Poxviruses such as monkeypox virus (MPXV) are emerging zoonotic diseases. Compared to MPXV, Vaccinia virus (VACV) has reduced pathogenicity in humans and can be used as a partially protective vaccine against MPXV. While most orthopoxviruses have E3 protein homologues with highly similar N-termini, the MPXV homologue, F3, has a start

Poxviruses such as monkeypox virus (MPXV) are emerging zoonotic diseases. Compared to MPXV, Vaccinia virus (VACV) has reduced pathogenicity in humans and can be used as a partially protective vaccine against MPXV. While most orthopoxviruses have E3 protein homologues with highly similar N-termini, the MPXV homologue, F3, has a start codon mutation leading to an N-terminal truncation of 37 amino acids. The VACV protein E3 consists of a dsRNA binding domain in its C-terminus which must be intact for pathogenicity in murine models and replication in cultured cells. The N-terminus of E3 contains a Z-form nucleic acid (ZNA) binding domain and is also required for pathogenicity in murine models. Poxviruses produce RNA transcripts that extend beyond the transcribed gene which can form double-stranded RNA (dsRNA). The innate immune system easily recognizes dsRNA through proteins such as protein kinase R (PKR). After comparing a vaccinia virus with a wild-type E3 protein (VACV WT) to one with an E3 N-terminal truncation of 37 amino acids (VACV E3Δ37N), phenotypic differences appeared in several cell lines. In HeLa cells and certain murine embryonic fibroblasts (MEFs), dsRNA recognition pathways such as PKR become activated during VACV E3Δ37N infections, unlike VACV WT. However, MPXV does not activate PKR in HeLa or MEF cells. Additional investigation determined that MPXV produces less dsRNA than VACV. VACV E3Δ37N was made more similar to MPXV by selecting mutants that produce less dsRNA. By producing less dsRNA, VACV E3Δ37N no longer activated PKR in HeLa or MEF cells, thus restoring the wild-type phenotype. Furthermore, in other cell lines such as L929 (also a murine fibroblast) VACV E3Δ37N, but not VACV WT infection leads to activation of DNA-dependent activator of IFN-regulatory factors (DAI) and induction of necroptotic cell death. The same low dsRNA mutants demonstrate that DAI activation and necroptotic induction is independent of classical dsRNA. Finally, investigations of spread in an animal model and replication in cell lines where both the PKR and DAI pathways are intact determined that inhibition of both pathways is required for VACV E3Δ37N to replicate.
ContributorsCotsmire, Samantha (Author) / Jacobs, Bertram L (Thesis advisor) / Varsani, Arvind (Committee member) / Hogue, Brenda (Committee member) / Haydel, Shelley (Committee member) / Arizona State University (Publisher)
Created2021
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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

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
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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.

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
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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,

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.
ContributorsSchmidlin, Kara (Author) / Varsani, Arvind (Thesis advisor) / Van Doorslaer, Koenraad (Committee member) / Stenglein, Mark (Committee member) / Arizona State University (Publisher)
Created2019
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Description
The family Cactaceae is extremely diverse and has a near global distribution yet very little has been described regarding the community of viruses that infect or are associated with cacti. This research characterizes the diversity of viruses associated with Cactaceae plants and their evolutionary aspects. Five viruses belonging to the

