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International audience; The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of “minimalist” histones in archaea and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones are essential for viral infectivity, localize to cytoplasmic viral factories after virus infection, and ultimately are found in the mature virions. Cryogenic electron microscopy (cryo-EM) structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of “organisms” that require nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.

SummaryMembers of Marseilleviridae, one family of icosahedral giant viruses classified in 2012 have been identified worldwide in all types of environments. The virion shows a characteristic internal membrane extrusion at the five-fold vertices of the capsid, but its structural details need to be elucidated. We now report the 4.4 Å cryo-electron microscopy structure of the Melbournevirus capsid. An atomic model of the major capsid protein (MCP) shows a unique cup structure on the trimer that accommodates additional proteins. A polyalanine model of the penton base protein shows internally extended N- and C-terminals, which indirectly connect to the internal membrane extrusion. The Marseilleviruses share the same orientational organisation of the MCPs as PBCV-1 and CroV, but do not appear to possess a protein akin to the “tape measure” of these viruses. Minor capsid proteins named PC-β, zipper, and scaffold are proposed to control the dimensions of the capsid during assembly.

Giant viruses have extraordinarily large dsDNA genomes, and exceptionally, they encode various components of the translation apparatus, including tRNAs, aminoacyl-tRNA synthetases and translation factors. Here, we focused on the elongation factor 1 (EF1) family of viral translational GTPases (trGTPases), using computational and functional approaches to shed light on their functions. Multiple sequence alignment indicated that these trGTPases clustered into two groups epitomized by members of Mimiviridae and Marseilleviridae, respectively. trGTPases in the first group were more closely related to GTP-binding protein 1 (GTPBP1), whereas trGTPases in the second group were closer to eEF1A, eRF3 and Hbs1. Functional characterization of representative GTPBP1-like trGTPases (encoded by Hirudovirus, Catovirus and Moumouvirus) using in vitro reconstitution revealed that they possess eEF1A-like activity and can deliver cognate aa-tRNAs to the ribosomal A site during translation elongation. By contrast, representative eEF1A/eRF3/Hbs1-like viral trGTPases, encoded by Marseillevirus and Lausannevirus, have eRF3-like termination activity and stimulate peptide release by eRF1. Our analysis identified specific aspects of the functioning of these viral trGTPases with eRF1 of human, amoebal and Marseillevirus origin.

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.

SummaryThe organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of ‘minimalist’ histones in archaea, and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones inMarseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones localize to the cytoplasmic viral factories after virus infection, and ultimately to mature virions. CryoEM structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes, despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of ‘organisms’ that have nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.One Sentence SummarySome large DNA viruses encode fused histone doublets that are targeted to viral factories and assemble into open nucleosome-like structures.

Marseilleviridae is a family of the new order of giant viruses, which exhibit a characteristic inner membrane. Here, we investigated the entire structure of tokyovirus, a species of Marseillevirus at 7.7 Å resolution using 1 MV high-voltage cryo-EM and single particle analysis. The minor capsid lattice formed by five proteins, shows a novel structure compared to other icosahedral giant viruses. Under the minor capsid proteins, scaffold proteins connect two five-fold vertices and interact with the inner membrane. Previously reported giant viruses utilise “tape measure” proteins, proposed to control its capsid size, which could not be identified in tokyovirus, but scaffold proteins appear to perform a similar role. A density on top of the major capsid protein was identified, which suggested to be a 14kDa glycoprotein. Our observations suggest that the icosahedral particle of Marseilleviridae is constructed with a novel capsid protein network, which allows the characteristic inner membrane structure.

