京都大学 / 新制・課程博士 / 博士(理学) / 甲第25148号 / 理博第5055号 / 新制||理||1721(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 望月 敦史, 教授 西山 朋子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Les amibes sont des protozoaires unicellulaires ubiquitaires dans l'environnement qui se nourrissent des microorganismes qui les entourent. Cependant plusieurs études ont déjà montré que certains de ces microorganismes (bactéries et virus) sont capables d'entrer dans les amibes et de s'y multiplier au lieu d'y être digérés. Cette capacité et l'analogie entre les amibes et les macrophages fait de ces microorganismes de potentiels pathogènes humains. L'utilisation de la technique de coculture sur amibes a donc été développée afin de permettre l'isolement de ces microorganismes. En 2001 elle a permis l'isolement d'un petit coque Gram positif qui sera identifié en 2003 comme étant le premier virus géant isolé : Acanthamoeba polyphaga Mimivirus, pour « mimiking microbe » en référence à sa coloration positive au Gram. La découverte de ce virus associé aux amibes et qualifié de « géant » il y a moins d'une décennie a bouleversé les définitions même de virus. Une taille exceptionnelle de 500 nm, un génome qui dépasse 1 Mb, une particule comportant des ADN et des ARN, autant de caractéristiques originales qui l'ont rendu exceptionnel. Depuis, d'autres virus géants ont été isolés, notamment Marseillevirus, et il est probable que la famille s'étende rapidement maintenant que l'on sait que leur isolement était impossible à cause de leur caractère non filtrable. Des études en métagénomique dans l'environnement ont suggéré une grande ubiquité de ces virus dans l'environnement, notamment hydrique. / Free living amoebas are ubiquitous in the environment and feed on microorganisms. However, several studies have already shown that some of these microorganisms (bacteria and virus) are able to enter the amoeba and multiply within the cell. This capacity and the analogy between amoebas and macrophages allow to think that these microorganisms are potentially human pathogens. The use of the amoeba coculture method was thus developed to allow the isolation of these microorganisms. In 2001 it allow the isolation of a small Gram positive coccus which will be characterized in 2003 as the first isolated giant virus : Acanthamoeba polyphaga Mimivirus. The discovery of this virus less than a decade ago challenged the definition of viruses. An exceptional size of 500 nm, a genome which exceeds 1 Mb, a particle containing DNA and ARN, so many original characteristics which made it exceptional. Since, other giant viruses were isolated, in particular Marseillevirus, and it's likely that the family quickly extends now that we know that their isolation was impossible because of their not filterable character. Environmental metagenomic studies suggested a big ubiquity, particularly into hydric environment. Furthermore the description of virophages, small viruses capable of infecting Mimivirus and behaving towards him as a bacteriophage, contribute to debate on the nature of viruses as well as on their place in the evolution.

L’objectif de cette thèse était d’isoler de nouveaux Mimiviridae dans l’environnement marin à l’aide d’amibes, hôtes potentiels de ces derniers et surtout organismes ubiquistes phagocytaires et connus pour être parasités par des microorganismes pathogènes. Après la mise en place et la validation de protocoles d’échantillonnages et de mise en culture de prélèvements environnementaux, plusieurs échantillons ont été analysés. Un nouveau membre de la famille des Mimiviridae a été isolé à partir d’un échantillon marin côtier provenant du Chili, stocké dans un milieu enrichi en amidon (milieu riz, propice à la conservation voire à la production de virus) puis mis en coculture dans une souche d’amibe Acanthamoeba griffini. Le séquençage de son génome révèle 1260 kb, codant pour 1120 protéines putatives, ce qui en fait le plus grand génome viral connu. Nommé megavirus chiliensis, sa capside est icosaédrique et possède également des fibrilles comme Acanthamoeba polyphaga mimivirus tout en étant plus grande (diamètre apparent 520 nm vs 450 nm). Bien que les morphologies des deux virus soient similaires et que de nombreuses particularités de mimivirus soient conservées chez megavirus (stargate), 23 % des protéines de megavirus n’ont pas d’homologues chez mimivirus, et les 594 gènes orthologues partagés présentent une identité résiduelle moyenne de 50 %. De plus, megavirus présente 3 amino-acyl-ARNt-synthètases supplémentaires (IleRS, TrpRS et AsnRS) à celles de mimivirus. Ces résultats suggèrent que ces deux virus sont issus d’un génome cellulaire ancestral qui a évolué par réduction génomique. Un parasite intracellulaire obligatoire a également été isolé à partir d’échantillons de sédiments marins de la côte chilienne. Des observations au microscope électronique à transmission indiquent une forme ressemblant à une endospore, de taille très variable (de 400 nm jusqu’à 1 μm), avec une paroi multicouche épaisse (~60 nm) et un pore apical. Cependant, aucune évidence de division n’a encore été observée, laissant penser que cette entité capable de multiplication pourrait être un virus, sans ressembler aux morphologies connues à ce jour. / In order to isolate new Mimiviridae from the marine environment, we used amoeba -ubiquitous phagotrophic protozoa - as potential host for these viruses. Different sampling protocols were tested and validated before carrying out co-cultures with amoeba and environmentals samples. A new Mimiviridae giant virus was isolated from Chilean coastal seawater completed with rice media (enriched incubation). Produced in Acanthamoeba griffini, its genome sequence has 1,260 Mbp and encodes for 1120 putative proteins, making it the largest known viral genome and thus named Megavirus chiliensis. Its icosaedral capsid is covered with fibrils and its size is bigger than that of Acanthamoeba polyphaga mimivirus (520 nm vs 450 nm). Although both virions are very similar and most of the mimivirus idiosyncrasies are conserved in megavirus (stargate), 23 % of megavirus putative proteins have no mimivirus homologs. Both viruses share 594 orthologous proteins exhibiting an average identity of 50 %. Moreover, megavirus contains 3 additional amino-acyl tRNA synthetases (IleRS, TrpRS and AsnRS) compare to mimivirus. These results suggest that these viruses have evolved from an ancestral cellular genome by reductive evolution. In addition, an amoeba obligatory intracellular parasite was isolated from marine sediments from Chilean coast. Transmission electron microscopic images show a particule like endospore with variable size (from 400 nm to 1 µm), a multilayered outer wall (~60 nm) and an apical pore. No evidence of division was observed, suggesting that the multiplication of this endoparasite occurs without division, suggesting that it could be a virus without any similarity to those described today.

Marseilleviridae é uma família de vírus gigantes cujos membros infectam amebas de vida livre. Esses vírus têm sido encontrados em amostras ambientais de água, larvas de inseto, torres de resfriamento e mais recentemente em amostras humanas. Eles possuem capsídeo icosaédrico medindo entre 190-250 nm e genoma de DNA dupla fita circular ou linear. Sua replicação ocorre no citoplasma amebiano onde observam-se fábricas virais. Este trabalho tem como objetivo investigar a presença de vírus gigantes em mexilhões-dourados (Limnoperna fortunei) que habitam o Lago Guaíba, Porto Alegre, Brasil. Quarenta indivíduos foram coletados e agrupados em pools de 5 amostras (água interna e corpo, totalizando dezesseis pools). Os pools foram homogeneizados em tampão fosfato, centrifugados e o sobrenadante filtrado em membrana de 0,45μM. Foram cultivadas amebas da espécie Acanthamoeba polyphaga em meio PYG em microplacas de 24 poços, inoculadas com os sobrenadantes, incubadas a 30ºC e examinadas diariamente em busca de efeito citopático (ECP) até 72 horas após a inoculação. Quando CPE era evidente, os sobrenadantes foram coletados e ultracentrifugados através de um gradiente de sacarose de 25%. Um dos dezesseis pools induzindo CPE claro foi submetido à extração de DNA e sequenciamento do genoma viral completo um sequenciador de nova geração (Illumina MiSeq). O genoma do vírus chamado Golden mussel marseillevirus consiste de uma única molécula de DNA de 360610 pb, com um teor de G+C de 43,1%. A análise da sequência de nucleotídeos traduzida revelou a presença de proteínas virais que apresentam homologia com proteínas de outros membros da família Marseilleviridae, como Lausanevirus e vírus Insectomime, porém grande parte do seu genoma não apresenta identidade com proteínas depositadas no banco de dados. A análise filogenética do gene D6/D11 Helicase sugere que este vírus faça parte de uma nova linhagem de marseillevirus. Este é o primeiro estudo que demonstra o isolamento de um vírus gigante a partir de tecidos de mexilhão-dourado e infere que estes vírus estão distribuídos amplamente no meio ambiente. / Marseilleviridae is a