The family Cactaceae is extremely diverse and has a near global distribution yet very little has been described regarding the community of viruses that infect or are associated with cacti. This research characterizes the diversity of viruses associated with Cactaceae plants and their evolutionary aspects. Five viruses belonging to the economically relevant plant virus family Geminiviridae were identified, initially, two novel divergent geminiviruses named Opuntia virus 1 (OpV1) and Opuntia virus 2 (OpV2) and Opuntia becurtovirus, a new strain within the genus Becurtovirus. These three viruses were also found in co-infection. In addition, two known geminiviruses, the squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WCSV) were identified infecting Cactaceae plants and other non-cactus plants in the USA and Mexico. Both SLCV and WCSV are known to cause severe disease in cultivated Cucurbitaceae plants in the USA and Middle East, respectively. This study shows that WCSV was introduced in the America two times, and it is the first identification of this virus in the USA, demonstrating is likely more widespread in North America. These findings along with the Opuntia becurtovirus are probable events of spill-over in agro-ecological interfaces. A novel circular DNA possibly bipartite plant-infecting virus that encodes protein similar to those of geminiviruses was also identified in an Opuntia discolor plant in Brazil, named utkilio virus, but it is evolutionary distinct likely belonging to a new taxon. Viruses belonging to the ssDNA viral family Genomoviridae are also described and those thus far been associated with fungi hosts, so it is likely the ones identified in plants are associated with their phytobiome. Overall, the results of this project provide a molecular and biological characterization of novel geminiviruses and genomoviruses associated with cacti as well as demonstrate the impact of agro-ecological interfaces in the spread of viruses from or to native plants. It also highlights the importance of viral metagenomics studies in exploring virus diversity and evolution given then amount of virus diversity identified. This is important for conservation and management of cacti in a global scale, including the relevance of controlled movement of plants within countries.
ContributorsSalgado Fontenele, Rafaela (Author) / Varsani, Arvind (Thesis advisor) / Wilson, Melissa (Committee member) / Majure, Lucas (Committee member) / Van Doorslaer, Koenraad (Committee member) / Wojciechowski, Martin (Committee member) / Arizona State University (Publisher)
Created2021
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Description
Viruses infect organisms in all domains of life and are abundant entities in ecosystems. In particular, single-stranded DNA viruses have been found in a wide variety of hosts and ecosystems. Using a metagenomic approach, novel circular viruses have been identified in multiple environmental samples. This thesis focuses on viruses and

Viruses infect organisms in all domains of life and are abundant entities in ecosystems. In particular, single-stranded DNA viruses have been found in a wide variety of hosts and ecosystems. Using a metagenomic approach, novel circular viruses have been identified in multiple environmental samples. This thesis focuses on viruses and virus dynamics from avian sources. As part of this thesis, a novel phapecoctavirus was identified in a pigeon cloacal swab. The phapecoctavirus is most closely related to Klebsiella phage ZCKP1, identified from a freshwater sample. Beyond this, this thesis addresses circoviruses, which are of interest due to disease they cause to avian species. Evolution of circovirus recombination was studied in a closed system of uninfected and infected pigeons. 178 genomes of pigeon circovirus were sequenced, and patterns of recombination determined. Seven genotypes were present in the population and genotype 4 was shown to be present in a majority of samples after the experiment was finished. Circoviruses were also identified in waterfowl feces and the ten genomes recovered represent two new circovirus species. Overall, the research described in this thesis helped to gain a deeper understanding of the diversity and evolution of circular DNA viruses associated with avian species.
ContributorsKhalifeh, Anthony (Author) / Varsani, Arvind (Thesis advisor) / Kraberger, Simona J (Committee member) / Dolby, Greer (Committee member) / Arizona State University (Publisher)
Created2021
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Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically

Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
ContributorsPradhan, Swechchha (Author) / Hariadi, Rizal (Thesis advisor) / Hogue, Ian (Committee member) / Varsani, Arvind (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2022
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Description
Monkeypox virus (MPXV) is an orthopoxvirus that causes smallpox-like disease and has up to a 10% mortality rate, depending on the infectious strain. The global eradication of the smallpox virus has led to the decrease in smallpox vaccinations, which has led to a drastic increase in the number of human