Marseilleviridae is a family of large double-stranded DNA viruses that is currently divided into five subgroups, lineages A-E. Hokutovirus and kashiwazakivirus, both of which belong to lineage B, have been reported to induce host acanthamoeba cells to form aggregations called "bunches". This putatively results in increased opportunities to infect acanthamoeba cells, in contrast to lineage A, which has been reported to not form "bunches". In the present study, we isolated 14 virus strains of the family Marseilleviridae from several Japanese water samples, 11 of which were identified as lineage B viruses. All 11 lineage B strains caused infected amoeba cells to form bunches. We then investigated the involvement of monosaccharides in bunch formation by amoeba cells infected with hokutovirus. Galactose inhibited bunch formation, thereby allowing amoeba cells to delay the process, whereas mannose and glucose did not. A kinetic image analysis of hokutovirus-infected amoeba cells confirmed the inhibition of bunch formation by galactose. The number of hokutovirus-infected amoeba cells increased more rapidly than that of tokyovirus-infected cells, which belongs to lineage A. This result suggests that bunch formation by infected amoeba cells is advantageous for lineage B viruses.

Marseilleviridae is a family of large double-stranded DNA viruses that is currently divided into five subgroups, lineages A-E. Hokutovirus and kashiwazakivirus, both of which belong to lineage B, have been reported to induce host acanthamoeba cells to form aggregations called "bunches". This putatively results in increased opportunities to infect acanthamoeba cells, in contrast to lineage A, which has been reported to not form "bunches". In the present study, we isolated 14 virus strains of the family Marseilleviridae from several Japanese water samples, 11 of which were identified as lineage B viruses. All 11 lineage B strains caused infected amoeba cells to form bunches. We then investigated the involvement of monosaccharides in bunch formation by amoeba cells infected with hokutovirus. Galactose inhibited bunch formation, thereby allowing amoeba cells to delay the process, whereas mannose and glucose did not. A kinetic image analysis of hokutovirus-infected amoeba cells confirmed the inhibition of bunch formation by galactose. The number of hokutovirus-infected amoeba cells increased more rapidly than that of tokyovirus-infected cells, which belongs to lineage A. This result suggests that bunch formation by infected amoeba cells is advantageous for lineage B viruses.

Marseilleviridae members are large dsDNA viruses with icosahedral particles 250 nm in diameter infecting Acanthamoeba. Their 340 to 390 kb genomes encode 450 to 550 protein-coding genes. Since the discovery of marseillevirus (the prototype of the family) in 2009, several strains were isolated from various locations, among which 13 are now fully sequenced. This allows the organization of their genomes to be deciphered through comparative genomics. Here, we first experimentally demonstrate that the Marseilleviridae genomes are circular. We then acknowledge a strong bias in sequence conservation, revealing two distinct genomic regions. One gathers most Marseilleviridae paralogs and has undergone genomic rearrangements, while the other, enriched in core genes, exhibits the opposite pattern. Most of the genes whose protein products compose the viral particles are located in the conserved region. They are also strongly biased toward a late gene expression pattern. We finally discuss the potential advantages of Marseilleviridae having a circular genome, and the possible link between the biased distribution of their genes and the transcription as well as DNA replication mechanisms that remain to be characterized.

Highlights • This study is the first ever report on the virus diversity among the hot springs of Sikkim, India. • The study revealed the dominance of Siphoviridae, Myoviridae and Phycodnaviridae in the two hot spring solfataric mud sediments. • The metavirome ecology of the two hot springs have dsDNA viromes in abundance. • Giant DNA viruses such as Pandoravirus and Pithovirus were found through metagenomic approach., Viruses are the most prodigious repertory of the genetic material on the earth. They are elusive, breakneck, evolutionary life particles that constitute a riveting concealed world. Environmental viruses have been obscurely explored, and hence, such an intriguing world of viruses was studied in the Himalayan Geothermal Belt of Indian peninsula at Sikkim corridor through hot springs. The hot springs located at the North Sikkim district were selected for the current study. The solfataric mud sediment samples were pooled from both the hot springs. The virus community showed significant diversity among the two hot springs of Yume Samdung. Reads for viruses among the mud sediments at Old Yume Samdung hot springs (OYS) was observed to be 11% and in the case of New Yume Samdung hot springs (NYS) it was 6%. Both the hot springs were abundant in dsDNA viromes. The metavirome reads in both the OYS and NYS hot spring mud sediments showed the predominance of Caudovirales; Herpesvirales; Ortervirales among which viral reads from Siphoviridae, Myoviridae, Phycodnaviridae and Podoviridae were abundantly present. Other viral communities belonged to families like Baculoviridae, Mimiviridae, Parvoviridae, Marseilleviridae etc. Interestingly, in the case of NYS, the unassigned group reads belonged to some unclassified giant DNA viruses like genera Pandoravirus and Pithovirus. Other interesting findings were – reads for Badnavirus having ds (RT-DNA) was exclusively found in NYS whereas Rubulavirus having ss(-)RNA was exclusively found in OYS sample. This is the first ever report on viruses from any hot springs of Sikkim till date., Graphical abstract Image, graphical abstract