family of giant viruses whose members infect free living amoebae. These viruses have been found in environmental samples of water, insect larvae, cooling towers and, more recently, in human samples. They have icosahedral capsids measuring between 190-250 nm and their genome is double-stranded circular or linear DNA. Replication occurs in the host cell cytoplasm, inside the viral factories. This study aims to investigate the presence of giant viruses in tissues of golden mussels (Limnoperna fortunei) that inhabit the Guaiba Lake, Porto Alegre, Brazil. Forty specimens were pooled in groups of 5 specimens (internal water and body, totalizing sixteen pools). The pools were homogenized with phosphate buffer, centrifuged and the supernatant was filtered in 0,45μM. Monolayers of Acanthamoeba polyphaga were cultivated with PYG medium in 24-well microplates, inoculated with the pooled samples, incubated at 30 ºC and examined daily in search for cytopathic effect (CPE) up to 72 hours after inoculation. When CPE was evident, the supernatants were collected, clarified and ultra centrifuged through a 25% sucrose cushion. One out of the sixteen CPE positive pools was submitted to DNA extraction and complete sequencing of the viral genome in a NGS apparatus (Illumina MiSeq). The genome of the virus named Golden mussel marseillevirus consists of a single DNA molecule of 360,610 bp, with a G+C content of 43.1%. The analysis of the translated nucleotide sequence reveals the presence of proteins which are homologs to proteins predicted in other members of the family Marseilleviridae like, e.g. Lausannevirus and Isectomime virus. However, part of the viral genome has no identity with the nucleotide sequences available at the database. The phylogenetic analysis of the D6/D11 Helicase gene suggests that this virus is part of a new lineage of marseillevirus. This is the first study where the isolation of a giant virus from golden mussel tissues is achieved, suggesting that these viruses are widely distributed in the environment.

Marseilleviridae é uma família de vírus gigantes cujos membros infectam amebas de vida livre. Esses vírus têm sido encontrados em amostras ambientais de água, larvas de inseto, torres de resfriamento e mais recentemente em amostras humanas. Eles possuem capsídeo icosaédrico medindo entre 190-250 nm e genoma de DNA dupla fita circular ou linear. Sua replicação ocorre no citoplasma amebiano onde observam-se fábricas virais. Este trabalho tem como objetivo investigar a presença de vírus gigantes em mexilhões-dourados (Limnoperna fortunei) que habitam o Lago Guaíba, Porto Alegre, Brasil. Quarenta indivíduos foram coletados e agrupados em pools de 5 amostras (água interna e corpo, totalizando dezesseis pools). Os pools foram homogeneizados em tampão fosfato, centrifugados e o sobrenadante filtrado em membrana de 0,45μM. Foram cultivadas amebas da espécie Acanthamoeba polyphaga em meio PYG em microplacas de 24 poços, inoculadas com os sobrenadantes, incubadas a 30ºC e examinadas diariamente em busca de efeito citopático (ECP) até 72 horas após a inoculação. Quando CPE era evidente, os sobrenadantes foram coletados e ultracentrifugados através de um gradiente de sacarose de 25%. Um dos dezesseis pools induzindo CPE claro foi submetido à extração de DNA e sequenciamento do genoma viral completo um sequenciador de nova geração (Illumina MiSeq). O genoma do vírus chamado Golden mussel marseillevirus consiste de uma única molécula de DNA de 360610 pb, com um teor de G+C de 43,1%. A análise da sequência de nucleotídeos traduzida revelou a presença de proteínas virais que apresentam homologia com proteínas de outros membros da família Marseilleviridae, como Lausanevirus e vírus Insectomime, porém grande parte do seu genoma não apresenta identidade com proteínas depositadas no banco de dados. A análise filogenética do gene D6/D11 Helicase sugere que este vírus faça parte de uma nova linhagem de marseillevirus. Este é o primeiro estudo que demonstra o isolamento de um vírus gigante a partir de tecidos de mexilhão-dourado e infere que estes vírus estão distribuídos amplamente no meio ambiente. / Marseilleviridae is a family of giant viruses whose members infect free living amoebae. These viruses have been found in environmental samples of water, insect larvae, cooling towers and, more recently, in human samples. They have icosahedral capsids measuring between 190-250 nm and their genome is double-stranded circular or linear DNA. Replication occurs in the host cell cytoplasm, inside the viral