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Item Honey bee host-virus interactions at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) McMenamin, Alexander James; Chairperson, Graduate Committee: Michelle Flenniken; This is a manuscript style paper that includes co-authored chapters.Honey bees are important pollinators of the fruits, nuts and vegetable crops that feed our growing population. Unfortunately, honey bee colony losses have averaged 38% from 2008-2018. These losses are due to a variety of factors, including reduced quality forage, pesticide exposure in agricultural fields, parasites like the Varroa destructor mite, and pathogens. The most diverse group of pathogens effecting honey bees are small RNA viruses. Honey bees have evolved numerous strategies to restrict virus infection, including the RNA interference (RNAi) pathway. Bees infected with a model virus, Sindbis-GFP (SINV) have differential expression of hundreds of genes, including RNAi genes and several heat shock protein (HSP) encoding genes. Therefore, we hypothesized that heat shock proteins are antiviral in honey bees. To induce the heat shock response (HSR), SINV-infected bees were heat shocked at 42°C for 4 hours. Heat shock resulted in a 74-90% reduction in SINV RNA copies as compared to bees maintained at 32°C. Heat shocked and/or virus-infected bees had increased expression of several core HSR protein-encoding genes, but heat shock did not consistently result in the increased expression of RNAi genes (argonaute-2 and dcr-like). This indicates that heat shock proteins are contributing to an antiviral response. SINV-infected bees also had higher expression of a recently identified antiviral gene - bee antiviral protein-1 (bap1). Therefore, we further characterized bap1 using computation approaches including phylogenetic analysis, which determined that this gene is taxonomically restricted to Hymenoptera and Blatella germanica (the German cockroach). Structural predication programs indicated that bap1 is a highly disordered protein. Intriguingly, transcriptome and correlation analyses determined that bap1 was coexpressed with several genes implicated in antiviral immunity (i.e., ago2, tudor-sn and TEP7). Although the precise antiviral function of bap1 remains to be elucidated, we further developed experimental tools that will enable more incisive investigation of bap1 and other antiviral genes. Primary cultures of larval hemocytes (immune cells) and mixed-cell pupal tissue cultures supported productive replication of sacbrood virus, deformed wing virus, and Flock House virus. Infected pupal cell cultures exhibited virus-specific transcriptional responses in bap1, ago2, and dcr-like expression. Together, these data further elucidate honey bee antiviral immunity and provide new tools for studying honey bee host-virus interactions.Item Honey bee antiviral defense mechanisms at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) Parekh, Fenali Mukesh; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Chemical stimulants and stressors impact the outcome of virus infection and immune gene expression in honey bees (Apis mellifera)' in the journal 'Frontiers in immunology' which is contained within this dissertation.; Alexander J. McMenamin was an author and Verana Lawrence and Michelle L. Flennikenwas were co-authors of the article, 'Investigating virus-host interactions in cultured primary honey bee cells' in the journal 'Insects' which is contained within this dissertation.; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Honey bee antiviral response to flock house virus infection' which is contained within this dissertation.; This dissertation contains an article of which Fenali Mukesh Parekh is not the main author.Honey bees are important pollinators of fruit, nut, and vegetable crops that constitute a large proportion of the human diet. Unfortunately, annual honey bee colony losses are high, averaging 38% from 2008-2018 in the United States. Honey bee colony losses are attributed to multiple factors, including pathogens and chemical exposure. Virus incidence and abundance have been associated with colony losses. The majority of honey bee viruses are positive-sense single stranded RNA viruses. Honey bees antiviral defense include RNA interference (RNAi), a double-stranded RNA (dsRNA) triggered sequence-specific post-transcriptional silencing mechanism and a non-sequence specific dsRNA-triggered pathway. In addition, signal transduction cascades include the Toll, Imd, and Jak/STAT pathways that promote the expression of honey bee immune response genes that are also induced in response to virus infections. To investigate the impact of chemical exposure on honey bee immune responses and virus infections, we infected bees with a panel of viruses including two model viruses (i.e., Flock House virus (FHV) and Sindbis-GFP) and a naturally infecting honey bee virus, deformed wing virus (DWV) and fed them sucrose syrup containing either thyme oil, a beekeeper applied fungicide Fumagilin-B ®, or the insecticide clothianidin. We determined that bees fed thyme oil augmented sucrose syrup exhibited greater expression of key immune genes, i.e., ago2, dcr-like, abaecin, hymenoptaecin, and