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We report the detection of genomic signatures of giant viruses (GVs) in the metagenomes of three environment samples from Mumbai, India, namely, a pre-filter of a household water purifier, a sludge sample from wastewater treatment plant (WWTP), and a drying bed sample of the same WWTP. The de novo assembled contigs of each sample yielded 700 to 2000 maximum unique matches with the GV genomic database. In all three samples, the maximum number of reads aligned to Pandoraviridae, followed by Phycodnaviridae, Mimiviridae, Iridoviridae, and other Megaviruses. We also isolated GVs from every environmental sample (n = 20) we tested using co-culture of the sample with Acanthomoeba castellanii. From this, four randomly selected GVs were subjected to the genomic characterization that showed remarkable cladistic homology with the three GV families viz., Mimivirirdae (Mimivirus Bombay [MVB]), Megaviruses (Powai lake megavirus [PLMV] and Bandra megavius [BAV]), and Marseilleviridae (Kurlavirus [KV]). All 4 isolates exhibited remarkable genomic identity with respective GV families. Functionally, the genomes were indistinguishable from other previously reported GVs, encoding nearly all COGs across extant family members. Further, the uncanny genomic homogeneity exhibited by individual GV families across distant geographies indicate their yet to be ascertained ecological significance.

Microbes trapped in permanently frozen paleosoils (permafrost) are the focus of increasing researches in the context of global warming. Our previous investigations led to the discovery and reactivation of two Acanthamoeba-infecting giant viruses, Mollivirus sibericum and Pithovirus sibericum from a 30,000-year old permafrost layer. While several modern pithovirus strains have since been isolated, no contemporary mollivirus relative was found. We now describe Mollivirus kamchatka, a close relative to M. sibericum, isolated from surface soil sampled on the bank of the Kronotsky river in Kamchatka. This discovery confirms that molliviruses have not gone extinct and are at least present in a distant subarctic continental location. This modern isolate exhibits a nucleo-cytoplasmic replication cycle identical to that of M. sibericum. Its spherical particle (0.6-μm in diameter) encloses a 648-kb GC-rich double stranded DNA genome coding for 480 proteins of which 61 % are unique to these two molliviruses. The 461 homologous proteins are highly conserved (92 % identical residues in average) despite the presumed stasis of M. sibericum for the last 30,000 years. Selection pressure analyses show that most of these proteins contribute to the virus fitness. The comparison of these first two molliviruses clarify their evolutionary relationship with the pandoraviruses, supporting their provisional classification in a distinct family, the Molliviridae, pending the eventual discovery of intermediary missing links better demonstrating their common ancestry.ImportanceVirology has long been viewed through the prism of human, cattle or plant diseases leading to a largely incomplete picture of the viral world. The serendipitous discovery of the first giant virus visible under light microscopy (i.e., >0.3μm in diameter), mimivirus, opened a new era of environmental virology, now incorporating protozoan-infecting viruses. Planet-wide isolation studies and metagenomes analyses have shown the presence of giant viruses in most terrestrial and aquatic environments including upper Pleistocene frozen soils. Those systematic surveys have led authors to propose several new distinct families, including the Mimiviridae, Marseilleviridae, Faustoviridae, Pandoraviridae, and Pithoviridae. We now propose to introduce one additional family, the Molliviridae, following the description of M. kamchatka, the first modern relative of M. sibericum, previously isolated from 30,000-year old arctic permafrost.

International audience; Almost fifteen years ago, the discovery of Acanthamoeba polyphaga mimivirus, the first giant virus, changed how we define a virus. It was discovered incidentally in a process of isolating Legionella sp. from environmental samples in the context of pneumonia epidemics using a co-culture system with Acanthamoeba. Since then, much effort and improvement has been put into the original technique. In addition to the known families of Mimiviridae and Marseilleviridae, four new proposed families of giant viruses have been isolated: Pandoravirus, Pithovirus, Faustovirus and Mollivirus. Major improvements were based on enrichment systems, targeted use of antibiotics and high-throughput methods. The most recent development, using flow cytometry for isolation and presumptive identification systems, opens a path to large environmental surveys that may discover new giant virus families in new protozoa supports used for culture support.

More than a century ago, the term 'virus' was introduced to describe infectious agents that are invisible by light microscopy and capable of passing through sterilizing filters. In addition to their extremely small size, most viruses have minimal genomes and gene contents, and rely almost entirely on host cell-encoded functions to multiply. Unexpectedly, four different families of eukaryotic 'giant viruses' have been discovered over the past 10 years with genome sizes, gene contents and particle dimensions overlapping with that of cellular microbes. Their ongoing analyses are challenging accepted ideas about the diversity, evolution and origin of DNA viruses.