factories. This study aims to investigate the presence of giant viruses in tissues of golden mussels (Limnoperna fortunei) that inhabit the Guaiba Lake, Porto Alegre, Brazil. Forty specimens were pooled in groups of 5 specimens (internal water and body, totalizing sixteen pools). The pools were homogenized with phosphate buffer, centrifuged and the supernatant was filtered in 0,45μM. Monolayers of Acanthamoeba polyphaga were cultivated with PYG medium in 24-well microplates, inoculated with the pooled samples, incubated at 30 ºC and examined daily in search for cytopathic effect (CPE) up to 72 hours after inoculation. When CPE was evident, the supernatants were collected, clarified and ultra centrifuged through a 25% sucrose cushion. One out of the sixteen CPE positive pools was submitted to DNA extraction and complete sequencing of the viral genome in a NGS apparatus (Illumina MiSeq). The genome of the virus named Golden mussel marseillevirus consists of a single DNA molecule of 360,610 bp, with a G+C content of 43.1%. The analysis of the translated nucleotide sequence reveals the presence of proteins which are homologs to proteins predicted in other members of the family Marseilleviridae like, e.g. Lausannevirus and Isectomime virus. However, part of the viral genome has no identity with the nucleotide sequences available at the database. The phylogenetic analysis of the D6/D11 Helicase gene suggests that this virus is part of a new lineage of marseillevirus. This is the first study where the isolation of a giant virus from golden mussel tissues is achieved, suggesting that these viruses are widely distributed in the environment.

Marseilleviridae é uma família de vírus gigantes cujos membros infectam amebas de vida livre. Esses vírus têm sido encontrados em amostras ambientais de água, larvas de inseto, torres de resfriamento e mais recentemente em amostras humanas. Eles possuem capsídeo icosaédrico medindo entre 190-250 nm e genoma de DNA dupla fita circular ou linear. Sua replicação ocorre no citoplasma amebiano onde observam-se fábricas virais. Este trabalho tem como objetivo investigar a presença de vírus gigantes em mexilhões-dourados (Limnoperna fortunei) que habitam o Lago Guaíba, Porto Alegre, Brasil. Quarenta indivíduos foram coletados e agrupados em pools de 5 amostras (água interna e corpo, totalizando dezesseis pools). Os pools foram homogeneizados em tampão fosfato, centrifugados e o sobrenadante filtrado em membrana de 0,45μM. Foram cultivadas amebas da espécie Acanthamoeba polyphaga em meio PYG em microplacas de 24 poços, inoculadas com os sobrenadantes, incubadas a 30ºC e examinadas diariamente em busca de efeito citopático (ECP) até 72 horas após a inoculação. Quando CPE era evidente, os sobrenadantes foram coletados e ultracentrifugados através de um gradiente de sacarose de 25%. Um dos dezesseis pools induzindo CPE claro foi submetido à extração de DNA e sequenciamento do genoma viral completo um sequenciador de nova geração (Illumina MiSeq). O genoma do vírus chamado Golden mussel marseillevirus consiste de uma única molécula de DNA de 360610 pb, com um teor de G+C de 43,1%. A análise da sequência de nucleotídeos traduzida revelou a presença de proteínas virais que apresentam homologia com proteínas de outros membros da família Marseilleviridae, como Lausanevirus e vírus Insectomime, porém grande parte do seu genoma não apresenta identidade com proteínas depositadas no banco de dados. A análise filogenética do gene D6/D11 Helicase sugere que este vírus faça parte de uma nova linhagem de marseillevirus. Este é o primeiro estudo que demonstra o isolamento de um vírus gigante a partir de tecidos de mexilhão-dourado e infere que estes vírus estão distribuídos amplamente no meio ambiente. / Marseilleviridae is a family of giant viruses whose members infect free living amoebae. These viruses have been found in environmental samples of water, insect larvae, cooling towers and, more recently, in human samples. They have icosahedral capsids measuring between 190-250 nm and their genome is double-stranded circular or linear DNA. Replication occurs in the host cell cytoplasm, inside the viral factories. This study aims to investigate the presence of giant viruses in tissues of golden mussels (Limnoperna fortunei) that inhabit the Guaiba Lake, Porto Alegre, Brazil. Forty specimens were pooled in groups of 5 specimens (internal water and body, totalizing sixteen pools). The pools were homogenized with phosphate buffer, centrifuged and the supernatant was filtered in 0,45μM. Monolayers