[Show abstract][Hide abstract] ABSTRACT: Since the discovery of Mimivirus, the founding member of the family Mimiviridae, three lineages, A–C, have been delineated among the mimiviruses of amoebae. To date, all giant viruses with annotated genomes have been isolated from water samples. Here, we describe the genome of two mimiviruses, Terra1 virus and Terra2 virus, which were recovered by co-culturing on Acanthamoeba spp. from soil samples. These genomes are predicted to harbor 1055 and 890 genes, respectively. Comparative genomics and phylogenomics show that Terra1 virus and Terra2 virus are classified within lineages C and A of the amoebae-associated mimiviruses, respectively. The genomic architecture of both viruses show conserved collinear central regions flanked by less conserved areas towards the extremities, when compared with other mimivirus genomes. A cluster of genes that are orthologous to bacterial genes and have no counterpart in other viral genomes except in lineage C mimiviruses was identified in Terra1 virus.
[Show abstract][Hide abstract] ABSTRACT: Giant viruses of amoebae were discovered 10 years ago and led to the description of two new viral families: Mimiviridae and Marseilleviridae. These viruses exhibit remarkable features, including large capsids and genomes that are similar in size to those of small bacteria and their large genetic repertoires include genes that are unique among viruses. The family Mimiviridae has grown during the past decade since the discovery of its initial member, Mimivirus, and continues to expand. Here, we describe the genome of a new giant virus that infects Acanthamoeba spp., Courdo11 virus, isolated in 2010 by inoculating Acanthamoeba spp. with freshwater collected from a river in southeastern France. The Courdo11 virus genome is a double stranded DNA molecule composed of 1,245,674 nucleotides. The comparative analyses of Courdo11 virus with the genomes of other giant viruses showed that it belongs to lineage C of mimiviruses of amoebae, being most closely related to Megavirus chilensis and LBA 111, the first mimivirus isolated from a human. Major characteristics of the M. chilensis genome were identified in the Courdo11 virus genome, found to encode three more tRNAs. Genomic architecture comparisons mirrored previous findings that showed conservation of collinear regions in the middle part of the genome and diversity towards the extremities. Finally, fourteen ORFans were identified in the Courdo11 virus genome, suggesting that the pan-genome of mimiviruses of amoeba might reach a plateau.
[Show abstract][Hide abstract] ABSTRACT: Marseillevirus is a giant virus that was isolated in 2007 by culturing water collected from a cooling tower in Paris, France, on Acanthamoeba polyphaga. Since then, five other marseilleviruses have been detected in environmental or human samples. The genomes of two of the six marseilleviruses have been described in detail. We describe herein the genome of Cannes 8 virus, a new member of the proposed family "Marseilleviridae." Cannes 8 virus was isolated from water collected from a cooling tower in Cannes in southeastern France. Its genome is a circular double-stranded DNA molecule with 374,041 base pairs, larger than the Marseillevirus and Lausannevirus genomes. This genome harbors 484 open reading frames predicted to encode proteins with sizes ranging from 50 to 1,537 amino acids, among which 380 (79 %) and 272 (56 %) are bona fide orthologs of Marseillevirus and Lausannevirus proteins, respectively. In addition, 407 and 336 predicted proteins have significant hits against Marseillevirus and Lausannevirus proteins, respectively, and 294 proteins are shared by all three marseilleviruses. The Cannes 8 virus genome has a high level of collinearity (for 96 % of orthologs) with the Marseillevirus genome. About two-thirds of the Cannes 8 virus gene repertoire is composed of family ORFans. The description and annotation of the genomes of new marseilleviruses that will undoubtedly be recovered from environmental or clinical samples will be helpful to increase our knowledge of the pan-genome of the family "Marseilleviridae."
[Show abstract][Hide abstract] ABSTRACT: Background:
Mimiviridae Mimivirus, including the largest known viruses, multiply in amoebae. Mimiviruses have been linked to pneumonia, but they have never been isolated from patients. To further understand the pathogenic role of these viruses, we aimed to isolate them from a patient presenting with pneumonia.
