Les virus sont ubiquitaires et abondants dans l’environnement. Ils influent fondamentalement sur l’écologie de l’ensemble des écosystèmes et du microbiote humain. Dès 2002, avec la découverte de virus géants d’amibes, la virologie s’est complexifiée. Les Megavirales (nouvel ordre au sein des grands virus nucléocytoplasmiques) ont 10% de gènes homologues aux cellules eucaryotes, et ont la caractéristique singulière d’être infectés par des virophages.Avec l’avènement de la métagénomique, le nombre de métagénomes produits ne cesse de croître de manière exponentielle. C’est ainsi que la virologie a connu un nouvel essor et a pu mieux être étudiée en s’affranchissant des difficultés de culture et d’isolement des virus dans les conditions artificielles de laboratoire. La métagénomique permet d’étudier les communautés microbiennes mais également de découvrir de nouveaux microbes. La bioinformatique est devenue incontournable dans le domaine de la biologie et essentielle pour les biologistes afin de traiter les masses de données et en extraire toute la richesse de l’information biologique nécessaire. La première partie de cette thèse consiste en une revue de la littérature décrivant la bioinformatique au service de la métagénomique virale. La deuxième partie présente la création d’un nouvel outil « MG-Digger » dédié à l’analyse rapide et automatisée de séquences d’intérêts spécifiques dans les métagénomes. La dernière partie se concentre sur l’utilisation de cet outil sur des données issues de projets métagénomiques afin de répondre à des questions biologiques précises, notamment sur les données de l’expédition scientifique TARA à travers les océans. / Viruses are ubiquitous and abundant in the environment and can influence all ecosystems ecology and the human microbiota.Since 2002, with the discovery of giant viruses of amoeba, virology has become more complex and the definition of virus has been called into question, not only because of their phenotypic sizes similar to those of bacteria but also their genomic content exceeding thousand genes. Megavirales, also known as nucleocytoplasmic large DNA viruses, have 10% homologous genes to eukaryotic cells and interestingly can be infected by virophages. With the advent of metagenomic, the number of metagenomes produced is exponentially increasing as well as our understanding of virology which has been studied. Metagenomics studies showed an efficient way to study microbial communities and identify novel viruses without the difficulties of culture and isolation of viruses in artificial laboratory conditions.Metagenomic requires considerable computational and storage resources (Big data processing). Therefore, bioinformatics has become an integral part of research and development in the biomedical sciences by providing tools that handle complex datasets and finally giving the necessary biological information. The first part of this thesis consists of an exhaustive review of the literature describing bioinformatics and viral metagenomics. The second part presents a new "MG-Digger" tool dedicated to the rapid and automated analysis of specific interest sequences in metagenomes. The third part focuses on the use of this tool on metagenomic data to answer to specific biological questions, including data from the TARA scientific expedition across the oceans.

Brown algae are important primary producers and habitat formers in coastal environments and are believed to have evolved multicellularity independently of the other eukaryotes. The phaeoviruses that infect them form a stable lysogenic relationship with their host via genome integration, but have only been extensively studied in two genera: Ectocarpus and Feldmannia. In this study I aim to improve our understanding of the genetic diversity, host range and distribution of phaeoviruses. Sequencing and phylogenetic analysis of amplified fragments of three core phaeoviral genes (encoding major capsid protein (MCP), DNA polymerase and superfamily III helicase) of phaeovirus infected algae confirmed the suspected phaeoviral identity of viruses infecting E. fasciculatus, F. simplex, Pilayella littoralis, Myriotrichia clavaeformis and Hincksia hincksiae. Furthermore, this approach revealed multiple virus sequence variants within individual strains, and moreover that the variants formed two distinct subgroups. Subgroup A was highly conserved and observed in multiple algal genera, whereas subgroup B was much more diverse, but only found in Feldmannia species. Transcriptome sequencing of an actively infected F. irregularis strain revealed polymorphisms within key viral genes, suggesting that multiple variants were indeed active within this strain. High resolution melt curve (HRM) technology was used to develop a high throughput screening method for detecting phaeoviral MCP as a proxy for detection of phaeoviruses. This technique was also able to assign 88% of those detected to one of the subgroups, based on their differing melting temperature distributions. This was then applied to 1034 Ectocarpus isolates collected from around Europe and South America, and in accordance with previous studies of phaeoviral infection, 43-79% of strains contain virus sequence (depending on species). 