vitellogenin and reduced virus abundance compared to virus-infected bees fed sucrose syrup. Whereas, virus-infected honey bees fed diets containing fumagillin or clothianidin exhibited reduced expression of key immune genes and higher virus abundance suggesting that chemical stressors act as immunosuppressors in honey bees. To understand the interplay of viruses and host cell gene expression more precisely, we cultured primary honey bee cells derived from larvae (i.e., hemocytes, immune cells) or pupae (i.e., mixed cell population including epithelial cells, adipocytes, muscle cells, hemocytes) and demonstrated that these cells supported replication of sacbrood virus, DWV, and FHV. Expression of select immune genes, including bap1, ago2, and dcr-like, in virus-infected honey bee cells was similar to expression in individual bees and varied for each virus. Together, these data further our understanding of the honey bee antiviral defense network and provide new tools for studying honey bee host-virus interactions.Item Understanding resistance and transcriptional responses to potato virus Y infection in potato plants(Montana State University - Bozeman, College of Agriculture, 2021) Ross, Brian Thomas; Chairperson, Graduate Committee: Michelle Flenniken; Nina Zidack and Michelle L. Flenniken were co-authors of the article, 'Extreme resistance to viruses in potato and soybean' in the journal 'Frontiers in plant science' which is contained within this dissertation.; Nina Zidack and Michelle L. Flenniken were co-authors of the article, 'Transcriptional responses to potato virus Y infection in resistant and susceptible potato cultivars' submitted to the journal 'Cultivars' which is contained within this dissertation.The potato is one of the world's most important crops. Cultivation of potatoes occurs on every continent except Antarctica and in a wide variety of climates. Potatoes are susceptible to a multitude of pathogens that can decrease yield and market quality. Viruses are particularly problematic for potato growers, as most potato production involves the replanting of tubers grown the previous year. Because virus-infected potato plants can harbor virus in their tubers, these tubers can in turn be the source of infection in the next generation of plants. Strains of Potato virus Y are the most economically burdensome viruses for potato growers worldwide. In field settings, Potato virus Y is primarily transmitted to plant by aphids feeding on leaves, but PVY can also be transmitted mechanically through infected plant sap. The use of insecticides and the application of mineral oil to leaves can help limit aphid populations and prevent infection to an extent but are generally both less effective and more environmentally impactful than genetic antiviral resistance mechanisms. The incorporation of genes that provide durable resistance to Potato virus Y into commercial potatoes is a major focus of potato breeders. One form of resistance, called extreme resistance, is characterized by a lack of symptoms and little to no virus replication occurring at the site of infection, but the molecular mechanisms of this response are not well understood. A comprehensive analysis of the extreme resistance literature indicates that movement of the resistance protein from the cytoplasm to the nucleus of the cell directly after virus infection may be a key aspect of this immune response. The downstream, transcriptional aspects of the extreme resistance response are also not well understood. We analyzed the gene expression from a Potato virus Y-resistant potato variety, Payette Russet, and a commonly grown susceptible variety, Russet Burbank, at a series of time points after virus infection using RNA sequencing. Results of these analyses indicate that an immune response likely occurs in Payette Russet quickly after virus inoculation. These analyses also indicate that the virus-susceptible variety, Russet Burbank, exhibits changes in gene expression that are similar to other susceptible potato varieties during asymptomatic or tolerant infection. Furthering our understanding of the molecular mechanisms controlling resistance and severity of virus infections will help inform future breeding and genetic engineering efforts, which require detailed knowledge of the mechanisms of virus resistance.Item Virus host interactions at the single cell level in hot springs of Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2019) Munson-McGee, Jacob Hampton; Chairperson, Graduate Committee: Mark J. Young; Jamie C. Snyder and Mark J. Young were co-authors of the article, 'Introduction to archaeal viruses' in the journal 'Genes' which is contained within this dissertation.; Ross Hartman was an author and Mark J. Young were co-authors of the article, 'vFish for the quantification of viral infection in natural environments' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.; Erin K. Field, Mary Bateson, Colleen Rooney, Ramunas Stepanauskas and Mark J. Young were co-authors of the article, 'The identification and characterization of a nanoarchaeota, its cellular host and a nanoarchaeal virus across Yellowstone National Park hot springs' which is contained within this dissertation.; Colleen Rooney