We cultured, on Acanthamoeba polyphaga amoebae, pulmonary samples from 196 Tunisian patients with community-acquired pneumonia during the period 2009-2010. An improved technique was used for Mimivirus isolation, which used agar plates where the growth of giant viruses is revealed by the formation of lysis plaques. Mimivirus serology was tested by microimmunofluorescence and by bidimensional immunoproteomic analysis using Mimivirus strains, to identify specific immunoreactive proteins. The new Mimivirus strain genome sequencing was performed on Roche 454 GS FLX Titanium, then AB SOLiD instruments.
We successfully isolated a Mimivirus (LBA111), the largest virus ever isolated in a human sample, from a 72-year-old woman presenting with pneumonia. Electron microscopy revealed a Mimivirus-like virion with a size of 554 ± 10 nm. The LBA111 genome is 1.23 megabases, and it is closely related to that of Megavirus chilensis. Furthermore, the serum from the patient reacted specifically to the virus compared to controls.
This is the first Mimivirus isolated from a human specimen. The findings presented above together with previous works establish that mimiviruses can be associated with pneumonia. The common occurrence of these viruses in water and soil makes them probable global agents that are worthy of investigation.
Full-text · Article · May 2013 · Clinical Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Background:
Megavirales is a proposed new virus order composed of Mimivirus, Marseillevirus and closely related viruses, as well as members of the families Poxviridae, Iridoviridae, Ascoviridae, Phycodnaviridae and Asfarviridae. The Megavirales virome, which we refer to as the megavirome, has been largely neglected until now because of the use of technical procedures that have jeopardized the discovery of giant viruses, particularly the use of filters with pore sizes in the 0.2-0.45-μm range. Concurrently, there has been accumulating evidence supporting the role of Mimivirus, discovered while investigating a pneumonia outbreak using amoebal coculture, as a causative agent in pneumonia.
In this paper, we describe the detection of sequences related to Mimivirus and Marseillevirus in the gut microbiota from a young Senegalese man. We also searched for sequences related to Megavirales in human metagenomes publicly available in sequence databases.
We serendipitously detected Mimivirus- and Marseillevirus-like sequences while using a new metagenomic approach targeting bacterial DNA that subsequently led to the isolation of a new member of the family Marseilleviridae, named Senegalvirus, from human stools. This discovery demonstrates the possibility of the presence of giant viruses of amoebae in humans. In addition, we detected sequences related to Megavirales members in several human metagenomes, which adds to previous findings by several groups.
Overall, we present convergent evidence of the presence of mimiviruses and marseilleviruses in humans. Our findings suggest that we should re-evaluate the human megavirome and investigate the prevalence, diversity and potential pathogenicity of giant viruses in humans.
Full-text · Article · May 2013 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Giant viruses of the Megavirales order have been recently isolated from aquatic environments and have long been neglected because they are removed from samples during viral purification for viral metagenomic studies. Due to bacterial overgrowth and susceptibility to high concentrations of antibiotics, isolation by amoeba co-culture has a low efficiency and is highly time-consuming. Thus, few environments have been exhaustively investigated to date, although the ubiquitous distribution of the Acanthamoeba sp. suggests that these viruses could also be ubiquitous. In this work, we have implemented a high-throughput method to detect amoebae lysis on agar plates that allows the testing of hundreds of samples in a few days. Using this procedure, a total of 11 new Marseilleviridae strains and four new Mimiviridae strains, including a virus infected with a virophage, were isolated from 1000 environmental samples from Tunisia. Of these, four corresponded to new genotypic variants. These isolates are the first African environmental isolates identified from these two families, and several samples were obtained from a hypersaline aquatic environment. These results demonstrate that this technique can be used for the evaluation and characterization of large collections of giant viruses to provide insight into understanding their ecology.
