First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis

Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection

Zamilon, a Novel Virophage with <i>Mimiviridae</i> Host Specificity

<div><p>Virophages, which are potentially important ecological regulators, have been discovered in association with members of the order <i>Megavirales</i>. Sputnik virophages target the <i>Mimiviridae</i>, Mavirus was identified with the <i>Cafeteria roenbergensis</i> virus, and virophage genomes reconstructed by metagenomic analyses may be associated with the <i>Phycodnaviridae</i>. Despite the fact that the Sputnik virophages were isolated with viruses belonging to group A of the <i>Mimiviridae</i>, they can grow in amoebae infected by <i>Mimiviridae</i> from groups A, B or C. In this study we describe Zamilon, the first virophage isolated with a member of group C of the <i>Mimiviridae</i> family. By co-culturing amoebae with purified Zamilon, we found that the virophage is able to multiply with members of groups B and C of the <i>Mimiviridae</i> family but not with viruses from group A. Zamilon has a 17,276 bp DNA genome that potentially encodes 20 genes. Most of these genes are closely related to genes from the Sputnik virophage, yet two are more related to <i>Megavirus chiliensis</i> genes, a group B <i>Mimiviridae</i>, and one to Moumouvirus monve transpoviron.</p></div

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

The Zamilon genome.

<p>The Zamilon genome, with predicted coding sequences on the forward strand (blue) and the reverse strand (red). Phylogenetic analyses of ORF6, 9, 11, 12, 18 and 19 are included with bootstrap values indicated (cutoff ≥50). * indicates the best hit (<i>E</i>-values: 0, 0, 8⁻⁸⁰, 2⁻⁹⁰, 5⁻¹⁴⁷, and 5⁻³⁴, respectively).</p

Closest homologs of the Zamilon open reading frames (ORFs).

<p>Best hit for Zamilon's ORFs obtained with BlastP against the non-redundant (nr) NCBI database.</p><p>Hypothetical functions were determined by homology and conservation of protein domains. * indicates ORFs with no significant homology in the nr database. These ORFs were aligned directly to the Sputnik virophage.</p

Impact of the Zamilon virophage.

<p>(<b>A–B</b>) Transmission electron microscopy images of abnormal Mont1 virus particles (arrows) produced from the virus factory with (<b>A</b>; scale bar 0.1 µm) and without (<b>B</b>; scale bar 0.1 µm) Zamilon. (<b>C</b>) Kinetics of survival of amoebae infected with Mont1 or Mamavirus, with or without the Zamilon virophage (Blue: Mont1, Red: Mont1 and Zamilon, Green: Mamavirus, Purple: Mamavirus and Zamilon, Turquoise: negative control). The x-axis shows the time points, and the y-axis shows the number of amoebae per milliliter (×10⁵ cells/mL). (<b>D</b>) Histogram of Courdo11 virus growth alone (blue), with Sputnik (red) and with Zamilon (green). The difference in the Cycle threshold (Ct) between time points H0 and H24 is shown.</p