Les membres des familles Mimiviridae et Marseilleviridae, qui infectent et se répliquent dans Acanthamoeba spp. et d’autres protistes phagocytaires, ont été découverts au cours de la dernière décennie et rattachés à un groupe monophylétique de virus nommés les grands virus à ADN nucléocytoplasmiques (NCLDVs), qui infectent un large éventail d’eukaryotes y compris différents organismes unicellulaires. Récemment, il a été proposé de reclasser les NCLDVs dans un nouvel ordre viral nommé les Megavirales. Plusieurs dizaines de virus géants des amibes ont été isolés, mais le génome de peu d’entre eux a été étudié de façon approfondie. Nous avons étudié les génomes de ces virus géants d'amibe afin d’acquérir une meilleure compréhension de leur répertoire de gènes et leur importance évolutionnaire. L'analyse phylogénétique des virus géants d'amibe distingue clairement trois lignées, nommées A, B et C. Nous avons étudié en détail le génome de Acanthamoeba polyphaga moumouvirus, le membre fondateur de la lignée B et avons déchiffré son contenu en gènes et sa relation évolutive avec d'autres organismes. Nous avons également étudié les génomes de Terra1 virus et Terra2 virus, qui appartiennent respectivement aux lignées C et A, et ont été isolés à partir d'échantillons de sol alors que les mimivirus décrits aupravant ont été isolés à partir d'eau douce ou de mer. En outre, nous avons décrit le génome du virus Courdo11, qui appartient à la lignée C, et est étroitement lié au premier Mimivirus isolé d'un humain, qui présentait une pneumonie inexpliquée. / The members of families Mimiviridae and Marseilleviridae, which infect and replicate in Acanthamoeba spp. and other phagocytic protists, were discovered during the past decade and linked to a monophyletic group of viruses named the Nucleocytoplasmic Large DNA viruses (NCLDVs), which infect a broad variety of eukaryotes including diverse unicellular organisms. Recently, it has been proposed to reclassify the NCLDVs into a new viral order named the Megavirales. Several dozens of giant viruses of amoeba have been isolated but the genome of very few has been extensively studied. We studied the genomes of these giant viruses of amoeba to gain a better understanding of their gene repertoire and evolutionary importance. The phylogenetic analysis of giant viruses of amoeba clearly distinguished three lineages, named lineages A, B and C. We studied in detail the genome of Acanthamoeba polyphaga moumouvirus, the leader member of lineage B to decipher its gene content and its evolutionary relationship with other organisms. We further studied the genomes of Terra1 virus and Terra2 virus, which belong to lineages C and A, respectively, and were isolated from soil samples whereas previously described mimiviruses of amoeba were isolated from fresh or marine water. Furthermore, we described the genome of Courdo11 virus, which belongs to lineage C, and is closely related to the first mimivirus isolated from a human, who exhibited unexplained pneumonia.

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.

Nucleocytoplasmic large DNA viruses (NCLDVs) have genome sizes that range from around 100 kilobases (kb) to up to 2.5 megabases, and virion sizes that can reach up to 1.5 μm. Their large size in both of these contexts is atypical and defies the traditional view that viruses are streamlined, "filterable infectious agents". NCLDVs include many diverse groups, including Poxviruses, Asfarviruses, Iridoviruses, Mimiviruses, and Marseilleviruses. Poxviruses are perhaps the most well-studied; these viruses have 135-360 kbp genomes with about half of the genes encoding essential replication genes and the other half encoding genes related to host-virus interactions. Many of the genes involved in host-virus interactions are involved in immunomodulatory processes and have homology to proteins encoded by the host. These viral genes, often referred to as "mimics", are therefore believed to be the result of host-to-virus gene transfer. In this study I sought to examine if common poxvirus immunomodulatory genes were found in other NCLDV lineages, and if so, to analyze the evolutionary history of these genes. I identified 5 protein families of immunomodulatory genes that were found in both poxviruses and other NCLDV lineages, and I used phylogenetic tools to compare viral immunomodulatory genes of NCLDVs to their eukaryotic orthologs to evaluate the number of times different NCLDV lineages have acquired these genes. Our phylogenetic analyses showed that several viral immunomodulatory genes were acquired multiple times by different NCLDV lineages, while others appear to have been transferred between viral groups. Interestingly, some NCLDV genes clustered together with homologs from the unrelated Herpesviridae family, suggesting that inter-viral gene exchange can traverse vast evolutionary distances. The vast diversity of hosts infected by different NCLDV lineages suggests that these immunomodulatory genes play key roles that are useful to viruses in a variety of contexts. This research provides insight into how giant viruses acquire host genes, which contribute to their large genome size, and how those genes evolved to subvert antiviral defenses. / Master of Science / Giant viruses are a relatively recent discovery, from the beginning of this century. Nucleocytoplasmic large DNA viruses (NCLDVs) are a classification of multiple giant virus families. These viruses have large genomes from around 100 kilobases to 2.5 megabases of DNA. For reference, the genome size of the flu virus is approximately 13 kilobases. Most viruses cannot be seen by the human eye, even with microscopes, but giant viruses can get as big as bacteria, which can be seen with microscopes. It is unknown how or why these viruses get so large. One explanation is that they steal genes from their host and those genes evolve to work against the host. In this thesis, I explored some of the genes that these viruses have picked up. I curated a set of 49 previously characterized viral genes to analyze in this context. These genes have to do with modulating the host immune system and are known as "immunomodulatory genes". Viral immunomodulatory genes are often mimics of the host genes which function to help the immune system. However, a virus evolves faster than a host and the virus mimic gene can evolve to work against the immune system. This change can be visualized using phylogenetic tools; the viral genes will be more similar to each other than to the host genes and cluster separately on a phylogenetic tree. About half of the genes of Poxviruses, a giant virus family that has viruses that infect humans, are related to virus-host interactions, and include viral mimic genes. Poxviruses have been far better studied than other NCLDV families because of their public health importance. Variola virus, the virus that causes smallpox, is a poxvirus. Other NCLDV infect animals, algae, and amoeba. Though their hosts are different, their genomes have similar features. I set out to discover whether some of these previously characterized viral immunomodulatory genes that exist in poxviruses also exist in other NCLDV families. I utilized phylogenetic tools and a database of giant virus sequences to figure out which genes are being picked up by which family of NCLDV. I also sought to determine whether the individual NCLDV families have their own acquired immunomodulatory gene or have a gene very similar to all other families, suggesting an ancient acquisition. If the gene is very similar, it suggests that an ancestor of the NCLDV acquired the gene and it has stuck around as the group diverged into families. It is also interesting if different families stole the same type of gene multiple times because that indicates the importance of that gene in subverting the antiviral immune system for viral replication. This work provides insight into how giant viruses acquire host genes, which contribute to their large genome size, and how they evolved those genes to subvert antiviral defenses.