of Acanthamoeba polyphaga were cultivated with PYG medium in 24-well microplates, inoculated with the pooled samples, incubated at 30 ºC and examined daily in search for cytopathic effect (CPE) up to 72 hours after inoculation. When CPE was evident, the supernatants were collected, clarified and ultra centrifuged through a 25% sucrose cushion. One out of the sixteen CPE positive pools was submitted to DNA extraction and complete sequencing of the viral genome in a NGS apparatus (Illumina MiSeq). The genome of the virus named Golden mussel marseillevirus consists of a single DNA molecule of 360,610 bp, with a G+C content of 43.1%. The analysis of the translated nucleotide sequence reveals the presence of proteins which are homologs to proteins predicted in other members of the family Marseilleviridae like, e.g. Lausannevirus and Isectomime virus. However, part of the viral genome has no identity with the nucleotide sequences available at the database. The phylogenetic analysis of the D6/D11 Helicase gene suggests that this virus is part of a new lineage of marseillevirus. This is the first study where the isolation of a giant virus from golden mussel tissues is achieved, suggesting that these viruses are widely distributed in the environment.

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.

Les virus géants sont un groupe de virus ADN double brin caractérisés par une taille géante du virion et du génome, et un répertoire de gènes qui comprend environ 450 à 2500 gènes prédits. Une proportion importante de ces gènes (jusqu'à 93%) sont des 'ORFans', ou codent pour des protéines de fonction inconnue. Acanthamoeba polyphaga mimivirus est le premier virus géant découvert, il y a une décennie, par co-culture sur Acanthamoeba spp. Il est le membre prototype de la famille Mimiviridae. Le génome de Mimivirus code pour environ 1000 protéines, parmi lesquelles ~50% n'ont pas d'homologue connu dans les banques de séquences publiques. La capside de Mimivirus a un diamètre d'environ 500 nm et est couverte par une couche dense de fibres, à l'exception de l'un de ses sommets. Ces fibres sont d'environ 130 nm de longueur et se composent d'une tige souple et d'une tête de forme globulaire.Dans ce travail de thèse, nous avons cherché à étudier les gènes impliqués dans la formation des fibres de Mimivirus. Dans ce but, nous avons notamment exprimé des gènes candidats dans E. coli, et nous avons mis au point une stratégie qui a utilisé l'interférence ARN afin d'étudier la fonction et la structure des protéines de Mimivirus. Nous avons annoté quatre protéines associées aux fibres. La stratégie utilisant les petits ARN interférant appliquée ici est originale et a été utilisée pour la première fois pour les virus géants qui infectent les amibes. Elle pourrait permettre de décrypter la fonction des gènes des mimivirus et d'annoter potentiellement des centaines de protéines présentes dans les bases de données publiques, et de différencier l'ADN poubelle des gènes réellement utilisés. / Giant viruses are a group of double stranded DNA viruses that are characterized by a giant virion and genome size, and gene repertoires encompassing approximately 450 to 2500 predicted genes. A substantial proportion of these genes (up to 93%) consists in ORFans, or encodes proteins with unknown functions. Acanthamoeba polyphaga mimivirus is the first giant virus that was discovered, a decade ago, after co-culturing on Acanthamoeba spp. It is the prototype member of the family Mimiviridae. Mimivirus encodes about 1000 proteins, among which ~50% have no known homolog in public sequence databases. The Mimivirus capsid is about 500 nm in diameter and is covered by a dense layer of fibers, except at one of its vertices. These fibers are about 130 nm in length and consist of a soft shaft and a globular shaped head.In this thesis work, we aimed to study the genes involved in the formation of the Mimivirus fibers. For this purpose, we have expressed candidate genes in E. coli, and implemented a strategy that used RNA interference to study the function and structure of Mimivirus proteins. We then succeeded in annotating four proteins as fiber associated proteins. The short interfering RNA strategy that we applied here is original and has been used for the first time in giant viruses that infect amoeba. It could allow deciphering the function of the mimivirus gene repertoires and help annotating hundreds of proteins without known function found in public databases and differentiate between junk DNA and truly used genes.

