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Giant viruses have brought new perspectives on the virosphere. They have been increasingly described in humans, including in several metagenomic studies. Here, we searched into the metagenome of the 5300-year-old Otzi mummy for the presence of giant virus-related sequences using MG-Digger pipeline. We found 19 reads (0.00006% of the total read number) that best matched (mean ± standard deviation (range) for e-values of 5.0E-6 ± 1.4E-6 (6.0E-5–4.0E-10) and for amino acid identity of 69.9 ± 8.7% (46.4–84.9%) and most significantly with sequences from various giant viruses, including mostly mimiviruses. This expands current knowledge on the ubiquity and relationship with humans of giant viruses.

Made available in DSpace on 2022-04-29T08:38:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Mimiviruses are giant viruses of amoeba that can be found in association with virophages. These satellite-like viruses are dependent on the mimivirus viral factory to replicate. Mimiviruses can also be associated with linear DNA molecules called transpovirons. Transpovirons and virophages are important drivers of giant virus evolution although they are still poorly studied elements. Here, we describe the isolation and genomic characterization of a mimivirus/virophage/transpoviron tripartite system from Brazil. We analyzed transmission electron microscopy images and performed genome sequencing and assembly, gene annotation, and phylogenetic analysis. Our data confirm the isolation of a lineage A mimivirus (1.2 Mb/1012 ORFs), called mimivirus argentum, and a sputnik virophage (18,880 bp/20 ORFs). We also detected a third sequence corresponding to a transpoviron from clade A (6365 bp/6 ORFs) that presents small terminal inverted repeats (77 nt). The main genomic features of mimivirus argentum and of its virophage/transpoviron elements corroborates with what is described for other known elements. This highlights that this triple genomic and biological interaction may be ancient and well-conserved. The results expand the basic knowledge about unique and little-known elements and pave the way to future studies that might contribute to a better understanding of this tripartite relationship. Laboratório de Vírus Departamento de Microbiologia Universidade Federal de Minas Gerais (UFMG), MG Laboratório de Virologia Departamento de Microbiologia e Imunologia Instituto de Biotecnologia Universidade Estadual Paulista (Unesp), Alameda das Tecomarias s/n, Chácara Capão Bonito, SP Laboratório de Virologia Departamento de Microbiologia e Imunologia Instituto de Biotecnologia Universidade Estadual Paulista (Unesp), Alameda das Tecomarias s/n, Chácara Capão Bonito, SP CNPq: 302081/2018-6

The fascinating discovery of the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), belonging to the family Mimiviridae in 2008, and its associated virophage, Sputnik, have left the world of microbiology awestruck. To date, about 18 virophages have been isolated from different environmental sources. With their unique feature of resisting host cell infection and lysis by giant viruses, analogous to bacteriophage, they have been assigned under the family Lavidaviridae. Genome of T-27, icosahedral-shaped, non-enveloped virophages, consist of dsDNA encoding four proteins, namely, major capsid protein, minor capsid protein, ATPase and cysteine protease, which are essential in the formation and assembly of new virophage particles during replication. A few virophage genomes have been observed to contain additional sequences like PolB, ZnR and S3H. Another interesting characteristic of virophage is that Mimivirus lineage A is immune to infection by the Zamilon virophage through a phenomenon termed MIMIVIRE, resembling the CRISPR-Cas mechanism in bacteria. Based on the fact that giant viruses have been found in clinical samples of hospital-acquired pneumonia and rheumatoid arthritis patients, virophages have opened a novel era in the search for cures of various diseases. This article aims to study the prospective role of virophages in the future of human therapeutics.

Even after two decades since the identification of the first giant virus, the Acanthamoeba polyphaga mimivirus (APMV), it still elude scientists. Their gigantic size and genome are unique in the whole virosphere, and many aspects of their biology are still unknown, including their possible hosts. They are cultivated in laboratories using Acanthamoeba cells as hosts, but little is known about the infectivity of these giant viruses in vertebrate cells. However, there is evidence of the possible involvement of APMV in pneumonia and activation of inflammatory pathways. Among the hundreds of prospected giant viruses members is Tupanvirus, isolated in Brazil. Its particles have a characteristically large size varying between 1.2 to 2 μm and are covered by fibrils. In the present work, we aim to study the consequences of the incubation of APMV and Tupanvirus with mammalian cells. These cells express Toll-like receptors (TLR) that are capable of recognizing lipopolysaccharides, favoring the internalization of the antigen and activation of the inflammatory system. We used a lineage of human lung adenocarcinoma cells (A549) to evaluate possible effects of TLR activation by the giant viruses and if we could detect the probable cause of the said giant-virus dependent pneumonia. Our results show that APMV and Tupanvirus (TPV) activate cellular receptors related to the Toll-like 4 type-induced inflammatory response and that the A549 cells are capable of internalizing the latter virus. Therefore, this study brings new insights into the possible interactions established between mimiviruses (here represented by APMV and Tupanvirus) and members of the innate cellular immune response.