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.

The brief review is devoted to description of the discovery of giant viruses belonging to the families of Mimiviridae and Marseilleviridae, as well as unassigned genera Pithoviruses, Pandoravirus, and Molliviruses. The review presents issues of their origin, evolution, and molecular-biological characteristics.В обзоре рассмотрены происхождение, эволюция, распространение, таксономические, структурно-морфологические и молекулярно-биологические характеристики гигантских вирусов, отнесённых к двум семействам Mimiviridae и Marseilleviridae и трем негруппированным родам Pithovirus, Pandoravirus и Mollivirus. Гигантские вирусы различимы под световым микроскопом, а количество генов в геноме сопоставимо с таковым у бактерий, что привело к переоценке признаков, отличающих вирусы от других микроорганизмов.

DNA methylation is an important epigenetic mark that contributes to various regulations in all domains of life. Giant viruses are widespread dsDNA viruses with gene contents overlapping the cellular world that also encode DNA methyltransferases. Yet, virtually nothing is known about the methylation of their DNA. Here, we use single-molecule real-time sequencing to study the complete methylome of a large spectrum of giant viruses. We show that DNA methylation is widespread, affecting 2/3 of the tested families, although unevenly distributed. We also identify the corresponding viral methyltransferases and show that they are subject to intricate gene transfers between bacteria, viruses and their eukaryotic host. Most methyltransferases are conserved, functional and under purifying selection, suggesting that they increase the viruses’ fitness. Some virally encoded methyltransferases are also paired with restriction endonucleases forming Restriction-Modification systems. Our data suggest that giant viruses’ methyltransferases are involved in diverse forms of virus-pathogens interactions during coinfections., DNA methylation is an epigenetic marker in all domains of life. Here, Jeudy et al., using single-molecule realtime sequencing, determine DNA methylation patterns in giant viruses and evolutionary analysis of virus encoded DNA methyltransferases suggests that they affect viral fitness.

Background Viruses directly affect the most important biological processes in the ocean via their regulation of prokaryotic and eukaryotic populations. Marine sponges form stable symbiotic partnerships with a wide diversity of microorganisms and this high symbiont complexity makes them an ideal model for studying viral ecology. Here, we used morphological and molecular approaches to illuminate the diversity and function of viruses inhabiting nine sponge species from the Great Barrier Reef and seven from the Red Sea. Results Viromic sequencing revealed host-specific and site-specific patterns in the viral assemblages, with all sponge species dominated by the bacteriophage order Caudovirales but also containing variable representation from the nucleocytoplasmic large DNA virus families Mimiviridae, Marseilleviridae, Phycodnaviridae, Ascoviridae, Iridoviridae, Asfarviridae and Poxviridae. Whilst core viral functions related to replication, infection and structure were largely consistent across the sponge viromes, functional profiles varied significantly between species and sites largely due to differential representation of putative auxiliary metabolic genes (AMGs) and accessory genes, including those associated with herbicide resistance, heavy metal resistance and nylon degradation. Furthermore, putative AMGs varied with the composition and abundance of the sponge-associated microbiome. For instance, genes associated with antimicrobial activity were enriched in low microbial abundance sponges, genes associated with nitrogen metabolism were enriched in high microbial abundance sponges and genes related to cellulose biosynthesis were enriched in species that host photosynthetic symbionts. Conclusions Our results highlight the diverse functional roles that viruses can play in marine sponges and are consistent with our current understanding of sponge ecology. Differential representation of putative viral AMGs and accessory genes across sponge species illustrate the diverse suite of beneficial roles viruses can play in the functional ecology of these complex reef holobionts.

