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Broad Spectrum of Mimiviridae Virophage Allows Its Isolation Using a Mimivirus Reporter Broad Spectrum of Mimiviridae Virophage Allows Its Isolation Using a Mimivirus Reporter Morgan Gaia, Isabelle Pagnier, Ange´lique Campocasso, Ghislain Fournous, Didier Raoult, Bernard La Scola* URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, Aix Marseille Universite, Marseille, France 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. Citation: Gaia M, Pagnier I, Campocasso A, Fournous G, Raoult D, et al. (2013) Broad Spectrum of Mimiviridae Virophage Allows Its Isolation Using a Mimivirus Reporter. PLoS ONE 8(4): e61912. doi:10.1371/journal.pone.0061912 Editor: John Parkinson, Hospital for Sick Children, Canada Received August 14, 2012; Accepted March 18, 2013; Published April 15, 2013 Copyright: ß 2013 Gaia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction Mimivirus growth. Later, we identified an isolate (Lentille) in our APM collection that was associated with a new strain of virophage; Free-living amoebas are ubiquitous protozoa that feed on we named the new virophage strain Sputnik 2 [9]. More recently, microorganisms in their environment through phagocytosis. another virophage was isolated with Cafeteria roenbergensis virus, a However, some microorganisms are able to resist digestion by giant virus that infects a marine phagocytic protist [10]. Using this predator after phagocytosis [1]. Thus, amoebas have been metaproteogenomic analysis, another virophage was detected in a used as a tool for the isolation of digestion-resistant environmental sample of a hypersaline meromictic lake in Antarctica; this bacteria, such as Legionella sp. [2]. This technique allowed the virophage is found in association with Phycodnaviridae, a giant fortuitous discovery of the first giant virus, Acanthamoeba polyphaga virus that infects algae [11]. The concept of virophages, small Mimivirus (APM) [3]. APM is a large icosahedral virus with a viruses that infect giant virus factories and lead to population 500 nm capsid [4] that is covered with surrounding fibrils and control of their hosts, is controversial [12–14]. However, this contains a 1.2 Mbp genome [5]. It belongs to the nucleocytoplas- emerging field justifies the exploration of the spectrum of mic large DNA virus (NCLDV) family in the recently proposed virophages in possible hosts and the design of new tools to order Megavirales [6], which also includes Iridoviridae, Phycod- complete their repertoire. naviridae, Asfarviridae, Ascoviridae and Poxviridae [7]. A second The first aim of our study was to investigate the replication closely related but slightly larger APM was later isolated, and the capacity of Sputnik 1 and Sputnik 2 in an A. polyphaga co-culture strain was called Mamavirus [8]. Since the isolation of the first two with each APM in our collection using quantitative real-time PCR giant viruses in our laboratory, we have improved the co-culture and, more notably, to investigate if virophages previously isolated technique, established a collection of APM and reported 18 with group A APM can infect group B or group C viruses. Noting isolates [9]. We have established a preliminary phylogenetic tree the inability to co-culture a group C virus with its virophage that based on partial polB sequences; the tree shows a repartition into was detectable by PCR, we decided to inoculate the sample on three groups: group A (includes the Mimivirus and Mamavirus), amoeba and Mimivirus to recover the virophage. group B (includes the Moumouvirus) and group C (includes the recently described Megavirus chilensis) [6]. Materials and Methods We isolated a small virus that co-isolates with Mamavirus and infects the Mamavirus virus factory. We named this new virus APM and virophage production Sputnik and classified it as the first virophage [8]. This icosahedral, Each virus in our collection (Table 1) was produced by the 50 nm virus with an 18 kb DNA genome is deleterious for inoculation of 10 ml of PYG medium with 1 ml each of the PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e61912 Mimiviridae Virophage individual viral suspension calibrated at 106 particles/ml and the Real-time PCR assay for evaluation of Sputnik 