The giant Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group of diverse viruses that contain double-stranded DNA (dsDNA) genomes that replicate primarily in eukaryotic hosts. Two members of the NCLDV, Vaccinia Virus (VACV) and African Swine Fever Virus (ASFV), both synthesize Nudix enzymes that have been shown to decap mRNA, a process thought to accelerate viral and host mRNA turnover and promote the shutoff of host protein synthesis. Mimivirus encodes two Nudix enzymes in its genome, denoted as L375 and L534. Importantly, L375 exhibits sequence similarity to ASFV-DP and eukaryotic Dcp2, two Nudix enzymes shown to possess mRNA decapping activity. In this work, we demonstrate that recombinant Mimivirus L375 cleaves the 5’ m7GpppN mRNA cap, releasing m7GDP as a product. L375 did not significantly cleave mRNAs containing an unmethylated 5’GpppN cap, indicating that this enzyme specifically hydrolyzes methylated-capped transcripts. A point mutation in the L375 Nudix motif completely eliminated cap hydrolysis, showing that decapping activity is dependent on this motif. Addition of methylated cap derivatives or uncapped RNA inhibited L375 decapping activity, suggesting that L375 recognizes its substrate through interaction with both the mRNA cap and RNA body.

Since 2003, various viruses from the subfamily Megavirinae in the family Mimiviridae have been isolated worldwide, including icosahedral mimiviruses and tailed tupanviruses. To date, the evolutionary relationship between tailed and nontailed mimiviruses has not been elucidated. Here, we present the genomic and morphological features of a newly isolated giant virus, Cotonvirus japonicus (cotonvirus), belonging to the family Mimiviridae. It contains a linear double-stranded DNA molecule of 1.47 Mb, the largest among the reported viruses in the subfamily Megavirinae, excluding tupanviruses. Among its 1,306 predicted open reading frames, 1,149 (88.0%) were homologous to those of the family Mimiviridae. Several nucleocytoplasmic large DNA virus (NCLDV) core genes, aminoacyl-tRNA synthetase genes, and the host specificity of cotonvirus were highly similar to those of Mimiviridae lineages A, B, and C; however, lineage A was slightly closer to cotonvirus than the others were. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and the period of its infection cycle, cotonvirus is the most similar to the tupanviruses among the icosahedral mimiviruses. Interestingly, the cotonvirus utilizes Golgi apparatus-like vesicles for virion factory (VF) formation. Overall, we showed that cotonvirus is a novel lineage of the subfamily Megavirinae. Our findings support the diversity of icosahedral mimiviruses and provide mechanistic insights into the replication, VF formation, and evolution of the subfamily Megavirinae. IMPORTANCE We have isolated a new virus of an independent lineage belonging to the family Mimiviridae, subfamily Megavirinae, from the fresh water of a canal in Japan, named Cotonvirus. In a proteomic tree, this new nucleocytoplasmic large DNA virus (NCLDV) is phylogenetically placed at the root of three lineages of the subfamily Megavirinae—lineages A (mimivirus), B (moumouvirus), and C (megavirus). Multiple genomic and phenotypic features of cotonvirus are more similar to those of tupanviruses than to those of the A, B, or C lineages, and other genomic features, while the host specificity of cotonvirus is more similar to those of the latter than of the former. These results suggest that cotonvirus is a unique virus that has chimeric features of existing viruses of Megavirinae and uses Golgi apparatus-like vesicles of the host cells for virion factory (VF) formation. Thus, cotonvirus can provide novel insights into the evolution of mimiviruses and the underlying mechanisms of VF formation.

One of the most curious findings associated with the discovery of Acanthamoeba polyphaga mimivirus (APMV) was the presence of many proteins and RNAs within the virion. Although some hypotheses on their role in Acanthamoeba infection have been put forward, none have been validated. In this study, we directly transfected mimivirus DNA with or without additional proteinase K treatment to extracted DNA into Acanthamoeba castellanii. In this way, it was possible to generate infectious APMV virions, but only without extra proteinase K treatment of extracted DNA. The virus genomes before and after transfection were identical. We searched for the remaining DNA-associated proteins that were digested by proteinase K and could visualize at least five putative proteins. Matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography–mass spectrometry comparison with protein databases allowed the identification of four hypothetical proteins—L442, L724, L829, and R387—and putative GMC-type oxidoreductase R135. We believe that L442 plays a major role in this protein–DNA interaction. In the future, expression in vectors and then diffraction of X-rays by protein crystals could help reveal the exact structure of this protein and its precise role.