International audience; Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer. The LDB is 30 nm in size and its density matches that of a genome/protein complex. The observed LDB reinforces the structural complexity of MelV, setting it apart from other NCLDVs.

Amoeba-resisting microorganisms raised a great interest during the last decade. Among them, some large DNA viruses present huge genomes up to 2.5 Mb long, exceeding the size of small bacterial genomes. The rate of genome evolution in terms of mutation, deletion, and gene acquisition in these genomes is yet unknown. Given the suspected high plasticity of viral genomes, the microevolution of the 346 kb genome of Lausannevirus, a member of Megavirales, was studied. Hence, Lausannevirus was co-cultured within the amoeba Acanthamoeba castellanii over one year. Despite a low number of mutations, the virus showed a genome reduction of 3.7% after 12 months. Lausannevirus genome evolution in sympatric conditions was investigated by its co-culture with Estrella lausannensis, an obligate intracellular bacterium, in the amoeba A. castellanii during one year. Cultures were split every 3 months. Genome sequencing revealed that in these conditions both, Lausannevirus and E. lausannensis, show stable genome, presenting no major rearrangement. In fact, after one year they acquired from 2 to 7 and from 4 to 10 mutations per culture for Lausannevirus and E. lausannensis, respectively. Interestingly, different mutations in the endonuclease encoding genes of Lausannevirus were observed in different subcultures, highlighting the importance of this gene product in the replication of Lausannevirus. Conversely, mutations in E. lausannensis were mainly located in a gene encoding for a phosphoenolpyruvate–protein phosphotransferase (PtsI), implicated in sugar metabolism. Moreover, in our conditions and with our analyses we detected no horizontal gene transfer during one year of co-culture.

Members of the Marseilleviridae family are large DNA viruses with icosahedral particle structures that infect Acanthamoeba cells. The first Marseillevirus to be discovered was isolated in 2009. Since then, several other members of the Marseilleviridae family have been reported, including Lausannevirus, Senegalvirus, Cannes 8 virus, Insectomime virus, Tunisvirus, Melbournevirus, Port-Miou virus, and Brazilian Marseillevirus, which have been isolated from Europe, Africa, Australia, and South America. The morphological and genomic properties of a new Marseilleviridae family member, Tokyovirus, discovered in a water/soil sample from a Japanese river in Tokyo, were described in the present study. Tokyovirus possesses icosahedral particles of up to 200 nm in diameter, as revealed by a transmission electron microscopy (TEM) analysis, which form a giant virion factory in Acanthamoeba cells. A preliminary genome analysis predicted 487 coding sequences. A dot plot analysis and phylogenetic analysis using family B DNA polymerase, proliferating cell nuclear antigen (PCNA), and DNA-directed RNA polymerase alpha subunit genes revealed that Tokyovirus shares similarities with Marseillevirus, Melbournevirus, and Cannes 8 virus (Marseilleviridae subclade A), but not with Lausannevirus and Port-Miou virus (subclade B), Tunisvirus and Insectomime virus (subclade C), or Brazilian Marseillevirus (subclade D), suggesting that Tokyovirus has evolved separately from the previously described Marseilleviridae members.

Marseilleviridae is a family of viruses which have only been propagated in acanthamoeba. Marseillevirus sequences have been recently detected in different human matrices by viral metagenomics. Single-center studies worldwide have estimated a low prevalence of marseillevirus both in symptomatic patients and in healthy donors but, to date, no informations are available on the prevalence of this giant virus in Italy. By a polymerase chain reaction targeting the ORF152 viral sequence, we tested sera from 197 immunosuppressed patients and 285 healthy donors, and 63 and 30 respiratory and cerebrospinal fluid samples, respectively, of patients with various clinical conditions and referring the Virology Division for diagnostic purposes. We observed no evidence of Marseillevirus DNA in all 575 samples tested. Marseillevirus probably does not cause infection in human.