and Mark J. Young were co-authors of the article, 'An uncultivated virus infecting a nanoarchaeal parasite in the hot springs of Yellowstone National Park' submitted to the journal 'Virology' which is contained within this dissertation.; Shengyun Peng, Samantha Dewerff, Ramunas Stepanauskas, Rachel J. Whitaker, Joshua Weitz and Mark J. Young were co-authors of the article, 'A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments' in the journal 'The ISME journal' which is contained within this dissertation.Viruses are the most abundant biological entities on the planet and virus-host interactions are some of the most important factors in shaping microbial community structure and function and global chemical cycling. The high temperature low pH hot spring of Yellowstone National Park contain simplified microbial communities of 8-10 Archaeal species, and comparatively simple viral communities. These idealized communities that contain only viruses and their Archaeal hosts represent a model natural environment for the study of viruses and their hosts. This work presented here builds on previous population level studies of the viral and microbial communities to examine virus-host interactions at the single cell level. The identification of viral infection has long been a scourge of environmental virologist. In order to identify viral infection in natural environments we have adapted Fluorescent in situ hybridization (FISH) techniques to directly identify viral sequences. We further advance this technique to be compatible with flow cytometry analysis for the rapid quantification of viral infection of uncharacterized viruses in natural environments. This technique is used to quantify viral infection of two different viruses, previously only characterized by metagenomic sequencing analysis, in four geographically separate low pH high temperature hot springs of Yellowstone National Park. Finally, we combine viral and cellular metagenomics with cellular transcriptomics and single cell genomics to identify virus host interactions at the single cell level and identify viruses that are replicating in the hot springs. This work suggests that a majority of cells in the hot springs are interacting with viruses and that a majority of the cells are interacting with multiple viruses at any given time. We also identify RNA sequences from a majority of the viral types present in the hot springs suggesting that viral replication is occurring and is an important force in determining the structure and function of the microbial communities in these hot springs. Together these works represent a significant advancement of our understanding of virus host interactions in natural environments as well as new techniques to be used in future studies.Item Biological and structural properties of wild-type and mutant mengoviruses(Montana State University - Bozeman, College of Agriculture, 1984) Anderson, KevinItem Evaluation of susceptibility to wheat streak mosaic virus among small grains and alternative hosts in the Great Plains(Montana State University - Bozeman, College of Agriculture, 2011) Ito, Dai; Chairperson, Graduate Committee: Mary Burrows.Wheat streak mosaic virus (WSMV), endemic in small grains production areas of the Great Plains, causes yield losses of wheat 2 to 5% annually. Yield loss in individual fields can reach 100%. Control relies on cultural practices to control the vector, the wheat curl mite (Aceria tosichella Keifer, WCM), and the use of resistant or tolerant varieties. WSMV and WCM depend on living tissue for survival and reproduction, including common grassy weeds. Little is known about the relative importance of these weeds as alternative hosts of WSMV. The purpose of these studies was to evaluate the risk of infection with WSMV in commonly grown wheat varieties and various grassy weed species, information useful to understanding WSMV epidemiology and control. Winter wheat, spring wheat and barley varieties in Montana were evaluated in the field by measuring the effect of fall vs. spring inoculation and variety on incidence, symptom severity, and yield components. Winter wheat varieties from five states, and spring wheat and barley varieties from Montana were tested for incidence and absorbance in greenhouse. Fall-inoculated winter wheat had less effect of WSMV inoculation compared to spring-inoculated winter wheat. Yields of spring wheat varieties were largely reduced by WSMV inoculation. There was no correlation between yield and incidence or symptom severity. In greenhouse studies, the highest incidence was observed in varieties from Idaho and Nebraska, whereas the highest relative absorbance was observed in varieties from Montana. In 2008 and 2009, surveys of common grassy weeds were conducted. Grass species from croplands in six states were selected and mechanically inoculated to determine the susceptibility to WSMV. Grassy weeds were also evaluated as a source of WSMV by measuring transmission efficiency with virulifeous WCM. Bromus tectorum was the most prevalent grassy weed and the most frequent viral host. Aegilops cylindrica, and Avena fatua had the highest incidence and relative absorbance. There were no differences in the susceptibility of grass species