We scanned the PubMed search database for literature on the incidence of nosocomial respiratory viral infections (NRVI) published over a ten-year period. Necessity to apply the standard case definition and the laboratory panel based on the multiplex polymerase chain reaction in frequency assessment was established. In general, predominance of rhinoviruses in the etiological structure was detected. Rationale was given for introduction of nonspecific epidemic prevention activities against a broad spectrum of other respiratory pathogens including high-priority respiratory syncytial viruses, metapneumoviruses, adenoviruses, influenza and parainfluenza viruses and coronaviruses. Bocaviruses and mimiviruses were designated as rare species. The biological diversity of the pathogens causing NRVI calls for active promotion of molecular genetics techniques in the work of the laboratory services of health facilities to perform quality etiological diagnosis, design relevant antiviral therapy regimens and effective prevention programs whose implementation will lead to significant reduction in the spread risk of these infections and the treatment costs.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

We conducted an exhaustive search for three-dimensional structural homologs to the proteins of 20 key phylogenetically distinct nucleocytoplasmic DNA viruses (NCLDV). Structural matches covered 429 known protein domain superfamilies, with the most highly represented being ankyrin repeat, P-loop NTPase, F-box, protein kinase, and membrane occupation and recognition nexus (MORN) repeat. Domain superfamily diversity correlated with genome size, but a diversity of around 200 superfamilies appeared to correlate with an abrupt switch to paralogization. Extensive structural homology was found across the range of eukaryotic RNA polymerase II subunits and their associated basal transcription factors, with the coordinated gain and loss of clusters of subunits on a virus-by-virus basis. The total number of predicted endonucleases across the 20 NCLDV was nearly quadrupled from 36 to 132, covering much of the structural and functional diversity of endonucleases throughout the biosphere in DNA restriction, repair, and homing. Unexpected findings included capsid protein-transcription factor chimeras; endonuclease chimeras; enzymes for detoxification; antimicrobial peptides and toxin-antitoxin systems associated with symbiosis, immunity, and addiction; and novel proteins for membrane abscission and protein turnover. IMPORTANCE We extended the known annotation space for the NCLDV by 46%, revealing high-probability structural matches for fully 45% of the 9,671 query proteins and confirming up to 98% of existing annotations per virus. The most prevalent protein families included ankyrin repeat- and MORN repeat-containing proteins, many of which included an F-box, suggesting extensive host cell modulation among the NCLDV. Regression suggested a minimum requirement for around 36 protein structural superfamilies for a viable NCLDV, and beyond around 200 superfamilies, genome expansion by the acquisition of new functions was abruptly replaced by paralogization. We found homologs to herpesvirus surface glycoprotein gB in cytoplasmic viruses. This study provided the first prediction of an endonuclease in 10 of the 20 viruses examined; the first report in a virus of a phenolic acid decarboxylase, proteasomal subunit, or cysteine knot (defensin) protein; and the first report of a prokaryotic-type ribosomal protein in a eukaryotic virus.

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.

Since its discovery, the first identified giant virus associated with amoebae, Acanthamoeba polyphaga mimivirus (APMV), has been rigorously studied to understand the structural and genomic complexity of this virus. In this work, we report the isolation and genomic characterization of a new mimivirus of lineage B, named "Borely moumouvirus". This new virus exhibits a structure and replicative cycle similar to those of other members of the family Mimiviridae. The genome of the new isolate is a linear double-strand DNA molecule of ~1.0 Mb, containing over 900 open reading frames. Genome annotation highlighted different translation system components encoded in the DNA of Borely moumouvirus, including aminoacyl-tRNA synthetases, translation factors, and tRNA molecules, in a distribution similar to that in other lineage B mimiviruses. Pan-genome analysis indicated an increase in the genetic arsenal of this group of viruses, showing that the family Mimiviridae is still expanding. Furthermore, phylogenetic analysis has shown that Borely moumouvirus is closely related to moumouvirus australiensis. This is the first mimivirus lineage B isolated from Brazilian territory to be characterized. Further prospecting studies are necessary for us to better understand the diversity of these viruses so a better classification system can be established.

The genus "Tupanvirus" is a new proposed taxon to be included in the family Mimiviridae. The two known tupanvirus isolates were isolated from soda lake and oceanic sediments samples collected in Brazil and were named "tupanvirus soda lake" and "tupanvirus deep ocean", respectively. These viruses exhibit similarities to amoeba-infecting mimiviruses, but there are also several differences that place them in a separate group within the family Mimiviridae. Their virions have a mean size of 1.2 µm, which include a mimivirus-like capsid and a large cylindrical tail, both covered by fibrils. The linear double-stranded DNA genomes of up to 1,516,267 base pairs encode over 1,200 genes, among which ~ 30% have no homologs in any database, including in other mimivirus genomes. Compared to other mimiviruses, tupanviruses exhibit a broader host range and cause a cytotoxic effect in host and non-host organisms, a phenotype that is not observed for other mimiviruses. Remarkably, these viruses possess the most complete gene set related to the protein synthesis process, including 20 aminoacyl-tRNA synthetases, 67-70 tRNAs, many translation factors, and genes involved in maturation and modification of tRNA and mRNA, among others. Moreover, diverse phylogenomic analyses put tupanviruses in a distinct group within the family Mimiviridae. In light of the set of different features observed for these giant viruses, we propose establishment of a new genus to allow proper classification of two known tupanviruses and possibly many more similar viruses yet to be characterized.