The family Marseilleviridae, defined as a group of icosahedral double-stranded DNA viruses with particle size of approximately 250 nm and genome size of 350–380 kbp, belongs to the nucleo-cytoplasmic family of large DNA viruses. The family Marseilleviridae is currently classified into lineages A–E. In this study, we isolated 12 or 15 new members of the family Marseilleviridae from three sampling locations in Japan. Molecular phylogenetic analysis of the MCP genes showed that the new viruses could be further classified into three groups, hokutoviruses, kashiwazakiviruses, and kyotoviruses. Hokutoviruses were closely related to lineage B, kyotoviruses were related to lineage A, and kashiwazakiviruses were also classified into lineage B but a new putative subgroup of lineage B, revealing the diversity of this lineage. Interestingly, more than two viruses with slightly different MCP genes were isolated from a single water sample from a single location, i.e., two hokutoviruses and one kashiwazakivirus were isolated from a small reservoir, five kashiwazakiviruses from the mouth of a river, and five kyotoviruses from fresh water of a river, suggesting that several milliliters of water samples contain several types of giant viruses. Amoeba cells infected with hokutoviruses or kashiwazakiviruses exhibited a “bunch” formation consisting of normal and infected cells similarly to a tupanvirus, whereas cells infected with kyotoviruses or tokyovirus did not. These results suggest the previously unrecognized local diversity of the family Marseilleviridae in aquatic environments.

Viral kinases are known to undergo autophosphorylation and also phosphorylate viral and host substrates. Viral kinases have been implicated in various diseases and are also known to acquire host kinases for mimicking cellular functions and exhibit virulence. Although substantial analyses have been reported in the literature on diversity of viral kinases, there is a gap in the understanding of sequence and structural similarity among kinases from different classes of viruses. In this study, we performed a comprehensive analysis of protein kinases encoded in viral genomes. Homology search methods have been used to identify kinases from 104,282 viral genomic datasets. Serine/threonine and tyrosine kinases are identified only in 390 viral genomes. Out of seven viral classes that are based on nature of genetic material, only viruses having double-stranded DNA and single-stranded RNA retroviruses are found to encode kinases. The 716 identified protein kinases are classified into 63 subfamilies based on their sequence similarity within each cluster, and sequence signatures have been identified for each subfamily. 11 clusters are well represented with at least 10 members in each of these clusters. Kinases from dsDNA viruses, Phycodnaviridae which infect green algae and Herpesvirales that infect vertebrates including human, form a major group. From our analysis, it has been observed that the protein kinases in viruses belonging to same taxonomic lineages form discrete clusters and the kinases encoded in alphaherpesvirus form host-specific clusters. A comprehensive sequence and structure-based analysis enabled us to identify the conserved residues or motifs in kinase catalytic domain regions across all viral kinases. Conserved sequence regions that are specific to a particular viral kinase cluster and the kinases that show close similarity to eukaryotic kinases were identified by using sequence and three-dimensional structural regions of eukaryotic kinases as reference. The regions specific to each viral kinase cluster can be used as signatures in the future in classifying uncharacterized viral kinases. We note that kinases from giant viruses Marseilleviridae have close similarity to viral oncogenes in the functional regions and in putative substrate binding regions indicating their possible role in cancer.

