The introduction of next-generation sequencing (NGS) technologies has dramatically changed the field of virology, with many significant discoveries of novel circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses. Traditionally, most research into CRESS DNA viruses has often focused on investigating plant and animal pathogens that are of significant economic importance. This research has led to the discovery and establishment of three different CRESS DNA families including Geminiviridae, Nanoviridae and Circoviridae, which infect eukaryotes. CRESS DNA viruses can have single or multicomponent genomes, with the latter requiring all components for infection. CRESS DNA viruses have circular single-stranded DNA (ssDNA) genomes with at least one protein encoding a Rep which is responsible for viral replication. It has been shown that CRESS DNA viruses are able to evolve rapidly with nucleotide substitution rates that are similar to those observed in RNA viruses. The Rep gene has conserved regions known as motifs which are often used to determine relatedness between CRESS DNA virus. NGS has expanded our knowledge on the diversity of novel CRESS DNA viruses. Viral genomes are now routinely recovered from different sample types without any prior knowledge of the viral sequence. This has led to the development of the field of viral ecology. This field places an emphasis on viruses being one of the most abundant organisms on earth, and are therefore likely to play a major role in ecosystems. Environmental metagenomic studies have isolated CRESS DNA viruses from sea water, freshwater, faecal matter from various animals, soil, the atmosphere, sediments and sewage; dramatically increasing the known CRESS DNA viral genomes in the public domain. These studies are shedding light on the distribution of CRESS DNA viruses, as well as providing baseline data for future studies to examine virus-host interactions, community structure and ultimately viral evolution. Vector enable metagenomics (VEM) is another novel approach utilising NGS techniques for discovering CRESS DNA viruses. As many plant-infecting CRESS DNA viruses such as geminiviruses and nanoviruses are vectored by insects, this approach exploits this mechanism by using insect vectors as a surveillance tool to monitor and survey these viruses circulating in ecosystems. Recent studies have used these methods to identify known viral plant pathogens as well as novel viruses circulating in insect vectors such as whiteflies and other higher order insects such a mosquitoes and dragonflies. These approaches successfully demonstrated that VEM can be used as a unique method, with the first mastrevirus discovered in the new world being recovered from dragonfly species Erythrodiplax fusca using this approach. The research in this thesis uses metagenomics to survey CRESS DNA viral diversity in different organisms and environments. Two hundred and sixty eight novel CRESS DNA viruses were recovered and verified in this study from a range of sample types (adult Odonata, Odonata larvae, Mollusca, benthic sediment, water, Oligochaeta and Chironomidae) collected in the United States of America, Australia and New Zealand. All viral genomes isolated had two major proteins encoding for a putative Rep and coat protein (CP), with major Rep motifs identified in most Reps. Phylogenetic analysis of the Reps encoded by the viral genomes highlighted that most were extremely diverse falling outside of the previously described ssDNA viral families. A top-down approach was implemented to recover CRESS DNA viruses and possible viral pathogens from Odonata and their larvae. Thirty six viral genomes were recovered from terrestrial adult dragonflies as well as the twenty four from aquatic larvae. Dragonfly cycloviruses were isolated from the some adult Odonata species which were closely related to the isolates previously described by Rosario et al. (2012). The viruses isolated in the aquatic and terrestrial ecosystems differed substantially indicating that different CRESS DNA viromes exist in both land and water. The diversity of CRESS DNA viruses in seven different mollusc species (Amphibola crenata, Austrolvenus stutchburyi, Paphies subtriangulata, Musculium novazelandiae, Potamopyrgus antipodarum, Physella acuta and Echyridella menziesi) from Lake Sarah and the Avon-Heathcote estuary both in New Zealand, were also investigated. One hundred and forty nine novel viral genomes were recovered. Two CRESS DNA genomes were recovered from molluscs which have Rep-like sequences most closely related to those found in some bacterial genomes. Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) was originally isolated from fungal species Sclerotinia sclerotiorum in china and was later found in benthic sediments in New Zealand. As part of this study, SsHADV-1 was recovered from dragonflies (Erythemis simplicicollis, Ischnura ramburii and Pantala hymenaea) collected in Arizona and Oklahoma, USA suggesting a larger distribution of these viruses and not surprising given the near global distribution of S. sclerotiorum. Dragonfly larvae-associated circular DNA viruses (DflaCVs) that were originally isolated in Odonata larvae samples from three New Zealand lakes were later recovered from water, benthic sediment, worms and molluscs from one of the lakes initially sampled, suggesting that these viruses are ubiquitous in freshwater environments. This study has attempted to generate baseline data of CRESS DNA viruses in certain environments using NGS-informed approaches. This data was used to try and establish whether viral distribution in different samples types can potentially be explained by the food web interactions between different samples types. Although the analysis did not show any significant relationships between sample type interactions and viral distribution a few common associations between Odonata larvae and benthic sediment were evident. This was expected as the larvae live within the sediment so it could be assumed that they potentially have similar CRESS DNA viral distribution. Although the distribution of viruses varied across sample types, molluscs proved the best sampling tool for isolating largest numbers of CRESS DNA viruses in an ecosystem with extensive diversity. Overall, this research demonstrates the applications of NGS for investigating the diversity of CRESS DNA viruses. It demonstrates that some sample types such as Odonata in terrestrial systems and molluscs in aquatic environments, can be used as effective sampling tool to determine the diversity of CRESS DNA viruses in different environments as well as detecting previously isolated viruses. The CRESS DNA viruses isolated in this body of work provides baseline data that can potentially be used in future research to investigate hosts of these viruses and their interactions with hosts and potential flow in their environments.

