Marseilleviridae est une famille de virus géants isolés initialement à partir de prélèvements environnementaux, dont Marseillevirus est le membre fondateur. La présence des marseillevirus chez l’Homme a été démontrée dans quelques études. Les objectifs sont de mieux documenter la présence des marseillevirus chez l’Homme, de modéliser l’infection par Marseillevirus chez la souris, et enfin, de décrire les génomes des marseillevirus. Nous rapportons un cas d’infection par Marseillevirus chez une patiente atteinte d’un cancer des ganglions, soulevant la question d’un éventuel lien entre Marseillevirus et cancer, à l’instar de l’association existant entre d’autres virus et les cancers. L’infection des souris par Marseillevirus montre que celui-ci persiste un mois au niveau des «amygdales», confirmant le portage pharyngé chronique observé chez un deuxième patient. Enfin, nous identifions deux nouveaux groupes au sein de la famille, soulignant l’importante diversité génétique de la famille. / Marseilleviridae is a new family of giant viruses primarily isolated from environmental samples and whose Marseillevirus is the founding member. The presence of marseilleviruses in humans has been demonstrated in few studies. The aims are to better document the presence of marseilleviruses in humans, to develop a model of infection of mice with Marseillevirus, and to describe the genomes of marseilleviruses. We report a first caes of infection by Marseillevirus in apatient with a lymph nodes cancer, raising the question of a potential link between Marseillevirus and cancer, as the well established association between some viruses and cancers. The infection of miceshows that Marseillevirus persist one month in the “tonsils”, confirming the chronic pharyngeal carriage reported in a second patient. Finally, we identify two new subgroups in the family, highlighting the considerable genetic diversity of the family.

Les virus sont présents dans tous les écosystèmes, et sont les entités les plus abondantes dans le milieu marin. Bien que nous associons systématiquement virus aux maladies, la plupart d'entre eux coexistent cependant en équilibre avec leur hôte. Les virus sont associés à tous les règnes de la vie, même les virus qui affectent d'autres virus(virophages). La définition aujourd'hui d'un virus chez les virologues, c'est qu'un virus est un parasite génétique qui utilise des systèmes cellulaires pour sa propre réplication. Les hôtes les plus couramment utilisés par les virus que nous avons étudiés sont principalement des protozoaires. Ainsi, les Amoebozoa font l'objet de nombreuses études et sont utilisés pour isoler de nouvelles espèces intracellulaires( virus, bactéries). Ces espèces ont évolué de manière à résister aux effets consécutifs à la phagocytose ou à l'ingestion dans des vacuoles, et restent viable dans le cytoplasme de l'amibe, et ont le potentiel de se multiplier dans les parasites. Dans cette étude, nous avons dans un premier temps étudier les diverses interactions existantes entre virus Acanthamoeba polypaghaga Mimivirus(APMV) et des bactéries au sein de l'amibe. Pour cela, nous avons choisi un système original basé sur la co-culture de l'APMV, soit seul ou en combinaison avec deux autres microorganismes isolés individuellement à partir de l'amibe. Il s'agit d'une bactérie intracellulaire stricte(BABL1) et le virophage de APMV (Sputnik). Cela nous a permis de mettre en évidence, d'une part la capacité du virophage à moduler la virulence d'APMV tout en révélant, d'autre part, la bataille qui a eu lieu entre eux au cours de l'infection de l'hôte. dans un deuxième temps, nous avons examiné l'activité virucide des biocides couramment utilisés en pratique clinique pour la désinfection des équipements hospitaliers. APMV et Marseillevirus montrent une grande résitance aux biocides chimiques, en particulier l'alcool. Seule la température de 75°C et le glutaraldéhyde ont réussi à réduire les titres d'APMV et Marseillevirus à des niveaux indétectables. Après dessiccation ou exposition aux rayonnements ultraviolets, APMV et marseillevirus ont démontré leur stabilité durable. Précédent le pré-traitement des échantillons de l'environnement par l'éthanol à 70°C, a permis la disparition des contaminants bactériens sans réduire la charge virale, permettant leur isolement sur amibe, sans avoir besoin d'utiliser des antibiotiques, qui peuvent avoir un effet délétère su les amibes. / In this study, we first examined the various interactions taking place between the virus Acanthamoeba polyphaga Mimivirus (APMV) and bacteria within the amoeba. We chose an original system based on a co-culture of APMV either alone or in combination with two other organisms isolated from amoeba, i.e a strict intracellular bacterium (BABL1) and the virophage of APMV (Sputnik). This allowed us to highlight, on the one hand, the possibility to modulate the virulence of APMV while revealing, on the other hand, the battle which occurs between them during the infection of the host. We then examined the virucidal activity of biocides commonly used in clinical practice for the disinfection of hospital equipment. APMV and Marseillevirus show high resistance to chemical biocides, especially to alcohol. Only a temperature of 75°C or glutaraldehyde were able to reduce APMV and Marseillevirus titres to undetectable levels. Whether dried or under ultraviolet, APMV and Marseillevirus demonstrated their lasting stability. Previous pre-treatment of environmental samples by ethanol 70° allowed disappearance of bacterial contaminating bacteria without reducing giant virus load allowing their isolation on amoeba without need the use of antibiotic that may have a deleterious effect on amoebae.

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 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.