17% of the isolates tested even contained sequence from both subgroups. 82 Laminariales strains, close relatives of the Ectocarpales, were also screened because they comprise commercially important kelp species but are not known to be infected by viruses. 10-17% of these tested positive for phaeoviral MCP, which when sequenced formed a separate group within the phaeoviruses. This finding could have a major impact on the kelp farming industry if the viruses are found to affect reproduction as happens in the Ectocarpales. The discovery of two subgroups is contrary to current beliefs that the phaeoviruses are a single monophyletic group, and that each species of alga has its own phaeovirus, casting doubt on the usefulness of the current convention of naming each phaeovirus after its host. It appears that the subgroup B viruses have begun to evolve away from the stable, K-selected subgroup A viruses towards a more r- type strategy with higher mutation and diversification. This study has identified potential mechanisms that may influence this shift, including mutations in a region of the DNA polymerase known to negatively affect DNA replication fidelity, combined with an active integrase and lack of a proofreading exonuclease, along with the observed infection of individuals with both phaeovirusal subgroups. The resulting mutations and recombinations could lead to the diversity observed here, and may provide a suitable model for the study of other emergent virus infections.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22294号 / 理博第4608号 / 新制||理||1661(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 緒方 博之, 教授 青山 卓史, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Les virus du genre Chlorovirus (phycoDNAviridae) sont des virus à ADN double-brin de grande taille, qui infectent des algues vertes eucaryotes unicellulaires vivant en eau douce nommées les chlorelles (Trebouxiophyceae). A l'aide d'approches bioinformatiques, j'ai consacré mon travail de thèse à l'étude de la diversité génomique des chlorovirus et de leur histoire évolutive, ainsi qu'à l'étude transcriptomique de l'infection virale chez son hôte.Dans le premier volet de ma thèse, j'ai procédé à l'assemblage et à l'annotation de 50 nouveaux génomes de chlorovirus récemment séquencés (Roche-454). J'ai ainsi été en mesure de mieux caractériser les différences de structure du génome et de contenu en gène des différents chlorovirus en fonction de leurs hôtes. J'ai également mis en évidence l'existence d'un quatrième sous-groupe de chlorovirus, repoussant les limites connues de la diversité de ces virus. Enfin j'ai montré que les Chlorovirus ne suivent pas le même schéma évolutif des autres NCLDV et que l'origine de leurs gènes est encore inconnue, bien que probablement virale.Dans un autre projet, j'ai également étudié les variations de la transcription des gènes de la chlorelle (C. varabilis NC64A) induites par l'infection par un chlorovirus (PBCV-1) grâce au séquençage profond (Illumina) des ARNm polyadénylés présent dans la cellule saine, puis après infection. J'ai ainsi pu montrer que les différentes fonctions cellulaires sont impactées de façon préférentielle par l'infection. / Giant viruses in the genus Chlorovirus (Phycodnaviridae) infect eukaryotic green microalgae known as Chlorella (Trebouxiophyceae). Using bioinformatic approaches, I dedicated my thesis on the study of the genomic diversity and evolutionnary history of the chlorovirus at the genus level, and the transcriptomic of the viral infection.In the first part on my work, I conducted the assembly and annotation of 50 new chlorovirus genomes recently sequenced (Roche-454). I was able to refine the known differences between chloroviruses, both in genome structure and gene content terms. Clues for the existence of a fourth subgroup of chloroviruses were also found. I was also able to show that the chlorovirus does not follow the same evolutionnary pattern as the other NCLDV, and that the origin of their genes is still largely unknown, but presumably of viral origin.In a second project, I studied the variation in the transcription of chlorella's genes (C. variabilis NC64A) during the infection by a chlorovirus (PBCV-1) using the deep-sequencing (Illumina) of all polyadenylated messenger RNA in the healthy or infected cell. This way, I was able to show that the various cellular functions are preferentially impacted by the infection.

