Broad spectrum of mimiviridae virophage allows its isolation using a mimivirus reporter

Abstract

The giant virus Mimiviridae family includes 3 groups of viruses: group A (includes Acanthamoeba polyphaga Mimivirus), group B (includes Moumouvirus) and group C (includes Megavirus chilensis). Virophages have been isolated with both group A Mimiviridae (the Mamavirus strain) and the related Cafeteria roenbergensis virus, and they have also been described by bioinformatic analysis of the Phycodnavirus. Here, we found that the first two strains of virophages isolated with group A Mimiviridae can multiply easily in groups B and C and play a role in gene transfer among these virus subgroups. To isolate new virophages and their Mimiviridae host in the environment, we used PCR to identify a sample with a virophage and a group C Mimiviridae that failed to grow on amoeba. Moreover, we showed that virophages reduce the pathogenic effect of Mimivirus (plaque formation), establishing its parasitic role on Mimivirus. We therefore developed a co-culture procedure using Acanthamoeba polyphaga and Mimivirus to recover the detected virophage and then sequenced the virophage's genome. We present this technique as a novel approach to isolating virophages. We demonstrated that the newly identified virophages replicate in the viral factories of all three groups of Mimiviridae, suggesting that the spectrum of virophages is not limited to their initial host.

Figure 1. Sputnik 1 and 2 growth in different giant viruses.

A histogram of Sputnik 1 and 2 growth in different giant viruses according to their phylogenetic position in the groups A, B and C of Mimiviridae based on Pol B gene sequences. The growth, measured by real-time PCR quantification, was calculated between day 0 and day 3 and corresponded with complete amoebal lysis.

Figure 2. Sputnik within viruses of the different groups of Mimiviridae.

(A) A DAPI and immunofluorescence labeling of Sputnik 1 within viruses of the groups A (Mimivirus), B (Moumouvirus) and C (Courdo11) of Mimiviridae at 6 h post infection. This figure shows the Sputnik particles labeled with mouse antibody serum (green), and the nucleic acids are indicated by DAPI stain (blue/purple). The virus factories are especially visible by the abundant green stain. (B) Aspect of Sputnik3 virophage produced in virus factory of group A Mimivirus (on the left; scale bar 1 µm), group B Moumouvirus (at the center; scale bar 200 nm) and group C Courdo11 (on the right; scale bar 2 µm).

Figure 3. Sputnik 3 virophage isolation.

(A) Schematic summarizing the protocol used in this study for the isolation of virophage. Observations of the virophage isolated in this study by (B) transmission electron microscopy of negatively stained particles (A, scale bar 200 nm) and (C) transmission electron microscopy of thin section of culture (B, scale bar 500 nm). (D) Genome of Sputnik 3.