Background. Viruses strongly influence microbial population dynamics and ecosystem functions However, our ability to quantitatively evaluate those viral impads is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenornes (vromes). This leaves the ecology of nondsDNA viruses nearly unlmovvn, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). Methods. Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results. Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were 1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryoteinfecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion. Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Mastreviruses (family Geminiviridae) are plant-infecting viruses with circular single-stranded (ss) DNA genomes (~2.7kb). The genus Mastrevirus is comprised of thirty-two species which are transmitted by leafhoppers belonging to the genus Cicadulina. Mastreviruses are widely distributed and have been found in the Middle East, Europe, Asia, Australia, Africa and surrounding islands. Only one species, dragonfly-associated mastrevirus has so far been identified in the Americas, isolated from a dragonfly in Puerto Rico. Species can be group based on the host(s) they infect, those which infect monocotyledonous (monocot) plants and those which infect dicotyledonous (dicot) plants. In recent years many new mastrevirus species have been discovered. Several of these new discoveries can largely been attributed to the development of new molecular tools. The current state of sequencing platforms has made it affordable and easier to characterise mastreviruses at a genome level thus allowing scientists to delve deeper into understanding the dynamics of mastreviruses. A few mastrevirus species have been identified as important agricultural pathogens and as a result have been the focus of much of the mastrevirus research. Maize streak virus, strain A (MSV-A) has been the most extensively studied due to the devastating impact it has on maize production in Africa. Studies have shown that MSV-A likely emerged as a pathogen of maize less than 250 years following introduction of maize in Africa by early European settlers. There is compelling evidence to suggest that MSV-A is likely the result of recombination events between wild grass adapted MSV strains. It therefore is equally important to monitor viruses infecting non-cultivated plants in order to gain a greater understanding of the epidemiological dynamics of mastreviruses, which in turn is essential for implementing disease management strategies. The objective of the research undertaken as part of this PhD thesis was to investigate global mastrevirus dynamics focusing on diversity, host and geographic ranges, mechanisms of evolution, phylogeography and possible origins of these viruses. In addition to this a viral metagenomic approach was used in order to identify novel mastreviruses or mastrevirus-like present in New Zealand. The dynamics of the monocot-infecting mastreviruses are investigated in Chapter Two and Three. The work described in these two chapters focus mainly on mastreviruses which infect non-cultivated grasses in Africa and Australia, a total of 161 full mastrevirus genomes were recovered collectively in the two studies. Chapter Two reveals a high level of mastrevirus diversity present in Australia with the discovery of four new species and several new strains of previously characterised species. An extensive sampling effort in Africa undertaken in Chapter Three reveals a broader host range and geographic distribution of the African monocot-infecting mastreviruses than previously documented. Mosaic patterns of recombination are evident among both the Australian and African monocot-infecting mastreviruses. In Chapters Four, Five and Six a comprehensive investigation was undertaken focusing on the dicot-infecting mastreviruses. The study undertaken in Chapter Four entailed the recovery of 49 full mastrevirus genomes from Australia, the Middle East, Africa, Turkey and the Indian Subcontinent to investigate the diversity of dicot-infecting mastreviruses from a global context. Analyses revealed a high degree of CpCDV strain diversity and extended the known geographic range of CpCDV. For the first time phylogeographic analysis was able to investigate the origins of the dicot-infecting mastreviruses. Results revealed the likely origin of the most recent common ancestor (MRCA) of these viruses is likely closer to Australia than anywhere else that dicot-infecting mastreviruses have been sampled and illuminated a supported series of historical movements following the emergence of the MRCA. In Chapter Five two novel mastreviruses Australian-like mastreviruses were isolated from chickpea material from Pakistan. A comprehensive analysis of CpCDV isolates in the major pulse growing regions of Sudan in Chapter Six reveals that this region harbours a high degree of strain diversity. Complex patterns of intra-species recombination indicate these strains are evidently circulating in these regions and infecting the same hosts, driving the emergence of new CpCDV strains. Collectively the results discussed in Chapters Two through Six extended the current knowledge of mastrevirus diversity. The natural host range of many mastreviruses has proven to be more extensive than previously documented, with many species having overlapping host ranges and hence these hosts could be acting as ‘mixing vessels’ enabling inter-species recombination. Patterns of recombination and selection were observed in both the monocot-infecting and the dicot-infecting mastreviruses further elucidating the mechanisms these viruses employ to evolve rapidly. Extensive sampling in a wide range of geographic regions provides insights into the true geographic range of species such as MSV and CpCDV. Given that mastreviruses have been able to move globally and Australia has been identified as a major mastrevirus diversity hotspot it is conceivable that mastreviruses are also present in New Zealand. In Chapter Seven and Eight this is explored by using a viral metagenomic approach to investigate the ssDNA viral populations associated with wild grasses and sewage material in New Zealand. Although no mastreviruses were recovered, this endeavour resulted in the discovery of more than 50 novel circular Rep-encoding ssDNA (CRESS DNA) viruses associated with non-cultivated grasses and treated sewage material, many of which are similar to mastreviruses and other geminiviruses. These discoveries expand current knowledge on the diversity of ssDNA viruses present in New Zealand and further highlight this viral metagenomic approach as an effective method for ssDNA virus discovery. Overall the results discussed in this thesis provide insights into mastrevirus diversity and dynamics as well as revealing a wealth of novel CRESS DNA viruses, some of which share similarities to geminiviruses.