Mimivirus' genome includes parts of 5S, 16S and 23S ribosomal RNAs encoded by Acanthamoeba's mitogenome, the giant virus' host. Two non-exclusive hypotheses for rRNA remnants in giant viruses are examined: 1. mitogenomes invade viral genomes as they do for nuclear chromosomes (producing numts); 2. megaviral genomes evolved from an ancestral mitogenome. Alignment analyses confirm mitochondrial, rather than alphaproteobacterial origins of megaviral rRNAs. Other mitogenes have likely megaviral homologues. These megaviral homologues coevolve to much larger extents than candidate rRNA homologues, suggesting rRNA decay in viruses. Megaviral mitogene homologues overall follow mitochondrial gene order, suggesting mitogenome ancestry. Ancestral synteny decreases with megaviral genome size, suggesting that subsequent mitogene insertions blur ancestral gene order. Putative defenses against DNA invasion conserve mitogene order in short megaviral genomes. Synteny between mitogenome and megaviral genomes confirms the RNA/DNA polymerase-homologies-based hypothesis that giant viruses have mitochondrial-like ancestors, viral rRNA remnants are corollary of mitogenomic origins of megaviral genomes. Note that giant viruses, mitochondria and bacterial spores all have double membranes, spores and viruses have protein coats. Mitochondria might occasionally form spore-like structures that drifted into megaviruses. These missing links could confirm mitogenome ancestry of giant viruses rather than giant virus ancestry of mitochondria.

Nucleo-cytoplasmic large DNA viruses are doubled stranded DNA viruses capable of infecting eukaryotic cells. Since the discovery of Mimivirus and Pandoravirus, there has been no doubt about their extraordinary features compared to “classic” viruses. Recently, we reported the expansion of the proposed family Pithoviridae, with the description of Cedratvirus and Orpheovirus, two new viruses related to Pithoviruses. Studying the major capsid protein of Orpheovirus, we detected a homologous sequence in a mine drainage metagenome. The in-depth exploration of this metagenome, using the MG-Digger program, enabled us to retrieve up to 10 contigs with clear evidence of viral sequences. Moreover, phylogenetic analyses further extended our screening with the discovery in another marine metagenome of a second virus closely related to Orpheovirus IHUMI-LCC2. This virus is a misidentified virus confused with and annotated as a Rickettsiales bacterium. It presents a partial genome size of about 170 kbp. Electronic supplementary material The online version of this article (10.1186/s12985-018-0976-9) contains supplementary material, which is available to authorized users.

Background: The giant amoebal viruses of Mimivirus and Marseillevirus are large DNA viruses and have been documented in water, soil, and sewage samples. The trend of discovering these giant amoebal viruses has been increasing throughout Asia with Japan, India, and Saudi Arabia being the latest countries to document the presence of these viruses. To date, there have been no reports of large amoebal viruses being isolated in South East Asia. Objective: In this study, we aim to discover these viruses from soil samples in an aboriginal village (Serendah village) in Peninsular ­Malaysia. Method and Results: We successfully detected and isolated both Mimivirus-like and Marseillevirus-like viruses using Acanthamoeba castellanii. Phylogeny analysis identified them as Mimivirus and Marseillevirus, respectively. Conclusion: The ubiquitous nature of both Mimivirus and Marseillevirus is further confirmed in our study as they are detected in higher quantity in soil that is near to water vicinities in an aboriginal village in Peninsular Malaysia. However, this study is limited by our inability to investigate the impact of Mimivirus and Marseillevirus on the aboriginal villagers. More studies on the potential impact of these viruses on human health, especially on the aborigines, are warranted.

The study of the gut microbiome and virome has developed dramatically since the beginning of the 21st century. Nevertheless, giant viruses of amoebae, which are emerging viruses first described in 2003, have been largely neglected in virome investigations because they are bigger than classical viruses and devoid of ribosomal DNA. Dozens of these viruses have been isolated between 2003 and 2016, which were classified in at least 7 lineages including 2 new recognized virus families. These viruses challenge previous paradigms on viruses and share many characteristics with intra-cellular microbes. We reviewed here findings about the presence of these giant viruses of amoebae in the human gut, whose microbiota has been extensively studied during the last decade. Contrasting with what is currently done for classical viruses, many studies investigating the presence of giant viruses of amoebae have been conducted by culture on amoebae in first intention. To date, a mimivirus and a marseillevirus have been isolated from human feces, which indicates that they can still replicate after a stay in the gut. Besides, sequences related to giant viruses of amoebae have been detected in several metagenomes generated from human feces. Water is a likely source of human exposure to giant viruses of amoebae. The clinical or biological significance of the presence of these viruses in the human gut remains to be determined. Taken together, available findings warrant searching more extensively and systematically for giant viruses of amoebae in the human gut, along with in other body sites. Keywords: Mimivirus, Giant virus, Marseillevirus, Megavirales, Amoebae, Gut, Digestive tract, Microbiota