Les virus géants d'amibes( Acanthamoeba) sont des virus à ADN double brin . Ces virus géants ont été isolés depuis 2008 essentiellement à partir de prélèvements d'eaux et sols) collectés dans diverses régions géographiques à travers le monde, ou à partir de prélèvements humains (selle, liquide broncho-alvéolaire et sang). Ils sont repartis en 4 familles virales dont les plus représentées sont les familles Mimiviridae et Marseilleviridae avec pour membres fondateurs respectifs Mimivirus et Marseillevirus et comptent à ce jour respectivement 44 et 20 isolats. Les virus géants d'amibes sont ubiquitaires dans notre biosphère, et les êtres humains y sont potentiellement exposés. Au cours de cette Thèse, nous avons premièrement écrit une revue de la littérature décrivant les outils de mise en évidence des virus géants d'amibes chez l'homme incluant la sérologie, la culture, la PCR ou l'hybridation de sondes fluorescentes in situ. Deuxièmement, nous avons conçu et évalué 5 systèmes de PCR en temps réel détectant les membres des groupes de mimivirus d'amibes, leurs virophages et les marseillevirus. Nous avons participé à un 3ème travail décrivant les différentes procédures d'isolement sur amibes utilisées jusqu'à présent dans notre laboratoire . Enfin, dans un 4ème travail préliminaire, nous avons recherché par PCR la présence des mimivirus et marseillevirus dans 701 plasmas de patients infectés par HIV-1.Au total, nos travaux ont décrit les mises au point, performances et limites des tests de PCR pour l'étude des virus géants, et ont contribué aux outils et fourni des éléments pour l'étude de l'implication des virus géants d'amibes en pathologie humaine. / The giant viruses of amoebas( Acanthamoeba) are double stranded DNA viruses. These giant viruses have been isolated essentially from water and soil samples collected in various geographic regions around the world or from human samples (stool, blood and bronchoalveolar fluid). These giant viruses are divided into four viral families among which those comprising the largest number of representatives are the Mimiviridae and Marseilleviridae families, whose respective founders are Mimivirus and Marseillevirus and comprise 44 and 20 representative members, respectively. Giant viruses of amoeba are ubiquitous in our biosphere, which means that humans can be exposed to them. In this Thesis, we initially wrote a review of the literature describing the tools to detect the present of these giant viruses in humans, including serology, culture isolation, PCR and fluorescence in situ hybridization (FISH). Secondly, we designed and evaluated the performance of five real-time PCR systems targeting the members of the 3 groups of mimiviruses of amoeba, their virophages and the marseilleviruses. We were involved in a third work that described the different isolation procedures on amoebae used so far in our laboratory for giant viruses. Finally, in a fourth preliminary work, we looked by PCR for the presence of mimiviruses and marseilleviruses DNA in 701 plasma from patients infected with HIV-1. In summary, our work described the developed PCR assays for the study of giant viruses, and their performance and limitations, and it contributed to the tools and evidence for the study of the involvement of the giant amoeba virus in human pathology.