Diseases in marine animals are emerging at an increasing rate. Disease forecasting enabled by virus surveillance presents a proactive solution for managing emerging diseases. Broad viral surveys aid in disease forecasting by providing baseline data on viral diversity associated with various hosts, including many that are not associated with disease. However, these viruses can become pathogens due to expansion in host or geographic range, as well as when changing conditions shift the balance between commensal viruses and the host immune system. Therefore, it is extremely valuable to identify and characterize viruses present in many different hosts in a variety of environments, regardless of whether the hosts are symptomatic or not. The lack of a universal gene shared by all viruses makes virus surveillance difficult, because no single assay exists that can detect the enormous diversity of viruses. Viral metagenomics circumvents this issue by purifying viral particles directly from host tissues and sequencing the nucleic acids, allowing for virus identification. However, virus identification is only the first step, which should ideally be followed by complete sequencing of the viral genome to identify genes of interest and develop assays to reveal viral prevalence, tropism, ecology, and pathogenicity. This dissertation focuses on the discovery of novel viruses in marine animals, characterization of complete viral genomes, and the development of subsequent diagnostic assays for further analysis of virus ecology. First, viral metagenomics was used to explore the viruses present in the healthy Weddell seal (Leptonychotes weddellii) population in Antarctica, which led to the discovery of highly prevalent small, circular single-stranded DNA (ssDNA) viruses. The lack of knowledge regarding the viruses of Antarctic wildlife warrants this study to determine baseline viral communities in healthy animals that can be used to survey changes over time. From the healthy Weddell seals, viral metagenomics led to the discovery of 152 novel anellovirus genomes, encompassing two anellovirus species. Characterizing these viruses is important for understanding the prevalence and diversity of ssDNA viruses, which have only recently been described in marine animals. Furthermore, since emerging diseases can be caused by changing conditions affecting host susceptibility to a virus that was previously not related to disease (opportunistic pathogen), having baseline data allows for quick identification of the pathogen. In addition to determining baseline data, viral metagenomics can explore the role of viruses in disease. A novel virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in the Atlantic sea star Asterias forbesi displaying symptoms of sea star wasting disease (SSWD). AfaCV was the first circular replicase-encoding ssDNA (CRESS-DNA) virus discovered in echinoderms, but it was only present in 10% of SSWD sea stars indicating it is not involved in the development of the disease. This dissertation also focuses on elucidating the role of two previously characterized viruses, chelonid fibropapillomatosis-associated herpesvirus (CHHV5; Chelonid herpesvirus 5, ChHV5) and Zalophus californianus anellovirus (ZcAV), in animal health. PCR amplicon sequencing was used to obtain large portions of the 132 kb genome of ChHV5, the putative etiological agent of the neoplastic sea turtle disease, fibropapillomatosis. Obtaining the genome of ChHV5 from Florida green, Kemp’s ridley, and loggerhead sea turtles provides data for phylogenetic analysis across geographic locations and sea turtle species, as well as a reference for designing downstream molecular assays to examine viral latency. ZcAV was first described from the lungs of captive sea lions involved in a mortality event. PCR could not detect ZcAV in the blood of infected animals, and since sea lions are a protected species, it is not possible to obtain lung biopsies from live sea lions to determine ZcAV prevalence or its role in sea lion health. To answer these important questions, an enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to ZcAV in serum from wild sea lion populations. This newly developed ELISA showed that sea lions mount an immune response to ZcAV, and was used to determine the prevalence of ZcAV among wild sea lion populations. This dissertation makes an important contribution to marine science through discovery and characterization of viruses present in healthy and diseased marine animals. Several different methods were used for virus whole-genome sequencing including viral metagenomics, PCR amplicon sequencing, and target enrichment. These findings were expanded upon by developing and using PCR assays and a serological assay to screen for virus prevalence. These methods have implications for viral surveillance and understanding the role of novel viruses in animal health.