to WSMV by their state of origin. WCM transmission study indicated infected grass species had lower transmission efficiency than from infected wheat. These studies will benefit producers in Montana to assess their risk of WSMV based on variety selection and the presence of grassy weeds.Item Crenarchaeal virus-host systems : structure-function studies of crenarchaeal viruses and prokaryotic adaptive immunity(Montana State University - Bozeman, College of Letters & Science, 2011) Lintner, Nathanael Greenleaf; Chairperson, Graduate Committee: C. Martin Lawrence; Valerie Copie (co-chair)Virus-host interactions are one of the most important drivers of microbial ecology and evolution. Among the three domains of life, the least is known about viruses infecting members of the domain Archaea. This work combines a traditional hypothesis-driven molecular and biochemical approach with a structural genomics-like approach of pursuing a large number of structural targets to gain a detailed molecular understanding of a model Crenarchaeal virus-host system, Sulfolobus turreted icosahedral virus (STIV) and its Sulfolobus host. This work specifically focuses on the viral protein, A81, a putative transcriptional regulator associated with the prokaryotic adaptive immune system, CRISPR-Cas, and a large protein-RNA complex involved in CRISPR-mediated DNA interference. A81 is a protein-of-unknown-function encoded by the STIV genome. The structure of STIV-A81 reveals a unique ring-shaped octameric assembly. While structural homology-based searches fail to reveal a function for A81, the central pore has a size and charge consistent with a potential interaction with single-stranded nucleic acid. The structure of the CRIPSR-associated protein, Csa3 reveals a putative 2-domain transcription factor. The N-terminal domain is a variation on the di-nucleotide binding-domain that orchestrates dimer formation. There is a conserved 2-fold symmetric pocket on the dimer axis that likely represents a regulatory ligand-binding site implying a small-molecule regulator of CRISPR/Cas. The C-terminal domain is a winged helix-turn-helix common among transcription factors. The domain architecture of Csa3 suggests a small molecule regulator of CRISPR/Cas in the Crenarchaea. The CRISPR-associated complex for antiviral defense (CASCADE) is predicted to be central to CRISPR-mediated DNA-interference in many bacteria and Archaea. We isolated components of an archaeal CASCADE from Sulfolobus solfataricus. Csa2 expressed in S. solfataricus co-purifies with Cas5a, Cas6, Csa5 and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein, forms a stable complex with Cas5a. A recombinant Csa2-Cas5a-complex is sufficient to bind crRNA and complementary ssDNA. Transmission electron microscopy reveals an extended helical complex of variable length, perhaps due to substochiometric amounts of capping factors. Csa2 displays a crescent-shaped fold including a modified RNA-recognition motif (RRM) plus two additional domains present as insertions into the RRM. A preliminary model for this and other CASCADEs is proposed.Item Archaeal host virus interactions(Montana State University - Bozeman, College of Letters & Science, 2011) Wirth, Jennifer Fulton; Chairperson, Graduate Committee: Mark J. YoungViruses are the most abundant biological entity on earth, and virus-host interactions are one of the most important factors shaping microbial populations (Suttle, 2007b). The study of both the cellular and viral members of the domain Archaea is a relatively new field. Thus, the viruses (and their cellular hosts) of Archaea are poorly understood as compared to viruses of Bacteria and Eukarya. This work has sought to expand our understanding of archaeal viruses by two general approaches. The first is by developing and implementing the use of a genetic system for a crenarchaeal virus, Sulfolobus turreted icosahedral virus (STIV), isolated from a hot (82°C) acidic (pH 2.2) pool in Yellowstone National Park, USA. The second approach has been to look at viral communities and their interactions with their cellular hosts in natural environments. We have developed a genetic tool, an infectious clone for STIV, which has allowed for genetic analysis of this virus. A number of viral genes have been knocked out, and their functions investigated using this tool. We have determined that at least three viral genes, A197, B345 and C381, are required for viral replication, while one gene, B116, is not essential. Work continues investigating function for other STIV genes as well as specific interactions with its host, Sulfolobus solfataricus. We have performed total community sequencing (metagenomics) for both the cellular and viral populations of several hot springs in Yellowstone National Park. We have been able to assemble a near full-length putative novel viral genome from one of these sites. We have also performed an in depth analysis of the function of a newly described bacterial and archaeal adaptive immune system (CRISPR/Cas) in a natural environment. This study has provided insights into the function of this immune system in a complex nutrient limited environment, which would not have been observed by studying cultured isolates in a laboratory.