5 Acanthamoeba polyphaga amoebal suspension at 5610 cells/ml multiplication (strain Linc AP-1). After the complete lysis of the amoebas, each After incubation, 200 ml of each co-culture was sampled for co-culture was centrifuged at 2000 rpm for 10 min to pellet the DNA extraction and real-time PCR at H1, H0 and Day 3 (to remaining fragments. The supernatant was filtrated through a observe the complete lysis of amoebas). The DNA extraction was 0.8 mm pore filter to remove residual amoebas and cysts. The performed with a Qiamp DNA extraction kit (Qiagen, Hilden, supernatants were frozen at 280uC before being used for viral co- Germany) according to the manufacturer’s instructions. Real-time culture inoculation. PCR was performed with the LightCycler 480 SYBR Green I Sputnik 1 [8] and Sputnik 2 [9] were produced in co-culture Master (Roche) according to the manufacturer’s instructions. with their natural hosts, Mamavirus and Lentille virus, respec- Sputnik was detected and quantified by using a primer pair tively, in 10 ml of PYG medium containing 1 ml of the amoeba Sputnik 5 targeting the ORF20 of encoding the major capsid protein suspension at 5610 cells/ml, 1 ml of giant virus suspension and (Forward primer 59-GAGATGCTGATGGAGCCAAT-39, Re- 1 ml of each Sputnik strain. After the complete lysis of the verse primer 59-CATCCCACAAGAAAGGAGGA-39). For each amoeba, the purification was performed as described above, but sample, the cycle threshold (Ct) was correlated to a reference scale the filtration was executed on three successive filters of 0.8-, 0.45- to allow the evaluation of the Sputnik concentration. A total of and 0.22 mm pore sizes to obtain a pure Sputnik suspension. This 200 ml of each co-culture was analyzed by real-time PCR directly suspension was prepared before each inoculation assay. These tests after inoculation to assess the quantities of Sputnik 1 and Sputnik were performed in triplicate. 2. Sputnik co-culture with APM collection Microscopic observations Each giant virus was inoculated separately into 10 ml of PYG m 5 After incubation, 200 l of each co-culture at H0 and H6 was medium containing 1 ml of amoeba suspension at 5610 cells/ml added to a Cytospin single chamber (Thermo), cytocentrifuged at and 1 ml of each Sputnik suspension. After 1 hour of incubation at 800 rpm for 10 min and fixed with methanol. Indirect immuno- 32uC, the supernatant was gently removed to eliminate the fluorescence microscopy was performed with mouse anti-Sputnik Sputnik particles that were not internalized by the amoebas, and serum as described previously. To label the virus factories and the 10 ml of fresh PYG medium was added. This time point was cell nucleus, we used 5 ml of DAPI (49,69-diamino-2-phenylindole, defined as H0. The Lentille virus, naturally infected by Sputnik 2, Molecular Probes). The viable trophozoites were counted using was used as a positive control. trypan blue (Oxoid) to assess the speed of the lysis of the amoebas by each virus. The preparation of selected samples for transmission electron microscopy was performed as described previously [15] to confirm the results of the quantitative PCR and immunofluores- cence assays. Table 1. The list of giant viruses and their classification into Pol B gene sequence and phylogeny genotype groups. The complete Pol B gene sequences for all the viruses used in this study were available from unfinished APM genome sequenc- ing in progress in our laboratory. The sequences of DNA Name Group GenBank polymerase B were aligned using MUSCLE and trimmed by APM A HQ336222 TrimAL after visual editing. The phylogenetic tree was built with a Mamavirus A JF979171 maximum likelihood using Phyml software. Lentille A JF979182 Environmental detection of APM and virophages Fauteuil A JF979168 Tentative detection of APM and virophages was performed on Pointerouge1 A JF979167 90 soil samples collected in Marseille and the surrounding areas Cher A JF979166 (south of France). To each 15 g sample, 150 mL of sterile water Longchamps A JF979169 was added. Decantation occurred overnight at 4uC, and then the Terra2 A GU265562 samples were filtered, first through Wattman’s paper and then Lactour A JF979173 through 0.1 mm membrane.
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