No preview · Article · Dec 2012 · Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: The family "Marseilleviridae" is a new proposed taxon for giant viruses that infect amoebae. Its first member, Acanthamoeba polyphaga marseillevirus (APMaV), was isolated in 2007 by culturing on amoebae a water sample collected from a cooling tower in Paris, France. APMaV has an icosahedral shape with a diameter of ≈250 nm. Its genome is a double-stranded circular DNA that is 368,454 base pairs (bp) in length. The genome has a GC content of 44.7 % and is predicted to encode 457 proteins. Phylogenetic reconstructions showed that APMaV belongs to a new viral family among nucleocytoplasmic large DNA viruses, a group of viruses that also includes Acanthamoeba polyphaga mimivirus (APMV) and the other members of the family Mimiviridae as well as the members of the families Poxviridae, Phycodnaviridae, Iridoviridae, Ascoviridae, and Asfarviridae. In 2011, Acanthamoeba castellanii lausannevirus (ACLaV), another close relative of APMaV, was isolated from river water in France. The ACLaV genome is 346,754 bp in size and encodes 450 genes, among which 320 have an APMaV protein as the closest homolog. Two other giant viruses closely related to APMaV and ACLaV have been recovered in our laboratory from a freshwater sample and a human stool sample using an amoebal co-culture method. The only currently identified hosts for "marseilleviruses" are Acanthamoeba spp. The prevalence of these viruses in the environment and in animals and humans remains to be determined.
Full-text · Article · Nov 2012 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ∼7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer.
Full-text · Article · Oct 2012 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Clin Microbiol Infect 2012; 18: 1185–1193
Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.
[Show abstract][Hide abstract] ABSTRACT: Interest in giant viruses has risen sharply since 2003, following the discovery of the Mimivirus and four other protist-infecting giant viruses that are linked to the nucleocytoplasmic large DNA viruses (NCLDVs). Despite considerable heterogeneity in hosts and genome sizes, the NCLDVs have been shown to be monophyletic based on analyses of their sequences and gene repertoires and recent studies have proposed that these viruses share a common ancient ancestor and compose a fourth domain of life. In addition, several characteristics of these giant viruses contradict or do not match the criteria used for the canonical definition of viruses, and the NCLDV denomination is not completely appropriate. We propose here to define a new viral order named Megavirales.
[Show abstract][Hide abstract] ABSTRACT: Two isolates of intra-amoeba-growing bacteria, LegAT and LegMT, were characterized on the basis of microscopic appearance, staining characteristics, axenic growth at different temperatures and the sequences of the mip, rpoB, 16S rRNA, and rnpb genes as well as the 23S-5S region. Phylogenetic analysis showed that these two isolates lay within the radius of the Legionellaceae. Furthermore, the analysis of these genes yielded congruent data that indicated that, although LegMT clusters specifically with L. feeleii and LegAT clusters with L. nautarum, the divergence observed between these species was greater than that observed between other members of the family. Taken together, these results support the proposal of these two isolates as new Legionella species, which we propose be named Legionella tunisiensis sp. nov. (for LegMT) and Legionella massiliensis sp. nov. (for LegAT).
Preview · Article · Feb 2012 · International Journal of Systematic and Evolutionary Microbiology
[Show abstract][Hide abstract] ABSTRACT: The genome sequence of the Mamavirus, a new Acanthamoeba polyphaga mimivirus strain, is reported. With 1,191,693 nt in length and 1,023 predicted protein-coding genes, the Mamavirus has the largest genome among the known viruses. The genomes of the Mamavirus and the previously described Mimivirus are highly similar in both the protein-coding genes and the intergenic regions. However, the Mamavirus contains an extra 5'-terminal segment that encompasses primarily disrupted duplicates of genes present elsewhere in the genome. The Mamavirus also has several unique genes including a small regulatory polyA polymerase subunit that is shared with poxviruses. Detailed analysis of the protein sequences of the two Mimiviruses led to a substantial amendment of the functional annotation of the viral genomes.
Full-text · Article · Jun 2011 · Genome Biology and Evolution
[Show abstract][Hide abstract] ABSTRACT: Most phagocytic protist viruses have large particles and genomes as well as many laterally acquired genes that may be associated with a sympatric intracellular life (a community-associated lifestyle with viruses, bacteria, and eukaryotes) and the presence of virophages. By subculturing Mimivirus 150 times in a germ-free amoebal host, we observed the emergence of a bald form of the virus that lacked surface fibers and replicated in a morphologically different type of viral factory. When studying a 0.40-μm filtered cloned particle, we found that its genome size shifted from 1.2 (M1) to 0.993 Mb (M4), mainly due to large deletions occurring at both ends of the genome. Some of the lost genes are encoding enzymes required for posttranslational modification of the structural viral proteins, such as glycosyltransferases and ankyrin repeat proteins. Proteomic analysis allowed identification of three proteins, probably required for the assembly of virus fibers. The genes for two of these were found to be deleted from the M4 virus genome. The proteins associated with fibers are highly antigenic and can be recognized by mouse and human antimimivirus antibodies. In addition, the bald strain (M4) was not able to propagate the sputnik virophage. Overall, the Mimivirus transition from a sympatric to an allopatric lifestyle was associated with a stepwise genome reduction and the production of a predominantly bald virophage resistant strain. The new axenic ecosystem allowed the allopatric Mimivirus to lose unnecessary genes that might be involved in the control of competitors.
Full-text · Article · Jun 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: A recent work has provided strong arguments in favor of a fourth domain of Life composed of nucleo-cytoplasmic large DNA viruses (NCLDVs). This hypothesis was supported by phylogenetic and phyletic analyses based on a common set of proteins conserved in Eukarya, Archaea, Bacteria, and viruses, and implicated in the functions of information storage and processing. Recently, the genome of a new NCLDV, Cafeteria roenbergensis virus (CroV), was released. The present work aimed to determine if CroV supports the fourth domain of Life hypothesis.
A consensus phylogenetic tree of NCLDVs including CroV was generated from a concatenated alignment of four universal proteins of NCLDVs. Some features of the gene complement of CroV and its distribution along the genome were further analyzed. Phylogenetic and phyletic analyses were performed using the previously identified common set of informational genes present in Eukarya, Archaea, Bacteria, and NCLDVs, including CroV.
Phylogenetic reconstructions indicated that CroV is clearly related to the Mimiviridae family. The comparison between the gene repertoires of CroV and Mimivirus showed similarities regarding the gene contents and genome organization. In addition, the phyletic clustering based on the comparison of informational gene repertoire between Eukarya, Archaea, Bacteria, and NCLDVs unambiguously classified CroV with other NCLDVs and clearly included it in a fourth domain of Life. Taken together, these data suggest that Mimiviridae, including CroV, may have inherited a common gene content probably acquired from a common Mimiviridae ancestor.
This further analysis of the gene repertoire of CroV consolidated the fourth domain of Life hypothesis and contributed to outline a functional pan-genome for giant viruses infecting phagocytic protistan grazers.
[Show abstract][Hide abstract] ABSTRACT: Metagenomic studies have revealed that Acanthamoeba polyphaga Mimivirus relatives are common in the environment; however, only three Acanthamoeba-growing giant viruses have been isolated from hundreds of environmental samples. We attempted herein to isolate new Acanthamoeba-growing giant viruses from environmental samples.
We inoculated 105 environmental samples by our usual procedure but with the addition of selected antibiotics to inhibit bacterial overgrowth.
We isolated 19 giant viruses with capsid sizes of 150 to 600 nm, including one associated with a virophage. For the first time some were isolated from saltwater and soil samples. Tentative characterization using the PolB gene sequence was possible for some of these viruses. They were closely related to each other but different from the two previous isolates of Acanthamoeba polyphaga Mimivirus. Results obtained by MALDI-TOF MS analysis of viral particles were congruent with that of PolB sequencing.
Our data confirm that Acanthamoeba-growing giant viruses are common in the environment. Additionally, MALDI-TOF MS analysis can be used for the initial screening of new viruses to avoid redundant analysis. However, due to their genetic variability, it is likely that the genome sequences of most of these viruses will have to be determined for accurate classification.
[Show abstract][Hide abstract] ABSTRACT: Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of "Marseille" virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are "melting pots" of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins.
Preview · Article · Dec 2009 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Staphylococcus aureus is a major human pathogen responsible for a variety of nosocomial and community-acquired infections. Recent reports show that the prevalence of Methicillin-Resistant S. aureus (MRSA) infections in cystic fibrosis (CF) patients is increasing. In 2006 in Marseille, France, we have detected an atypical MRSA strain with a specific antibiotic susceptibility profile and a unique growth phenotype. Because of the clinical importance of the spread of such strain among CF patients we decided to sequence the genome of one representative isolate (strain CF-Marseille) to compare this to the published genome sequences. We also conducted a retrospective epidemiological analysis on all S. aureus isolated from 2002 to 2007 in CF patients from our institution.
CF-Marseille is multidrug resistant, has a hetero-Glycopeptide-Intermediate resistance S. aureus phenotype, grows on Cepacia agar with intense orange pigmentation and has a thickened cell wall. Phylogenetic analyses using Complete Genome Hybridization and Multi Locus VNTR Assay showed that CF-Marseille was closely related to strain Mu50, representing vancomycin-resistant S. aureus. Analysis of CF-Marseille shows a similar core genome to that of previously sequenced MRSA strains but with a different genomic organization due to the presence of specific mobile genetic elements i.e. a new SCCmec type IV mosaic cassette that has integrated the pUB110 plasmid, and a new phage closely related to phiETA3. Moreover this phage could be seen by electron microscopy when mobilized with several antibiotics commonly used in CF patients including, tobramycin, ciprofloxacin, cotrimoxazole, or imipenem. Phylogenetic analysis of phenotypically similar h-GISA in our study also suggests that CF patients are colonized by polyclonal populations of MRSA that represents an incredible reservoir for lateral gene transfer.
In conclusion, we demonstrated the emergence and spreading of a new isolate of MRSA in CF patients in Marseille, France, that has probably been selected in the airways by antibiotic pressure. Antibiotic-mediated phage induction may result in high-frequency transfer and the unintended consequence of promoting the spread of virulence and/or antibiotic resistance determinants. The emergence of well-adapted MRSA is worrying in such population chronically colonized and receiving many antibiotics and represents a model for emergence of uncontrollable super bugs in a specific niche.
This article was reviewed by Eric Bapteste, Pierre Pontarotti, and Igor Zhulin. For the full reviews, please go to the Reviewers' comments section.
[Show abstract][Hide abstract] ABSTRACT: Viruses are obligate parasites of Eukarya, Archaea and Bacteria. Acanthamoeba polyphaga mimivirus (APMV) is the largest known virus; it grows only in amoeba and is visible under the optical microscope. Mimivirus possesses a 1,185-kilobase double-stranded linear chromosome whose coding capacity is greater than that of numerous bacteria and archaea1, 2, 3. Here we describe an icosahedral small virus, Sputnik, 50 nm in size, found associated with a new strain of APMV. Sputnik cannot multiply in Acanthamoeba castellanii but grows rapidly, after an eclipse phase, in the giant virus factory found in amoebae co-infected with APMV4. Sputnik growth is deleterious to APMV and results in the production of abortive forms and abnormal capsid assembly of the host virus. The Sputnik genome is an 18.343-kilobase circular double-stranded DNA and contains genes that are linked to viruses infecting each of the three domains of life Eukarya, Archaea and Bacteria. Of the 21 predicted protein-coding genes, eight encode proteins with detectable homologues, including three proteins apparently derived from APMV, a homologue of an archaeal virus integrase, a predicted primase-helicase, a packaging ATPase with homologues in bacteriophages and eukaryotic viruses, a distant homologue of bacterial insertion sequence transposase DNA-binding subunit, and a Zn-ribbon protein. The closest homologues of the last four of these proteins were detected in the Global Ocean Survey environmental data set5, suggesting that Sputnik represents a currently unknown family of viruses. Considering its functional analogy with bacteriophages, we classify this virus as a virophage. The virophage could be a vehicle mediating lateral gene transfer between giant viruses.