Giant viruses of amoebae were discovered serendipitously in 2003; they are visible via optical microscopy, making them bona fide microbes. Their lifestyle, structure, and genomes break the mold of classical viruses. Giant viruses of amoebae are complex microorganisms. Their genomes harbor between 444 and 2,544 genes, including many that are unique to viruses, and encode translation components; their virions contain >100 proteins as well as mRNAs. Mimiviruses have a specific mobilome, including virophages, provirophages, and transpovirons, and can resist virophages through a system known as MIMIVIRE (mimivirus virophage resistance element). Giant viruses of amoebae bring upheaval to the definition of viruses and tend to separate the current virosphere into two categories: very simple viruses and viruses with complexity similar to that of other microbes. This new paradigm is propitious for enhanced detection and characterization of giant viruses of amoebae, and a particular focus on their role in humans is warranted.

Most cancer forms known to be caused by viruses are increased among the immunosuppressed, but several cancer forms without established viral etiology are also increased, notably nonmelanoma skin carcinoma (NMSC). We followed all 13,429 solid organ transplantation patients in Sweden for cancer occurrence after transplantation. We requested these tumor specimens and sequenced the first 89 specimens received (62 NMSCs, 27 other cancers). The sequences were analyzed for viruses based on two bioinformatics algorithms (paracel-blast (sensitive for detection of known viruses) and hidden Markov model (HMM; sensitive for distantly related viruses)). Among the 62 NMSCs, the virus family detected in the largest proportion of specimens was Mimiviridae (9/62 NMSCs). The majority of the virus-related reads belonged to Papillomaviridae. The HMM analysis identified 86 additional previously not described viral contigs related to 11 virus families, with reads related to Mimiviridae being the most common (detected in 28/62 NMSCs) with the most prevalent contig (Mimivirus SE906, 1937 bp) detected in 17/62 NMSCs. Among the 27 other cancers, viral sequences were detected in only 5 specimens by blast analysis, compared to in all 27 specimens by HMM (Mimiviridae, Poxviridae, Phycodnaviridae and virus-related sequences yet unclassified to any family). 99% of the virus reads belonged to a single previously not described sequence (Mimivirus SE996, 911 bp). A multitude of viruses is readily detectable in specimens with cancers occurring among the immunosuppressed, with sequences related to Mimiviridae being the most prevalent. Further research would be needed to elucidate the biological significance of the viruses.

Acanthamoeba polyphaga mimivirus (APMV) is a member of the family of giant viruses, harboring a 1,200 kbp genome within its 700 nm-diameter viral particle. The R214 gene of the APMV genome was recently shown to encode a homologue of the Rab GTPases, molecular switch proteins known to play a pivotal role in the regulation of membrane trafficking that were considered to exist only in eukaryotes. Herein, we report the first crystal structures of GDP- and GTP-bound forms of APMV Rab GTPase, both of which were determined at high resolution. An in-depth structural comparison of APMV Rab with each other and with mammalian Rab homologues led to an atomic-level elucidation of the inactive-active conformational change upon GDP/GTP exchange. APMV Rab GTPase exhibited considerable structural similarity to human Rab5, as previously predicted based on its amino acid sequence. However, it also contains unique structural features differentiating it from mammalian homologues, such as the functional substitution of a phenylalanine residue for the stabilization of the nucleotide's guanine base.

Key Points Acanthamoeba polyphaga mimivirus (APMV) and subsequently discovered giant viruses of amoebae challenge the previous definition of viruses and their classification.The replication cycle, structure, genomic make-up and plasticity of giant viruses differ from those of traditional viruses. They extend the definition of viruses into a broader range of biological entities, some of which are very simple and others of which have a complexity that is comparable to that of other microorganisms.Giant viruses of amoebae have virus particles as large as some microorganisms that are visible by light microscopy and that have a stunning level of complexity. Their genomes are mosaics and contain large repertoires of genes, some of which are hallmarks of cellular organisms, although the majority of which have unknown functions.Mimiviruses are associated with a specific mobilome and are parasitized by viruses that they can defend against.Several hypotheses on the ancient origin and evolutionary relationship between cellular organisms and giant viruses of amoebae have been proposed, and these topics continue to be debated.The detection of giant viruses of amoebae in humans and the study of their potential pathogenicity are emerging fields. Supplementary information The online version of this article (doi:10.1038/nrmicro.2016.197) contains supplementary material, which is available to authorized users., The discovery of the giant amoebal virus mimivirus, in 2003, opened up a new area of virology. Extended studies, including those of mimiviruses, have since revealed that these viruses have genetic, proteomic and structural features that are more complex than those of conventional viruses. Supplementary information The online version of this article (doi:10.1038/nrmicro.2016.197) contains supplementary material, which is available to authorized users., The accidental discovery of the giant virus of amoeba — Acanthamoeba polyphaga mimivirus (APMV; more commonly known as mimivirus) — in 2003 changed the field of virology. Viruses were previously defined by their submicroscopic size, which probably prevented the search for giant viruses, which are visible by light microscopy. Extended studies of giant viruses of amoebae revealed that they have genetic, proteomic and structural complexities that were not thought to exist among viruses and that are comparable to those of bacteria, archaea and small eukaryotes. The giant virus particles contain mRNA and more than 100 proteins, they have gene repertoires that are broader than those of other viruses and, notably, some encode translation components. The infection cycles of giant viruses of amoebae involve virus entry by amoebal phagocytosis and replication in viral factories. In addition, mimiviruses are infected by virophages, defend against them through the mimivirus virophage resistance element (MIMIVIRE) system and have a unique mobilome. Overall, giant viruses of amoebae, including mimiviruses, marseilleviruses, pandoraviruses, pithoviruses, faustoviruses and molliviruses, challenge the definition and classification of viruses, and have increasingly been detected in humans. Supplementary information The online version of this article (doi:10.1038/nrmicro.2016.197) contains supplementary material, which is available to authorized users.