The discovery of large DNA viruses has challenged the traditional perception of viral complexity due to their enormous genome size and physical dimensions. Previously, viruses were considered small, filterable agents until the discovery of large DNA viruses. Among large DNA viruses, the phylum Nucleocytoviricota and its members, which are often called "giant viruses" have large genome sizes (up to 2.5 Mbp) and virion sizes (up to 1.5 um). Due to having large virion and genome sizes, these viruses were often excluded from viral surveys and remained understudied for years. Luckily, the advancement of metagenomic analysis has facilitated the study of large DNA viruses by analyzing them directly from their environment without cultivating them in the lab, which could be challenging for viruses. In the first chapter of the thesis, I investigated 11 metagenome-assembled genomes (MAGs) of giant viruses previously surveyed from Station ALOHA in the Pacific Ocean. St. ALOHA is located near Hawaii and represents oligotrophic gyres which the majority of the ocean is made of them. I focused on 11 MAGs of giant viruses to get insight into their phylogenetic characteristics, genomic repertoire, and global distribution patterns. Despite the fact that metagenomic analysis has facilitated the study of genetic materials of microbes and viruses on a huge scale, it is essential to benchmark the performance of metagenomic tools and understand the associated biases, particularly in viral metagenomics. In the second chapter, I evaluated the performance of metagenomic tools (contigs assembler and binning tool) in recovering viral genomes using annotated dataset. We used a metagenome simulator (CAMISIM) to generate simulated short reads with known composition to assess these processes. Moreover, I emphasized the importance of binning contigs for viral genomes to fully recover the genomes of viruses along with discussing how diversity metrics were differed for contigs, bins populations. / Master of Science / Viruses are generally thought to be small biological agents with small genome (genetic material) sizes and tiny physical structures; for instance, the genome length of a Human Immunodeficiency Virus (HIV) is around 10 kilobase pair (a unit for measuring genetic material in an organism), and the virion size (physical dimension of a virus) can go up to 120 nm. The discovery of large DNA viruses has challenged the idea of considering viruses as small biological entities, as their genome sizes and physical dimensions can be up to 2.5 megabase pairs and 1500 nm, respectively. Famous members of large DNA viruses from the phylum Nucleocytoviricota are often known as "Giant Viruses'' because they have enormous genome sizes and physical dimensions. Due to having large viral particles, these viruses may usually be excluded from viral surveys. For instance, in field studies, samples must be filtered through a fraction (e.g., 0.2 um) to eliminate bacterial and archaeal genomes and cellular debris, which also results in excluding larger viruses. Since these viruses remain understudied for several years because of biases associated with having large viral particles, there is a solid need to discover and investigate more about them. Growing and cultivating viruses in the laboratory may be challenging, as they need specific hosts to be dependent on to produce more viral progeny and some specific laboratory environments. Luckily, with the advancement of biotechnology, scientists could find ways to evade the need for cultivating viruses in the lab and study them with computational tools such as metagenomic analysis and bioinformatic tools. Metagenomics analysis helps to study the genetic materials of microbial or viral populations directly from their habitat without growing them in a laboratory. In short, metagenomic analysis has multiple steps, including collecting and filtering samples, fragmenting DNA within the samples, generating short DNA sequences (short-read sequences) with NGS (Next Generation Sequencing) technology, assembling short-read sequences into large DNA fragments which can be contigs (contiguous DNA fragments) and metagenome-assembled genome (MAGs). With metagenomic analysis, we can recover the genome of multiple organisms, and we name the recovered genome as metagenome-assembled genome (MAGs) as it is generated through metagenomic processes. The metagenomic analysis will allow us to study microbes and viruses in their environment and gain insight into their taxonomic details, genomic content, and how widespread they are. In the first chapter, I studied 11 MAGs of giant viruses previously surveyed from St. ALOHA, Hawaii. St. ALOHA is a good field site for examining microbial processes and diversity and a good representative of oligotrophic waters (low in nutrients). I examined 11 MAGs of giant viruses to investigate their taxonomic characteristics to clarify which order they belong to within their phylum, their genomic content, and their global distribution pattern. Although studies have successfully recovered the genome of large DNA viruses from their habitats and then analyzed them, all these metagenomic processes need to be evaluated so the results will be valid to consider as the genome of our interested organisms. In the second chapter, I developed a workflow for viral metagenomic analysis to assess metagenomic tools' performance in recovering reliable viral genomes, particularly for large DNA viruses. Most of these benchmarking workflows are done for bacterial and archaeal genomes, and in this thesis, I used these metagenomic tools and applied them to recover large DNA viruses genomes. Also, I emphasized the importance of using binning tools to fully recover large DNA viruses genomes, as due to their large genome size, their genomes might remain fragmented into different contigs, which are longer sequences than reads but shorter than MAGs.