Monkeypox virus (MPXV) is an orthopoxvirus that causes smallpox-like disease and has up to a 10% mortality rate, depending on the infectious strain. The global eradication of the smallpox virus has led to the decrease in smallpox vaccinations, which has led to a drastic increase in the number of human MPXV cases. MPXV has been named the most important orthopoxvirus to infect humans since the eradication of smallpox and has been the causative agent of the 2022 world-wide MPXV outbreak. Despite being highly pathogenic, MPXV contains a natural truncation at the N-terminus of its E3 homologue. Vaccinia virus (VACV) E3 protein has two domains: an N- terminus Z-form nucleic acid binding domain (Z-BD) and a C-terminus double stranded RNA binding domain (dsRBD). Both domains are required for pathogenesis, interferon (IFN) resistance, and protein kinase R (PKR) inhibition. The N-terminus is required for evasion of Z-DNA binding protein 1 (ZBP1)-dependent necroptosis. ZBP1 binding to Z- form deoxyribonucleic acid/ribonucleic acid (Z-DNA/RNA) leads to activation of receptor-interacting protein kinase 3 (RIPK3) leading to mixed lineage kinase domain- like (MLKL) phosphorylation, aggregation and cell death. This study investigated how different cell lines combat MPXV infection and how MPXV has evolved ways to circumvent the host response. MPXV is shown to inhibit necroptosis in L929 cells by degrading RIPK3 through the viral inducer of RIPK3 degradation (vIRD) and by inhibiting MLKL aggregation. Additionally, the data shows that IFN treatment efficiently inhibits MPXV replication in a ZBP1-, RIPK3-, and MLKL- dependent manner, but independent of necroptosis. Also, the data suggests that an IFN inducer with a pancaspase or proteasome inhibitor could potentially be a beneficial treatment against MPXV infections. Furthermore, it reveals a link between PKR and pathogen-induced necroptosis that has not been previously described.
ContributorsWilliams, Jacqueline (Author) / Jacobs, Bertram (Thesis advisor) / Langland, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2022
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Description
Despite the prevalence of coyotes (Canis latrans) little is known about the viruses associated with this species. To assess the extent of viral research that has been conducted on coyotes, a literature review was performed. Over the last six decades, there have been many viruses that have been identified infecting

Despite the prevalence of coyotes (Canis latrans) little is known about the viruses associated with this species. To assess the extent of viral research that has been conducted on coyotes, a literature review was performed. Over the last six decades, there have been many viruses that have been identified infecting coyotes. The pathology of some cases implies that infection is rare and lethal while others have been demonstrated to be endemic to coyotes. In addition, the majority of the prior analyses were done through serological assays that were limited to investigating target viruses. To help expand what is known about coyote-virus dynamics, viral assays were conducted on coyote scat. The samples were collected as part of transects established along the Salt River near Phoenix, Arizona, United States (USA). The recovered viral genomes were clustered with other deoxynucleic acid (DNA) viruses and analyzed to determine phylogeny and genetic identity. From the recovered viral genomes, there are two novel circoviruses, one novel naryavirus, five unclassified cressdnaviruses, and two previously identified species of anelloviruses from the Wawtorquevirus genus. For these viruses, new phylogenies for their groups and pairwise identity plots have been generated. These figures give insight into the potential hosts and the evolutionary history. In the case of the anelloviruses, they likely derived from a wood rat (Neotoma) host, given the anellovirus family’s host specificity and its similarity to another viral genome derived from a wood rat in Arizona, USA. Of the recovered circovirus genomes, one is associated with a viral isolate collected from a dust sample in Arizona, USA. The second circovirus species identified is within a clade that consists of rodent associated circoviruses and canine circovirus. Other recovered genomes expand clusters of unclassified cressdnaviruses. The recovered genomes support further genomic analysis. These findings help support the notion that there is a wealth of viral information to be identified from animals like coyotes. By understanding the viruses that coyotes are associated with, it is possible to better understand the viral impact on the urban environment, domesticated animals, and wildlife in general.
ContributorsHess, Savage Cree (Author) / Varsani, Arvind (Thesis advisor) / Kraberger, Simona (Committee member) / Upham, Nathan S (Committee member) / Arizona State University (Publisher)
Created2023
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DescriptionA
ContributorsLund, Michael (Author) / Varsani, Arvind (Thesis advisor) / Upham, Nathan (Committee member) / Harris, Robin (Committee member) / Arizona State University (Publisher)
Created2023