Viral infection had not been observed for amoebae, until the Acanthamoeba polyphaga mimivirus (APMV) was discovered in 2003. APMV belongs to the nucleocytoplasmatic large DNA virus (NCLDV) family and infects not only A. polyphaga, but also other professional phagocytes. Here, we review the Megavirales to give an overview of the current members of the Mimi- and Marseilleviridae families and their structural features during amoebal infection. We summarize the different steps of their infection cycle in A. polyphaga and Acanthamoeba castellani. Furthermore, we dive into the emerging field of virophages, which parasitize upon viral factories of the Megavirales family. The discovery of virophages in 2008 and research in recent years revealed an increasingly complex network of interactions between cell, giant virus, and virophage. Virophages seem to be highly abundant in the environment and occupy the same niches as the Mimiviridae and their hosts. Establishment of metagenomic and co-culture approaches rapidly increased the number of detected virophages over the recent years. Genetic interaction of cell and virophage might constitute a potent defense machinery against giant viruses and seems to be important for survival of the infected cell during mimivirus infections. Nonetheless, the molecular events during co-infection and the interactions of cell, giant virus, and virophage have not been elucidated, yet. However, the genetic interactions of these three, suggest an intricate, multilayered network during amoebal (co-)infections. Understanding these interactions could elucidate molecular events essential for proper viral factory activity and could implicate new ways of treating viruses that form viral factories.

The complete genome sequence of Kurlavirus, a new member of the family Marseilleviridae is reported. The Kurlavirus genome was found to encode a remarkable complement of genes homologous to those of other members of the family Marseilleviridae. Interestingly, the Kurlavirus genome contains 71 fewer ORFs than that of Marseillevirus, even though their genome sizes are comparable.

The family Marseilleviridae encompasses giant viruses that replicate in free-living Acanthamoeba amoebae. Since the discovery of the founding member Marseillevirus in 2007, 7 new marseilleviruses have been observed, including 3 from environmental freshwater, one from a dipteran, and two from symptom-free humans. Marseilleviruses have ≈250-nm-large icosahedral capsids and 346–386-kb-long mosaic genomes that encode 444–497 predicted proteins. They share a small set of core genes with Mimivirus and other large and giant DNA viruses that compose a monophyletic group, first described in 2001. Comparative genomics analyses indicate that the family Marseilleviridae currently includes three lineages and a pan-genome composed of ≈600 genes. Antibodies against marseilleviruses and viral DNA have been observed in a significant proportion of asymptomatic individuals and in the blood and lymph nodes of a child with adenitis; these observations suggest that these giant viruses may be blood borne and question if they may be pathogenic in humans.

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.

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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Objective: Following the isolation of a Marseillevirus from the stool of a healthy young Senegalese and a Mimivirus from a Tunisian patient with pneumonia, we attempted to isolate other giant viruses of amoebae from a large human stool collection. Methods: During the period 2010-2011, a total of 1,605 stool samples, including 115 from Tunisian patients with pneumonia, were cultured on amoebae. We used a recently developed high-throughput isolation system to detect amoebae plaque lysis on agar plates; this method allows for the testing of 100 samples per plate per week. The giant virus was identified by sequencing of genes conserved in Megavirales. Results: A single giant virus, called Shan, was isolated from the stool of a Tunisian patient with pneumonia who responded poorly to antibiotics. This virus has an icosahedral shape typical of members of the family Mimiviridae and a size of 640 ± 10 nm. Phylogenetic analyses showed that Shan virus was classified as a member of Mimivirus lineage C that infects amoebae. Conclusion: Only one isolate was obtained in this study, suggesting that giant viruses of amoebae are rare in human stool. The isolation of Shan virus from a patient with pneumonia brings into question the etiological role of this virus and its subsequent release in stool.

Giant viruses that infect amoebae, including mimiviruses and marseilleviruses, were first described in 2003. Virophages were subsequently described that infect mimiviruses. Culture isolation with Acanthamoeba spp. and metagenomic studies have shown that these giant viruses are common inhabitants of our biosphere and have enabled the recent detection of these viruses in human samples. However, the genomes of these viruses display substantial genetic diversity, making it a challenge to examine their presence in environmental and clinical samples using conventional and real-time PCR. We designed and evaluated the performance of PCR systems capable of detecting all currently isolated mimiviruses, marseilleviruses and virophages to assess their prevalence in various samples. Our real-time PCR assays accurately detected all or most of the members of the currently delineated lineages of giant viruses infecting acanthamoebae as well as the mimivirus virophages, and enabled accurate classification of the mimiviruses of amoebae in lineages A, B or C. We were able to detect four new mimiviruses directly from environmental samples and correctly classified these viruses within mimivirus lineage C. This was subsequently confirmed by culture on amoebae followed by partial Sanger sequencing. PCR systems such as those implemented here may contribute to an improved understanding of the prevalence of mimiviruses, their virophages and marseilleviruses in humans.