Les Megavirales appartiennent à des familles de virus géants infectant un grand nombre d'hôtes eucaryotes. Leurs génomes ont des tailles variant de 100 kb to 2.5 mb et leur composition a montré des caractéristiques surprenantes qui ont soulevées diverses questions sur l’origine et l’évolution de ces virus. Les études de métagénomique environnementale ont montré qu’il existe une «matière noire», composée de séquences reliées à aucun organisme connu. Cependant, l'identification des séquences a été principalement réalisée en utilisant les séquences ADN ribosomal (ADNr), ce qui conduit à ignorer les virus. D’autres gènes informationnels « cœur », incluant la DNA-dependant RNA polymerase (RNAP) constituent d'autres marqueurs qui apparaissent comme plus appropriés pour une classification plus exhaustive des séquences, puisqu’ils apparaissent conservés dans les organismes cellulaires ainsi que les mégavirus. Nous avons utilisé un petit ensemble de gènes universels conservés incluant la RNAP et avons reconstruit des séquences ancestrales pour rechercher des séquences reliées aux mégavirus dans les bases de données. Cela a permis d’identifier trois nouvelles séquences de megavirus qui avaient été mal annotées comme correspondant à des organismes cellulaires, ainsi que de nouveaux clades viraux dans les bases métagénomiques environnementales. De plus, nous avons montré que l’ordre Megavirales constituait une quatrième branche monophylogénétique ou « TRUC » (pour Things Resisting Uncompleted Classification). Nos analyses montrent également que la RNAP ainsi que quelques autres gènes utilisés dans nos études permettent de considérer un répertoire plus complet d’organismes que l’ADNr. / Nucleocytoplasmic large DNA viruses (NCLDVs), or representatives of order Megavirales, belong to families of giant viruses that infect a large number of eukaryotic hosts. These viruses genomes size ranges from 100 kb to 2.5 mb and compose surprising features, which raised various questions about their origin and evolution. Environmental metagenomic studies showed that there is a “dark matter”, composed of sequences not linked to any known organism. However sequence identification was mainly determined using ribosomal DNA (rDNA) sequences, which led therefore to ignore viruses, because they are devoid of such genes. Informational genes, including DNA-dependant RNA polymerase (RNAP), are other markers that appear as more appropriate for a comprehensive classification as they are conserved in cellular organisms (Bacteria, Archaea and Eukarya) and in Megavirales. We used a small set of universally conserved genes that included RNAP and reconstructed ancestral sequences to search for megavirus relatives in sequence databases and to perform phylogeny reconstructions. This allowed identified three megaviral sequences that were misannotated as cellular orgainsms, and new viral clades in environmental databases. In addition, we delineated Megavirales as a fourth monophylogenetic TRUC (things resisting uncompleted classification) aside cellular organisms. Moreover, we classified by phylogenetic and phyletic analyses based on informational genes new giant viruses as new bona fide members of the fourth TRUC. Our analyses shows that RNAP as well as a few other genes used in our studies allow a more comprehensive overview and classification of the biological diversity than rDNA.

Namao virus (NV) was associated with mortality in lake sturgeon Acipenser fulvescens reared as part of a conservation stocking program for this endangered species in Manitoba, Canada. The virus itself was large, doubly encapsidated and icosahedral-shaped. Phylogenetic analyses using the major capsid protein showed that NV and other epitheliotropic sturgeon nucleo-cytoplasmic large DNA viruses shared a common evolutionary past and formed a distinct evolutionary lineage within Megavirales. Three PCR tests were developed and their analytical performance was validated for detection of these viruses. Testing of wild sturgeon revealed that NV is endemic in the Nelson River water basin in Manitoba. Bath exposure resulted in transmission of NV to healthy sturgeon. The gills appeared to be the initial site of infection with virus persisting in the head skin tissue for up to 62 days. The molecular tests will be useful tools for disease management in sturgeon conservation stocking programs. / October 2015

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.