Viruses has a relatively recently been discovered in high abundances in aquatic systems. Their possible importance has therefore been largely overlooked. In aquatic food webs there should be large differences in carbon and nutrient fluxes depending on whether the main cause of bacterial mortality is viral infection or grazing, where lysis following viral infection should result in a release of organic carbon and nutrients from the lysed bacteria and hence not reach higher trophic levels. Recent research on aquatic viruses has mainly focused on marine environments and the number of studies on freshwater viral ecology is limited. Hence, there is a need for more studies on the importance and functioning of viruses in freshwater systems. The aims of this thesis were to explore the functioning and diversity of viruses that infects bacteria (phages) in freshwater systems. To effectively address this I conducted two experiments and three field studies in 23 lakes in different parts of Sweden. The results show that viral infection and subsequent lysis of the host cell can partly explain the formation of non-nucleoid-containing bacteria and further that viruses can respond to increases in phosphorus concentrations without any net increase in bacterial abundance. Generally, a larger part of bacterial production in lakes were grazed by flagellates than lysed by viruses, but a larger fraction of the total bacterial mortality can be attributed to viruses in hypolimnion compared to in epilimnion. Further, the largest impact of phages on bacterial production may be in humic lakes, which have a relatively high frequency of visibly infected bacterial cells, but low flagellate abundance. Community composition of bacteria and viruses were only weakly coupled in the studied systems. The most important factors for predicting viral community composition were temperature and concentrations of dissolved organic carbon, total phosphorus and soluble reactive phosphorus. The viral community composition changed over the season and temperate phages could be detected by incubations with mitomycin C showing that a large fraction of the viruses detected appeared to be temperate phages. The most important environmental factor co-varying with viral community composition was again concentrations of total phosphorus. To summarize, bacteriophages, as a bacterial mortality factor, are important in freshwater microbial food webs and phosphorus supply has a potential central function in the regulation of the importance of bacteriophages and additionally for viral diversity.

Viruses represent the most abundant biological entities on earth where, they are able to interact with all kingdoms of life. Yet their diversity, ecology and evolutionary aspects are only beginning to be fully elucidated, mainly due to technical limitations. The vast majority of the microbial world remains elusive to culture; more than 90% of genome sequenced viral isolates infect only 5 of the 54 prokaryotic phyla that are currently recognized. In contrast, viral metagenomics bypasses the need for cultures by directly sequencing fragmented genetic material of environmental viral communities. This dissertation uses viral metagenomics by applying well-tested bioinformatic protocols and expanding them to compare and contrast patterns of diversity, richness and specialization of large viral metagenomic datasets, in both local and global scales. First I demonstrate the utility of a functional-based perspective by adopting the protein cluster environment to estimate global viral diversity. Then, I use this PC approach to analyze metagenomes from two ecologically different environments, which by uncovering local gene specialization showcases the adequacy of a gene-centered workflow. Then I continue to expand upon this PC framework to study the Tara Oceans virome analyses of these data reveal patters of diversity that support a seed bank model. Finally, in search of a more meaningful ecological unit, I move from a gene-centered standpoint towards a population-based frame. We adopted a novel metagenomic technique that allowed me to uncover the discontinuity in the genomic sequence space, thus empirically defining a population. This final contribution will allow to sort and count viral communities, the first step to applying ecological and evolutionary theory.

Viruses are ubiquitous biological entities whose importance and role in aquatic habits is beginning to take form. However, several habitats have undergone limited to no examination with viral-geochemical parameters minimally examined and viral-mineral relationships in the natural environment and the role of mineralization on viral-host dynamic completely lacking. To further develop knowledge on the presence and abundances of viruses, how viruses impact aquatic systems, and how viral-host interactions can be impacted under mineralizing conditions, viruses were examined under a variety of habitats and experimental conditions. Water samples were collected from the deep subsurface (up to 450 m underground) and acid mine drainage (AMD) systems in order to determine the presence, abundance, and viral-geochemical relationships within the systems. Samples were also collected from a variety of freshwater habitats, which have undergone limited examination, to determine viral-geochemical and viral-mineral relationships. Lastly, bacteriophage-host dynamics were examined under authigenic mineral precipitation to determine how mineralization impacts this relationship. Results reveal that not only are viruses present in the deep subsurface and AMD systems, but they are abundant (up to 107 virus-like particles/mL) and morphogically diverse. Viruses are also the strongest predictor of prokaryotic abundance in southern Ontario freshwater systems where potential nutrients are rich. Geochemical variables, such as pH and Eh, were shown to have negative impacts of viral abundance indicting that AMD environments are detrimental for free viruses (i.e. not particle associated). Direct evidence of viral-mineral interactions was found using transmission electron microscopy as viral particles were shown attached to iron-bearing mineral phases (determined through elemental analysis). In addition, evidence of viral participation in mineralization events was found in both AMD and freshwater environments where inverse correlations were noted between viral abundance and jarosite saturation indices (r = -0.71 and r = -0.33, respectively), and goethite saturation indices were also noted to be the strongest predictor of VLP abundance in freshwater habitats explaining 78% of the variability in the data. Lastly, iron precipitation and/or metal ion binding to bacterial surfaces greatly reduced phage replication (~98%) revealing bacterial mineralization has a protective benefit strongly hindering viral replication.

Viruses are ubiquitous biological entities whose importance and role in aquatic habits is beginning to take form. However, several habitats have undergone limited to no examination with viral-geochemical parameters minimally examined and viral-mineral relationships in the natural environment and the role of mineralization on viral-host dynamic completely lacking. To further develop knowledge on the presence and abundances of viruses, how viruses impact aquatic systems, and how viral-host interactions can be impacted under mineralizing conditions, viruses were examined under a variety of habitats and experimental conditions. Water samples were collected from the deep subsurface (up to 450 m underground) and acid mine drainage (AMD) systems in order to determine the presence, abundance, and viral-geochemical relationships within the systems. Samples were also collected from a variety of freshwater habitats, which have undergone limited examination, to determine viral-geochemical and viral-mineral relationships. Lastly, bacteriophage-host dynamics were examined under authigenic mineral precipitation to determine how mineralization impacts this relationship. Results reveal that not only are viruses present in the deep subsurface and AMD systems, but they are abundant (up to 107 virus-like particles/mL) and morphogically diverse. Viruses are also the strongest predictor of prokaryotic abundance in southern Ontario freshwater systems where potential nutrients are rich. Geochemical variables, such as pH and Eh, were shown to have negative impacts of viral abundance indicting that AMD environments are detrimental for free viruses (i.e. not particle associated). Direct evidence of viral-mineral interactions was found using transmission electron microscopy as viral particles were shown attached to iron-bearing mineral phases (determined through elemental analysis). In addition, evidence of viral participation in mineralization events was found in both AMD and freshwater environments where inverse correlations were noted between viral abundance and jarosite saturation indices (r = -0.71 and r = -0.33, respectively), and goethite saturation indices were also noted to be the strongest predictor of VLP abundance in freshwater habitats explaining 78% of the variability in the data. Lastly, iron precipitation and/or metal ion binding to bacterial surfaces greatly reduced phage replication (~98%) revealing bacterial mineralization has a protective benefit strongly hindering viral replication.

No description available.