Mimivirus was the first discovered amoebal giant virus. The Mimivirus virions are covered by a dense layer of approximately 130 nm-long fibers, the length and shape of which diverge from those of other viruses. Here, we aimed at expressing the L725 protein to further confirm and study its role as a fiber-associated protein. We report Escherichia coli expression of the L725 protein, which is encoded by a Mimivirus ORFan, was previously identified by proteomics in purified viral fibers and demonstrated to be a fiber-associated protein by RNA-silencing experiments. The expressed protein was recognized by anti-Mimivirus fiber or anti-Mimivirus L725 polyclonal antibodies. This study is the only expression, to our knowledge, of a product from a Mimiviral ORFan gene.

Giant viruses, like pandoraviruses and mimiviruses, have been discovered from diverse environments, and their broad global distribution has been established. Here, we report two new isolates of Pandoravirus spp. and one Mimivirus sp., named Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. isolate styx, co-isolated from riverbank soil in Japan. We obtained nearly complete sequences of the family B DNA polymerase gene (polB) of P. hades and P. persephone, the former carried two known intein regions, while the latter had only one. Phylogenetic analysis revealed that the two new pandoravirus isolates are closely related to Pandoravirus dulcis. Furthermore, random amplified polymorphic DNA analysis revealed that P. hades and P. persephone might harbor different genome structures. Based on phylogenetic analysis of the partial polB sequence, Mimivirus sp. isolate styx belongs to mimivirus lineage A. DNA staining suggested that the Pandoravirus spp. asynchronously replicates in amoeba cells while Mimivirus sp. replicates synchronously. We also observed that P. persephone- or Mimivirus sp. isolate styx-infected amoeba cytoplasm is extruded by the cells. To the best of our knowledge, we are the first to report the isolation of pandoraviruses in Asia. In addition, our results emphasize the importance of virus isolation from soil to reveal the ecology of giant viruses.

The Acanthamoeba polyphaga mimivirus (APMV) was first isolated during a pneumonia outbreak in Bradford, England, and since its discovery many research groups devoted efforts to understand whether this virus could be associated to human diseases, in particular clinical signs and symptoms of pneumonia. In 2013, we observed cytopathic effect in amoebas (rounding and lysis) inoculated with APMV inoculated PBMCs (peripheral blood mononuclear cell) extracts, and at that point we interpreted those results as mimivirus replication in human PBMCs. Based on these results we decided to further investigate APMV replication in human PBMCs, by transmission electron microscopy (TEM) and qPCR. No viral factory was observed in APMV inoculated PBMCs, at any analyzed time and M.O.I.s (multiplicity of infection), by checking 550 cells per condition tested. We also measured the variation of viral DNA by qPCR targeting helicase gene during the course of the TEM experiment in PBMCs, but the DNA levels stayed the same as the first time-point post infection. In summary, our newest qPCR and TEM results do not support previous statements (including ours) that mimivirus is able to replicate in humans PBMCs. nonPeerReviewed

BackgroundAfter the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genusAcanthamoeba; however other giant viruses have been isolated inVermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated.MethodsIn this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays.ResultsWe observed, through optical and IF microscopy, morphological changes inV. vermiformiscells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant.ConclusionsThe results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Since the discovery of mimivirus, numerous giant viruses associated with free-living amoebae have been described. The genome of giant viruses can be more than 2.5 megabases, and virus particles can exceed the size of many bacteria. The unexpected characteristics of these viruses have made them intriguing research targets and, as a result, studies focusing on their interactions with their amoeba host have gained increased attention. Studies have shown that giant viruses can establish host–pathogen interactions, which have not been previously demonstrated, including the unprecedented interaction with a new group of small viruses, called virophages, that parasitize their viral factories. In this brief review, we present recent advances in virophage–giant virus–host interactions and highlight selected studies involving interactions between giant viruses and amoebae. These unprecedented interactions involve the giant viruses mimivirus, marseillevirus, tupanviruses and faustovirus, all of which modulate the amoeba environment, affecting both their replication and their spread to new hosts.

