Molecular tools for the detection of giant viruses of the Mimiviridae and Marseilleviridae families : application to environmental and human samples

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

A Decade of Improvements in Mimiviridae and Marseilleviridae Isolation from Amoeba

International audienceSince the isolation of the first giant virus, the Mimivirus, by T.J. Rowbotham in a cooling tower in Bradford, UK, and after its characterisation by our group in 2003, we have continued to develop novel strategies to isolate additional strains. By first focusing on cooling towers using our original time-consuming procedure, we were able to isolate a new lineage of giant virus called Marseillevirus and a new Mimivirus strain called Mamavirus. In the following years, we have accumulated the world's largest unique collection of giant viruses by improving the use of antibiotic combinations to avoid bacterial contamination of amoeba, developing strategies of preliminary screening of samples by molecular methods, and using a high-throughput isolation method developed by our group. Based on the inoculation of nearly 7,000 samples, our collection currently contains 43 strains of Mimiviridae (14 in lineage A, 6 in lineage B, and 23 in lineage C) and 17 strains of Marseilleviridae isolated from various environments, including 3 of human origin. This study details the procedures used to build this collection and paves the way for the high-throughput isolation of new isolates to improve the record of giant virus distribution in the environment and the determination of their pangenome

Tobacco mosaic virus in the lungs of mice following intra-tracheal inoculation.

Plant viruses are generally considered incapable of infecting vertebrates. Accordingly, they are not considered harmful for humans. However, a few studies questioned the certainty of this paradigm. Tobacco mosaic virus (TMV) RNA has been detected in human samples and TMV RNA translation has been described in animal cells. We sought to determine if TMV is detectable, persists, and remains viable in the lung tissues of mice following intratracheal inoculation, and we attempted to inoculate mouse macrophages with TMV. In the animal model, mice were intratracheally inoculated with 10(11) viral particles and were sacrificed at different time points. The virus was detected in the mouse lungs using immunohistochemistry, electron microscopy, real-time RT-PCR and sequencing, and its viability was studied with an infectivity assay on plants. In the cellular model, the culture medium of murine bone marrow derived macrophages (BMDM) was inoculated with different concentrations of TMV, and the virus was detected with real-time RT-PCR and immunofluorescence. In addition, anti-TMV antibodies were detected in mouse sera with ELISA. We showed that infectious TMV could enter and persist in mouse lungs via the intratracheal route. Over 14 days, the TMV RNA level decreased by 5 log(10) copies/ml in the mouse lungs and by 3.5 log(10) in macrophages recovered from bronchoalveolar lavage. TMV was localized to lung tissue, and its infectivity was observed on plants until 3 days after inoculation. In addition, anti-TMV antibody seroconversions were observed in the sera from mice 7 days after inoculation. In the cellular model, we observed that TMV persisted over 15 days after inoculation and it was visualized in the cytoplasm of the BMDM. This work shows that a plant virus, Tobacco mosaic virus, could persist and enter in cells in mammals, which raises questions about the potential interactions between TMV and human hosts

Anti-TMV antibody testing in mouse serum samples.

<p>Detection of anti-TMV total antibodies in serum samples of 10 TMV-inoculated and 5 control mice at day 0 (just before the operation), and 13 TMV-inoculated and 8 control mice at day 7 and day 14 after intratracheal inoculation. *Statistically significant.</p

Infectivity test for TMV RNA-positive lung samples on <i>Nicotiana benthamiana</i>.

<p>A: Leaves of a non-inoculated plant; B: Leaves of a plant inoculated with a TMV RNA positive sample (TMV-inoculated mouse number 1 at day 3). Arrows show signs of systemic infection with discoloration and deformation of young leaves.</p

TMV RNA quantification by real-time PCR in bronchoalveolar lavage macrophages of TMV-inoculated and control mice.

<p>The PCR systems targets the coat protein gene.</p

Lung sections from water-inoculated mouse (A and C), and TMV-inoculated mouse at day 3 (B and D) after intratracheal inoculation.

<p>Note the absence of inflammation in the lungs of control mice, whereas inflammatory infiltrates in interalveolar walls composed in part by macrophages were observed in lungs from TMV-inoculated mice. Hematoxylin-eosin staining was used. Magnification, 200X (A and B) and 400X (C and D). Arrows indicate the inter-alveolar walls inflammation.</p

TMV RNA quantification in mouse lungs by real-time RT-PCR.

<p>A: TMV coat protein gene system; B: TMV replicase gene system. Control, non-inoculated mice.</p

Probes and primers used for RT-PCR assays.

a<p>in reference to sequence GenBank accession no. AB369276; nt, nucleotide.</p

Infectivity test for TMV RNA-positive lung samples on <i>Nicotiana tabacum</i> Xanthi.

<p>A: Leaf of a non-inoculated plant; B: Leaf of a plant inoculated with a TMV RNA positive sample (TMV-inoculated mouse number 2 at day1); C: Leaf of a plant inoculated with a TMV RNA positive sample (TMV-inoculated mouse number 1 at day 3). On inoculated leaves, arrows show local necrotic lesions induced by the virus in the plant cells.</p