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.

International audience; During the 12 past years, five new or putative virus families encompassing several members, namely Mimiviridae, Marseilleviridae, pandoraviruses, faustoviruses, and virophages were described. In addition, Pithovirus sibericum and Mollivirus sibericum represent type strains of putative new giant virus families. All these viruses were isolated using amoebal coculture methods. These giant viruses were linked by phylogenomic analyses to other large DNA viruses. They were then proposed to be classified in a new viral order, the Megavirales, on the basis of their common origin, as shown by a set of ancestral genes encoding key viral functions, a common virion architecture, and shared major biological features including replication inside cytoplasmic factories. Megavirales is increasingly demonstrated to stand in the tree of life aside Bacteria, Archaea, and Eukarya, and the megavirus ancestor is suspected to be as ancient as cellular ancestors. In addition, giant amoebal viruses are visible under a light microscope and display many phenotypic and genomic features not found in other viruses, while they share other characteristics with parasitic microbes. Moreover, these organisms appear to be common inhabitants of our biosphere, and mimiviruses and marseilleviruses were isolated from human samples and associated to diseases. In the present review, we describe the main features and recent findings on these giant amoebal viruses and virophages.

International audience; In 2003, Acanthamoeba polyphaga mimivirus (APMV) was discovered as parasitizing Acanthamoeba. It was revealed to exhibit remarkable features, especially odd genomic characteristics, and founded viral family Mimiviridae. Subsequently, a second family of giant amoebal viruses was described, Marseilleviridae, whose prototype member is Marseillevirus, discovered in 2009. Currently, the genomes of seven different members of this family have been fully sequenced. Previous phylogenetic analysis suggested the existence of three Marseilleviridae lineages: A, B and C. Here, we describe a new member of this family, Brazilian Marseillevirus (BrMV), which was isolated from a Brazilian sample and whose genome was fully sequenced and analyzed. Surprisingly, data from phylogenetic analyses and comparative genomics, including mean amino acid identity between BrMV and other Marseilleviridae members and the analyses of the core genome and pan-genome of marseilleviruses, indicated that this virus can be assigned to a new Marseilleviridae lineage. Even if the BrMV genome is one of the smallest among Marseilleviridae members, it harbors the second largest gene content into this family. In addition, the BrMV genome encodes 29 ORFans. Here, we describe the isolation and genome analyses of the BrMV strain, and propose its classification as the prototype virus of a new lineage D within the family Marseilleviridae.

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.

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.

Large viruses infecting algae or amoebae belong to the NucleoCytoplasmic Large DNA Viruses (NCLDV) and present genotypic and phenotypic characteristics that have raised major interest among microbiologists. Here, we describe a new large virus discovered in Acanthamoeba castellanii co-culture of an environmental sample. The virus, referred to as Lausannevirus, has a very limited host range, infecting Acanthamoeba spp. but being unable to infect other amoebae and mammalian cell lines tested. Within A. castellanii, this icosahedral virus of about 200 nm exhibits a development cycle similar to Mimivirus, with an eclipse phase 2 h post infection and a logarithmic growth leading to amoebal lysis in less than 24 h. The 346 kb Lausannevirus genome presents similarities with the recently described Marseillevirus, sharing 89% of genes, and thus belongs to the same family as confirmed by core gene phylogeny. Interestingly, Lausannevirus and Marseillevirus genomes both encode three proteins with predicted histone folds, including two histone doublets, that present similarities to eukaryotic and archaeal histones. The discovery of Lausannevirus and the analysis of its genome provide some insight in the evolution of these large amoebae-infecting viruses.

Objective: 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. Methods: We inoculated 105 environmental samples by our usual procedure but with the addition of selected antibiotics to inhibit bacterial overgrowth. Results: 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. Conclusion: 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.

When viruses were discovered, they were accepted as missing links between the inert world and living organisms. However, this idea was soon abandoned as information about their molecular parasitic nature accumulated. Recently, the notion that viruses are living organisms that have had a role in the evolution of some essential features of cells has experienced a renaissance owing to the discovery of unusually large and complex viruses that possess typical cellular genes. Here, we contend that there is strong evidence against the notion that viruses are alive and represent ancient lineages of the tree of life.

International audience; Acanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-µm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of “omic” approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host’s ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses’ diversity remains to be fully explored.

International audience; Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases.

We recently described a giant double stranded DNA virus called Mimivirus, isolated from amoebae, which might represent a new pneumonia-associated human pathogen. Its unique morphological and genomic characteristics allowed us to propose Mimivirus as a member of a new distinct Nucleocytoplasmic Large DNA viruses family, the Mimiviridae. Mimivirus-specific features, namely its size and its genomic complexity, ranged it between viruses and cellular organisms. This paper reviews our current knowledge on Mimivirus structure, life cycle and genome analysis and discusses its putative evolutionary origin in the tree of species of the three domains of life.

In the environment, protozoa are predators of bacteria and feed on them. The possibility that some protozoa could be a source of human pathogens is consistent with the discovery that free-living amoebae were the reservoir of Legionella pneumophila, the agent of Legionnaires' disease. Later, while searching for Legionella in the environment using amoeba co-culture, the first giant virus, Acanthamoeba polyphaga mimivirus, was discovered. Since then, many other giant viruses have been isolated, including Marseilleviridae, Pithovirus sibericum, Cafeteria roenbergensis virus and Pandoravirus spp. The methods used to isolate all of these viruses are herein reviewed. By analogy to Legionella, it was originally suspected that these viruses could be human pathogens. After showing by indirect evidence, such as sero-epidemiologic studies, that it was possible for these viruses to be human pathogens, the recent isolation of some of these viruses (belonging to the Mimiviridae and Marseilleviridae families) in humans in the context of pathologic conditions shows that they are opportunistic human pathogens in some instances.

Aim: During the last decade it became obvious that viruses belonging to Mimiviridae and Marseilleviridae families (order Megavirales), may be potential causative agents of pneumonia. Thus, we have performed a review of the association of Mimiviridae, Marseilleviridae, and virophages with pneumonia, particularly healthcare-associated pneumonia, and other infections of the respiratory tract. Results and discussion: According to the analysis of the published articles, viruses belonging to Mimiviridae family can be potential agents of both community-acquired and healthcare-associated pneumonia. In particular, these viruses may be associated with poor outcome in patients of intensive care units. The exact mechanism of their pathogenicity, however, still remains unclear. The discrepancies between the results obtained by serological and genomic methods could be explained by the high polymorphism of nucleotide sequences of Mimiviridae family representatives. Further investigations on the Mimiviridae pathogenicity and on the determination of Mimiviridae-caused pneumonia risk groups are required. However, the pathogenicity of the viruses belonging to Marseilleviridae family and virophages is unclear up to now., GMS Hygiene and Infection Control; 9(2):Doc16; ISSN 2196-5226

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.

Giant viruses infecting phagocytic protists are composed of mimiviruses, the record holders of particle and genome size amongst viruses, and marseilleviruses. Since the discovery in 2003 at our laboratory of the first of these giant viruses, the Mimivirus, a growing body of data has revealed that they are common inhabitants of our biosphere. Moreover, from the outset, the story of Mimivirus has been linked to that of patients exhibiting pneumonia and it was shown that patients developed antibodies to this amoebal pathogen. Since then, there have been several proven cases of human infection or colonization with giant viruses of amoebae, which are known to host several bacteria that are human pathogens. Mimiviruses and marseilleviruses represent a major challenge in human pathology, as virological procedures implemented to date have not used appropriate media to allow their culture, and molecular techniques have used filtration steps that likely prevented their detection. Nevertheless, there is an increasing body of evidence that mimiviruses might cause pneumonia and that humans carry marseilleviruses, and re-analyses of metagenomic databases have provided evidence that these giant viruses can be common in human samples. The proportion of human infections related to these giant mimiviruses and marseilleviruses and the precise short- and long-term consequences of these infections have been scarcely investigated so far and should be the subject of future works.

Since 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.

Acanthamoeba polyphaga mimivirus was discovered in 2003 but was isolated two decades ago during the investigation of a pneumonia outbreak, while trying to isolate Legionella-like bacterial pathogens that infect and survive in amoebae. Acanthamoeba polyphaga marseillevirus was isolated five years thereafter. These two viruses are the founding members of two new viral families whose members are the largest known viruses based on the sizes of the capsid and the genome. Beyond being giant, mimiviruses and marseilleviruses exhibit several unexpected and remarkable features. Their genomes encode proteins involved in translation, these viruses harbour messenger ribonucleic acid in addition to their double-stranded deoxyribonucleic acid (DNA) genome, and their gene repertoire indicates that they have a common ancestral origin with other large DNA viruses. Finally, mimiviruses and marseilleviruses are likely to be common in our biosphere and have begun to be isolated from humans. Key Concepts: Mimiviruses and marseilleviruses are giant viruses that infect phagocytic protists (Acanthamoeba spp. in most cases). Mimiviruses are the largest known viruses, and their genomes are the largest among viruses and are larger than those of the smallest cellular life. Mimiviruses and marseilleviruses differ greatly from ‘classical’ viruses in several respects. Mimiviruses and marseilleviruses are linked by phylogeny based on certain genes and their core genomes to the members of the five families of nucleocytoplasmic large DNA viruses; these seven families were recently proposed to be assigned to a new viral order, Megavirales. The mosaic gene repertoires of mimiviruses and marseilleviruses are likely linked to their sympatric lifestyle within phagocytic protistan grazers. Mimiviruses and marseilleviruses are common in our biosphere. Mimiviruses may cause pneumonia in humans. Keywords: mimivirus; marseillevirus; megavirales; mimiviridae; marseilleviridae; giant viruses; nucleocytoplasmic large DNA viruses; fourth domain

The nucleocytoplasmic large DNA viruses (NCLDVs) comprise a monophyletic group of viruses that infect animals and diverse unicellular eukaryotes. The NCLDV group includes the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Mimiviridae and the proposed family "Marseilleviridae". The family Mimiviridae includes the largest known viruses, with genomes in excess of one megabase, whereas the genome size in the other NCLDV families varies from 100 to 400 kilobase pairs. Most of the NCLDVs replicate in the cytoplasm of infected cells, within so-called virus factories. The NCLDVs share a common ancient origin, as demonstrated by evolutionary reconstructions that trace approximately 50 genes encoding key proteins involved in viral replication and virion formation to the last common ancestor of all these viruses. Taken together, these characteristics lead us to propose assigning an official taxonomic rank to the NCLDVs as the order "Megavirales", in reference to the large size of the virions and genomes of these viruses.

Zeus' Revenge Sediment-dwelling amoebae appear to have an unhappy affinity for huge viruses. Giant icosahedral Mimiviruses with genomes of the order of 1 megabase (Mb) were first identified in Acanthamoeba. Digging into antipodean sediments has once again been fruitful where Philippe et al. (p. 281 ; see the cover) discovered some enormous viruses in Acanthamoeba, visible by light microscopy and having genomes up to 2.5 Mb. The Pandoraviruses are phagocytosed by target cells and, after fusing with the phagosome membrane, their contents are released into the cytoplasm where they wreak terrible havoc on its nucleus. These viruses are encased into a tegument-like envelope and lack genes for capsid proteins, and there are no genes for protein translation, adenosine triphosphate generation, or binary fission—confirming their classification as viruses.

a b s t r a c t 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-m 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. Objectives: 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. Results: 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. Conclusions: 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.

The 2003 discovery of Acanthamoeba polyphaga Mimivirus led to several breakthroughs and subsequent discussions related to the evolution, origin and definition of viruses and dramatically boosted scientific interest in giant viruses. Mimivirus was the largest virus with respect to particle size and genome length, and its analysis blurred the paradigms of the viral world. Since 2008, several new viruses have been recovered from a variety of phagocytic protists and water samples. All of the protist-associated giant viruses have been proposed to share an ancestral origin and to constitute a new domain of life distinct from Bacteria, Archaea, and Eukarya, and they differ in many respects from other viruses and strongly challenge the canonical virus paradigm. Mimiviridae have a capsid diameter of approximately 500 nm and large genomes that encode more than 1,000 predicted proteins. Mimivirus and Marseillevirus were shown to harbor mRNA. Moreover, the Mimiviridae and Marseilleviridae encode proteins involved in translation and the Mimiviridae are themselves susceptible to infection by other viruses. In addition, the genomes of these viruses are mosaics composed of genes related to eukaryotes, bacteria and archaea, and they harbor signs of considerable modifications resulting from horizontal gene transfer, and gene duplication. Importantly, a growing body of evidence indicates that Mimiviridae and Marseill­eviridae are widely distributed in the biosphere and there are several clues suggesting the pathogenicity of these giant viruses that infect phagocytic protists. In this chapter, we will summarize the current knowledge on the Mimiviridae and Marseilleviridae families.

The discovery of Mimivirus, with its very large genome content, made it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation, we found in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supports existence of a clade containing NCLDVs clearly distinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life.

Amoebae are unicellular phagocytes that feed on microorganisms in their environment. Some amoebae have the largest genome size currently known on earth. They phagocytose any inert particle larger than 0.5 µm. Phagocytic amoebae can harbor different bacteria, fungi and giant viruses within the same cell. There is evidence of lateral gene transfer between the amoeba and its microbiological hosts. There is also evidence of gene exchange between viruses and bacteria hosted in amoebae. Moreover, there is evidence of gene transfer between viruses, such as Mimivirus and the virophage. As a consequence, viruses and intracellular bacteria living in amoebae have a sympatric lifestyle and large chimeric genome repertoires. We conclude that phagocytic protists continuously generate new species with chimeric repertoires that may succeed later if adapted to the environmental conditions and selected in a specific niche.

International audience; The current paradigm on the nature of viruses is based on early work of the 'phage group' (the pro-phage concept) and molecular biologists working on tumour viruses (the proto-oncogene concept). It posits that viruses evolved from either prokaryotic or eukaryotic cellular genes that became infectious via their association with capsid genes. In this view, after their emergence viruses continued to evolve by stealing cellular genes (the escape model). This paradigm has been challenged recently by scientists who propose that viruses pre-dated modern cells. In particular, the discovery of Mimivirus has stimulated a lot of discussions on the nature of viruses. There are two major schools of thought, those who defend the escape model, suggesting that giant viruses are giant pickpockets (chimera), and those who emphasize their uniqueness and ancient origin. Comparative genomics of Mimivirus and related viruses (nucleo-cytoplasmic large DNA viruses) have produced a lot of data that have been interpreted according to the prejudices of the authors and thus failed until now to generate a consensus. I briefly review here the history of these debates and how they lead to new proposals, such as the definition of viruses as capsid-encoding organisms or else the recognition of their fundamentally cellular nature, the virocell concept.

Mimivirus, a giant DNA virus (i.e. "girus") infecting species of the genus Acanthamoeba, was first identified in 2003. With a particle size of 0.7microm in diameter, and a genome size of 1.2Mb encoding more than 900 proteins, it is the most complex virus described to date. Beyond its unusual size, the Mimivirus genome was found to contain the first viral homologues of many genes thought to be the trademark of cellular organisms, such as central components of the translation apparatus. These findings revived the debate on the origin of DNA viruses, and the role they might have played in the emergence of eukaryotes. Published and ongoing studies on Mimivirus continue to lead to unexpected findings concerning a variety of aspects, such as the structure of its particle, unique features of its replication cycle, or the distribution and abundance of Mimivirus relatives in the oceans. Following a summary of these recent findings, we present preliminary results suggesting that octocorals might have come in close contact with an ancestor of Mimivirus, and that modern sponges might be host to a yet unidentified, even larger, member of the Mimiviridae.

Background Acanthamoeba polyphaga mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the Acanthamoeba genus. The existence of mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data. Results We now further demonstrate the presence of numerous "mimivirus-like" sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (Chrysochromulina ericina, Phaeocystis pouchetii, Pyramimonas orientalis) are very closely related to their homolog in mimivirus. Conclusion Our results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of Mimiviridae.

During recent years, the usefulness of amoebal co-cultures as an alternative means of isolating and cultivating fastidious microorganisms has been increasingly recognized. While characterizing a collection of bacteria that had been isolated using this approach, we encountered an organism that, on preliminary analysis, appeared to be a gram-positive coccus. However, additional examination revealed that it was not a bacterium but rather, surprisingly, a virus. The dimensions of the virus particle (diameter, 0.8 microm) and its genome size (1.2 Mb) are far more akin to those of bacteria than to those of previously recognized viruses. These characteristics, together with such features as the breadth and complexity of its gene content, challenge the current definition of a "virus." Furthermore, the virus, now named "Mimivirus," has been implicated as an agent of pneumonia in humans and, thus, should be considered a putative emerging pathogen.

The aim of this epidemiological study was to determine the prevalence of respiratory viruses, including new viruses, in hospitalised children in Austria. Two hundred fourteen nasopharyngeal samples from hospitalised children were tested for the presence of viruses using cell culture and PCR and/or viral antigen assays. The results revealed a parainfluenza virus 1 (PIV1) outbreak that ended right before the onset of the influenza season, with nearly no overlapping, moderate respiratory syncytial virus (RSV) activity, and only a few adenoviruses. Human metapneumovirus (hMPV) was present in 14.5% of the total samples but was detected in combination with other viruses in only five cases: with PIV1 in three cases and with RSV in two cases. There were no cases of dual infection with hMPV and flu or adenovirus. This suggests that hMPV alone is a leading cause of hospitalisation in children under 1 year of age. Interestingly, hMPV, in contrast to RSV, coincided with PIV1 but was absent during the community outbreak of the flu. Samples were also tested for Mimiviridae, a group of newly described DNA viruses that are similar to Legionella spp., replicate in water amoebae, and also have been found in alveolar cells. However, mimivirus was detected neither in respiratory samples nor in amoebae-containing water samples, indicating that this particular type of virus is either not abundant or does not contribute to paediatric respiratory illnesses.

A previous comparative-genomic study of large nuclear and cytoplasmic DNA viruses (NCLDVs) of eukaryotes revealed the monophyletic origin of four viral families: poxviruses, asfarviruses, iridoviruses, and phycodnaviruses [Iyer, L.M., Aravind, L., Koonin, E.V., 2001. Common origin of four diverse families of large eukaryotic DNA viruses. J. Virol. 75 (23), 11720-11734]. Here we update this analysis by including the recently sequenced giant genome of the mimiviruses and several additional genomes of iridoviruses, phycodnaviruses, and poxviruses. The parsimonious reconstruction of the gene complement of the ancestral NCLDV shows that it was a complex virus with at least 41 genes that encoded the replication machinery, up to four RNA polymerase subunits, at least three transcription factors, capping and polyadenylation enzymes, the DNA packaging apparatus, and structural components of an icosahedral capsid and the viral membrane. The phylogeny of the NCLDVs is reconstructed by cladistic analysis of the viral gene complements, and it is shown that the two principal lineages of NCLDVs are comprised of poxviruses grouped with asfarviruses and iridoviruses grouped with phycodnaviruses-mimiviruses. The phycodna-mimivirus grouping was strongly supported by several derived shared characters, which seemed to rule out the previously suggested basal position of the mimivirus [Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., Claverie, J.M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350]. These results indicate that the divergence of the major NCLDV families occurred at an early stage of evolution, prior to the divergence of the major eukaryotic lineages. It is shown that subsequent evolution of the NCLDV genomes involved lineage-specific expansion of paralogous gene families and acquisition of numerous genes via horizontal gene transfer from the eukaryotic hosts, other viruses, and bacteria (primarily, endosymbionts and parasites). Amongst the expansions, there are multiple families of predicted virus-specific signaling and regulatory domains. Most NCLDVs have also acquired large arrays of genes related to ubiquitin signaling, and the animal viruses in particular have independently evolved several defenses against apoptosis and immune response, including growth factors and potential inhibitors of cytokine signaling. The mimivirus displays an enormous array of genes of bacterial provenance, including a representative of a new class of predicted papain-like peptidases. It is further demonstrated that a significant number of genes found in NCLDVs also have homologs in bacteriophages, although a vertical relationship between the NCLDVs and a particular bacteriophage group could not be established. On the basis of these observations, two alternative scenarios for the origin of the NCLDVs and other groups of large DNA viruses of eukaryotes are considered. One of these scenarios posits an early assembly of an already large DNA virus precursor from which various large DNA viruses diverged through an ongoing process of displacement of the original genes by xenologous or non-orthologous genes from various sources. The second scenario posits convergent emergence, on multiple occasions, of large DNA viruses from small plasmid-like precursors through independent accretion of similar sets of genes due to strong selective pressures imposed by their life cycles and hosts.

The discovery and genome analysis of Acanthamoeba polyphaga Mimivirus, the largest known DNA virus, challenged much of the accepted dogma regarding viruses. Its particle size (>400 nm), genome length (1.2 million bp) and huge gene repertoire (911 protein coding genes) all contribute to blur the established boundaries between viruses and the smallest parasitic cellular organisms. Phylogenetic analyses also suggested that the Mimivirus lineage could have emerged prior to the individualization of cellular organisms from the three established domains, triggering a debate that can only be resolved by generating and analyzing more data. The next step is then to seek some evidence that Mimivirus is not the only representative of its kind and determine where to look for new Mimiviridae. An exhaustive similarity search of all Mimivirus predicted proteins against all publicly available sequences identified many of their closest homologues among the Sargasso Sea environmental sequences. Subsequent phylogenetic analyses suggested that unknown large viruses evolutionarily closer to Mimivirus than to any presently characterized species exist in abundance in the Sargasso Sea. Their isolation and genome sequencing could prove invaluable in understanding the origin and diversity of large DNA viruses, and shed some light on the role they eventually played in the emergence of eukaryotes., see also http://www.giantvirus.org

tion of microbes [2] . As a matter of fact, in the 20th century, some giant viruses were considered for a long time to be bacteria or parasites because the size factor biased their initial viral classification. Indeed, the established definition of a virus during the 20th century considered that all viruses should have at least one dimension of less than 0.2 μm [2, 7] . Giant viruses pose a problem to traditional virology techniques because purification techniques prior to virome study involve a filtration that removes giant viruses [12–14] . We were recently able to show that, when avoiding 0.2-μm filtration, giant viruses belonging to the Marseillevirus family could be found circulating in the blood of asymptomatic patients [13, 14] . In addition, for now, PCR techniques have unfortunately demonstrated little sensitivity because, due to the high variability of these viruses, tools were lacking to identify them [2, 15] . Culture techniques have indeed enabled the isolation of these viruses for the first time in human stool samples, with a reference reported in this paper [16] . New strategies for culture, purification [17] and isolation techniques [16] are reported in the present issue. Also, for the first time, these techniques have enabled the isolation of Mimivirus from a respiratory sample [18, 19] and the stools of a patient with pneumonia [20] . However, it is likely that serology is more effective in the detection of specific antibodies against the Marseilleviridae family, as related in Gilbert Greub’s work in this journal [21] . These data confirm that both Mimiviridae and Marseilleviridae may be found in humans [19] . Mimivirus The world of giant viruses was brutally opened up at the beginning of the 21st century when a microorganism visible under the microscope, Gram stained, and which had been considered a bacterium for several years, was identified as the largest virus known at the time: it was Mimivirus [1, 2] . Its genome contained more than 1,200 genes and had the particularity, given the wealth of information it contained, to harbor genes common to bacteria, archaea and eukaryotes [3] . To me, this particular feature was the most striking, and I postulated immediately for the publication of the genome, believing that this virus might be representative of a fourth domain [3] . This caused various reactions, in spite of a subsequent classification into four branches using genes encoding for RNA polymerase showing four groups: giant viruses, archaea, bacteria and eukaryotes [4, 5] . For a while, I was the only one to believe in the existence of a fourth domain, which was challenged both by Jean-Michel Claverie and C.E. Woese [6] . We proposed later, based on these data, to create a new classification splitting organisms into capsidencoding organisms and ribosome-encoding organisms [7] . In this issue, the specificity of codon and amino acid use of this group of viruses is reported [8] . Since then, we have reported the existence of a new family of viruses, Marseilleviridae [9] , the first strain being Marseillevirus [10] . The recent discovery of a new giant virus, considered to be an intracellular eukaryotic parasite for 15 years [11] , shows that the world of giant viruses keeps expanding and challenges the traditional classificaPublished online: October 17, 2013

Phylogenetic mapping of metagenomics data reveals the taxonomic distribution of large DNA viruses in the sea, including giant viruses of the Mimiviridae family., Background Viruses are ubiquitous and the most abundant biological entities in marine environments. Metagenomics studies are increasingly revealing the huge genetic diversity of marine viruses. In this study, we used a new approach - 'phylogenetic mapping' - to obtain a comprehensive picture of the taxonomic distribution of large DNA viruses represented in the Sorcerer II Global Ocean Sampling Expedition metagenomic data set. Results Using DNA polymerase genes as a taxonomic marker, we identified 811 homologous sequences of likely viral origin. As expected, most of these sequences corresponded to phages. Interestingly, the second largest viral group corresponded to that containing mimivirus and three related algal viruses. We also identified several DNA polymerase homologs closely related to Asfarviridae, a viral family poorly represented among isolated viruses and, until now, limited to terrestrial animal hosts. Finally, our approach allowed the identification of a new combination of genes in 'viral-like' sequences. Conclusion Albeit only recently discovered, giant viruses of the Mimiviridae family appear to constitute a diverse, quantitatively important and ubiquitous component of the population of large eukaryotic DNA viruses in the sea.

The origins of viruses are shrouded in mystery, but advances in genomics and the discovery of highly complex giant DNA viruses have stimulated new hypotheses that DNA viruses were involved in the emergence of the eukaryotic cell nucleus, and that they are worthy of being considered as living organisms.

Raoult et al . ([ 1 ][1]) analyzed the complete genome sequence of Mimivirus, the largest known virus. One of the most exciting results of this analysis is the identification of seven conserved mimiviral proteins that are evolutionarily related to their eukaryotic homologs: arginyl-tRNA synthetase