BackgroundThe study of retroviruses dates back to the early 1900s during investigations on neoplastic diseases in chickens. Subsequently, Robert Gallo reported the first human retrovirus HLTV in 1980. What we report here is not an archetypal retrovirus, but the discovery of an oncogenic giant microbial agent with a mega-genome, where the transforming retroviral nature co-exists with multiple archaeal oncogenes.MethodsAfter their isolation from human T cells Leukaemia, these organisms were examined at electron microscopy, tested for reverse transcriptase activity, fully sequenced, used for transformation tests on NIH-3T3 cells in vitro and tumours formation in mice. Same type of particles were also isolated from Canine Transmissible Venereal Tumour (CTVT), the oldest contagious cancer in nature.ResultsEM showed the presence of giant viral particles displaying retroviral antigens. These microbial entities harbour in their mega-genome a transforming retroviral kinase, cell-based oncogenes and have reverse transcriptase activity. The purified viral particles transformed NIH-3T3 cells and induced metastatic tumours in nude mice, three weeks post infection. Ruling out the possible presence of filterable retroviruses, a filtered supernatant did not display RT activity and did not transforms.ConclusionsWe discovered an ancestral microbial agent, acutely transforming. For its giant dimensions, the ability to retain the Gram stain, the presence of a mega-genome and its retroviral nature, we tentatively named the agent Retro-Giant-Virus (RGV). However, distinct from amoeba giant Mimiviruses, this transforming human agent has a different nature and does not require for its isolation amoeba co-culture, since amoeba is not its natural host.The morphology, biology and genetic features allocate this mammalian giant microbe halfway in between a classic oncogenic virus and an infectious cancer cell. Its transforming nature goes with its constant ability to induce tumours formation in mice.

Acanthamoeba-infecting Mimiviridae belong to three clades: Mimiviruses (A), Moumouviruses (B) and Megaviruses (C). The uniquely complex mobilome of these giant viruses includes virophages and linear 7 kb-DNA molecules called “transpovirons”. We recently isolated a virophage (Zamilon vitis) and two transpovirons (maBtv and mvCtv) respectively associated to B-clade and C-clade mimiviruses. We used the capacity of the Zamilon virophage to replicate both on B-clade and C-clade host viruses to investigate the three partite interaction network governing the propagation of transpovirons. We found that the virophage could transfer both types of transpovirons to B-clade and C-clade host viruses provided they were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate both maBtv and mvCtv, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We performed proteomic comparisons of host viruses and virophage particles carrying or cleared of transpovirons in search of proteins involved in this adaptation process. These experiments revealed that transpoviron-encoded proteins are synthetized during the combined mimiviruses/virophage/transpoviron replication process and some of them are specifically incorporated into the virophage and giant virus particles together with the cognate transpoviron DNA. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate from one host virus to another and where the Zamilon virophage and the transpoviron depend on their host giant virus to replicate, this without affecting the giant virus infectious cycle, at least in laboratory conditions.Author SummaryThe Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build huge viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions (transcription and replication) take place. They are themselves the target of infections by 20 kb-dsDNA virophages, replicating in the giant virus factories. They can also be found associated with 7kb-DNA episomes, dubbed transpovirons. We investigated the relationship between these three players by combining competition experiments involving the newly isolated Zamilon vitis virophage as a vehicle for transpovirons of different origins with proteomics analyses of virophage and giant virus particles. Our results suggest that relationship of the virophage, the transpoviron, and their host giant virus, extend the concept of commensalism to the viral world.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

Acanthamoeba-infecting Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions take place. They are themselves the target of infections by 20-kb-dsDNA virophages, replicating in the giant virus factories and can also be found associated with 7-kb-DNA episomes, dubbed transpovirons. Here we isolated a virophage (Zamilon vitis) and two transpovirons respectively associated to B- and C-clade mimiviruses. We found that the virophage could transfer each transpoviron provided the host viruses were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron originally isolated from the corresponding virus was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate each transpoviron, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We analysed the proteomes of host viruses and virophage particles in search of proteins involved in this adaptation process. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate and where the Zamilon virophage and the transpoviron depend on the giant virus to replicate, without affecting its infectious cycle.

Aquatic viruses have been extensively studied over the past decade, yet fundamental aspects of freshwater virus communities remain poorly described. Our goal was to characterize virus communities captured in the &gt, 0.22 &micro, m size-fraction seasonally and spatially in a freshwater harbour. Community DNA was extracted from water samples and sequenced on an Illumina HiSeq platform. Assembled contigs were annotated as belonging to the virus groups (i.e., order or family) Caudovirales, Mimiviridae, Phycodnaviridae, and virophages (Lavidaviridae), or to other groups of undefined viruses. Virophages were often the most abundant group, and discrete virophage taxa were remarkably stable across sites and dates despite fluctuations in Mimiviridae community composition. Diverse Mimiviridae contigs were detected in the samples and the two sites contained distinct Mimiviridae communities, suggesting that Mimiviridae are important algal viruses in this system. Caudovirales and Phycodnaviridae were present at low abundances in most samples. Of the 18 environmental parameters tested, only chlorophyll a explained the variation in the data at the order or family level of classification. Overall, our findings provide insight into freshwater virus community assemblages by expanding the documented diversity of freshwater virus communities, highlighting the potential ecological importance of virophages, and revealing distinct communities over small spatial scales.

Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly, and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary three-dimensional (3D)-electron microscopy techniques, including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant, and DNA is packed within virions prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening, and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggest that the molecular mechanisms for cellular membrane remodeling and persistence are unique. IMPORTANCE Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the world and appear to be unique in several aspects. They belong to at least four viral families, and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of “Molliviridae,” which was isolated from a 30,000-year-old permafrost sample and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna, (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled, and (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding of how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.

Viruses are the most prevalent infectious agents, populating almost every ecosystem on earth. Most viruses carry only a handful of genes supporting their replication and the production of capsids. It came as a great surprise in 2003 when the first giant virus was discovered and found to have a >1 Mbp genome encoding almost a thousand proteins. Following this first discovery, dozens of giant virus strains across several viral families have been reported. Here, we provide an updated quantitative and qualitative view on giant viruses and elaborate on their shared and variable features. We review the complexity of giant viral proteomes, which include functions traditionally associated only with cellular organisms. These unprecedented functions include components of the translation machinery, DNA maintenance, and metabolic enzymes. We discuss the possible underlying evolutionary processes and mechanisms that might have shaped the diversity of giant viruses and their genomes, highlighting their remarkable capacity to hijack genes and genomic sequences from their hosts and environments. This leads us to examine prominent theories regarding the origin of giant viruses. Finally, we present the emerging ecological view of giant viruses, found across widespread habitats and ecological systems, with respect to the environment and human health.

Viruses are obligate intracellular parasites and thus cannot replicate outside a living cell. Whereas a susceptible and permissive host cell is sufficient for most viruses to produce infectious progeny, some viruses such as satellite viruses require two different biological entities for their replication. In contrast to most satellite viruses with small RNA-based genomes, the lavidaviruses (or virophages) that replicate in eukaryotic microbes (protists) are comparable to adenoviruses in terms of particle size and genetic complexity. Lavidaviruses only replicate during a co-infection with a giant DNA virus (GV; see, e.g., [1]), and the first described virophage, Sputnik, was serendipitously discovered during a search for new GVs [2].

The detailed mechanisms of replication initiation, termination and segregation events were not yet known in Acanthamoeba polyphaga mimivirus (APMV). Here, we show detailed bioinformatics-based analyses of chromosomal replication in APMV from initiation to termination mediated by proteins bound to specific DNA sequences. Using GC/AT skew and coding sequence skew analysis, we estimated that the replication origin is located at 382 kb in the APMV genome. We performed homology-modeling analysis of the gamma domain of APMV-FtsK (DNA translocase coordinating chromosome segregation) related to FtsK-orienting polar sequences (KOPS) binding, suggesting that there was an insertion in the gamma domain which maintains the structure of the DNA binding motif. Furthermore, UvrD/Rep-like helicase in APMV was homologous to Bacillus subtilis AddA, while the chi-like quartet sequence 5′-CCGC-3′ was frequently found in the estimated ori region, suggesting that chromosomal replication of APMV is initiated via chi-like sequence recognition by UvrD/Rep-like helicase. Therefore, the replication initiation, termination and segregation of APMV are presumably mediated by DNA repair machineries derived from gram-positive bacteria. Moreover, the other frequently observed quartet sequence 5′-CGGC-3′ in the ori region was homologous to the mitochondrial signal sequence of replication initiation, while the comparison of quartet sequence composition in APMV/Rickettsia-genome showed significantly similar values, suggesting that APMV also conserves the mitochondrial replication system acquired from an ancestral genome of mitochondria during eukaryogenesis.

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.

International audience; Since 1998, when Jim van Etten's team initiated its characterization, Paramecium bursaria Chlorella virus 1 (PBCV-1) had been the largest known DNA virus, both in terms of particle size and genome complexity. In 2003, the Acanthamoeba-infecting Mimivirus unexpectedly superseded PBCV-1, opening the era of giant viruses, i.e., with virions large enough to be visible by light microscopy and genomes encoding more proteins than many bacteria. During the following 15 years, the isolation of many Mimivirus relatives has made Mimiviridae one of the largest and most diverse families of eukaryotic viruses, most of which have been isolated from aquatic environments. Metagenomic studies of various ecosystems (including soils) suggest that many more remain to be isolated. As Mimiviridae members are found to infect an increasing range of phytoplankton species, their taxonomic position compared to the traditional Phycodnaviridae (i.e., etymologically "algal viruses") became a source of confusion in the literature. Following a quick historical review of the key discoveries that established the Mimiviridae family, we describe its current taxonomic structure and propose a set of operational criteria to help in the classification of future isolates.

The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5′ ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes.