Discovery of novel circular replication-associated protein encoding single-stranded DNA viruses in ecosystems using viral metagenomic approaches

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