The Nucleocytoplasmic Large DNA Viruses (NCLDV) of eukaryotes (proposed order "Megavirales") comprise an expansive group of eukaryotic viruses that consists of the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, Pithoviridae, and Mimiviridae, as well as Pandoraviruses, Molliviruses, and Faustoviruses that so far remain unaccounted by the official virus taxonomy. All these viruses have double-stranded DNA genomes that range in size from about 100 kilobases (kb) to more than 2.5 megabases. The viruses with genomes larger than 500kb are informally considered "giant," and the largest giant viruses surpass numerous bacteria and archaea in both particle and genome size. The discovery of giant viruses has been highly unexpected and has changed the perception of viral size and complexity, and even, arguably, the entire concept of a virus. Given that giant viruses encode multiple proteins that are universal among cellular life forms and are components of the translation system, the quintessential cellular molecular machinery, attempts have been made to incorporate these viruses in the evolutionary tree of cellular life. Moreover, evolutionary scenarios of the origin of giant viruses from a fourth, supposedly extinct domain of cellular life have been proposed. However, despite all the differences in the genome size and gene repertoire, the NCLDV can be confidently defined as monophyletic group, on the strength of the presence of about 40 genes that can be traced back to their last common ancestor. Using several most strongly conserved genes from this ancestral set, a well-resolved phylogenetic tree of the NCLDV was built and employed as the scaffold to reconstruct the history of gene gain and loss throughout the course of the evolution of this group of viruses. This reconstruction reveals extremely dynamic evolution that involved extensive gene gain and loss in many groups of viruses and indicates that giant viruses emerged independently in several clades of the NCLDV. Thus, these giants of the virus world evolved repeatedly from smaller and simpler viruses, rather than from a fourth domain of cellular life, and captured numerous genes, including those for translation system components, from eukaryotes, along with some bacterial genes. Even deeper evolutionary reconstructions reveal apparent links between the NCLDV and smaller viruses of eukaryotes, such as adenoviruses, and ultimately, derive all these viruses from tailless bacteriophages.

Genomics and evolution of giant viruses are two of the most vigorously developing areas of virus research. Lately, metagenomics has become the main source of new virus genomes. Here we describe a metagenomic analysis of the genomes of large and giant viruses from deep sea sediments. The assembled new virus genomes substantially expand the known diversity of the nucleocytoplasmic large DNA viruses of eukaryotes. The results support the concept of independent evolution of giant viruses from smaller ancestors in different virus branches., The nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes (proposed order, “Megavirales”) include the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, and Mimiviridae, as well as still unclassified pithoviruses, pandoraviruses, molliviruses, and faustoviruses. Several of these virus groups include giant viruses, with genome and particle sizes exceeding those of many bacterial and archaeal cells. We explored the diversity of the NCLDV in deep sea sediments from the Loki’s Castle hydrothermal vent area. Using metagenomics, we reconstructed 23 high-quality genomic bins of novel NCLDV, 15 of which are related to pithoviruses, 5 to marseilleviruses, 1 to iridoviruses, and 2 to klosneuviruses. Some of the identified pithovirus-like and marseillevirus-like genomes belong to deep branches in the phylogenetic tree of core NCLDV genes, substantially expanding the diversity and phylogenetic depth of the respective groups. The discovered viruses, including putative giant members of the family Marseilleviridae, have a broad range of apparent genome sizes, in agreement with the multiple, independent origins of gigantism in different branches of the NCLDV. Phylogenomic analysis reaffirms the monophyly of the pithovirus-iridovirus-marseillevirus branch of the NCLDV. Similarly to other giant viruses, the pithovirus-like viruses from Loki’s Castle encode translation systems components. Phylogenetic analysis of these genes indicates a greater bacterial contribution than had been detected previously. Genome comparison suggests extensive gene exchange between members of the pithovirus-like viruses and Mimiviridae. Further exploration of the genomic diversity of Megavirales in additional sediment samples is expected to yield new insights into the evolution of giant viruses and the composition of the ocean megavirome.

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.

Members of the Marseilleviridae family are large DNA viruses with icosahedral particle structures that infect Acanthamoeba cells. The first Marseillevirus to be discovered was isolated in 2009. Since then, several other members of the Marseilleviridae family have been reported, including Lausannevirus, Senegalvirus, Cannes 8 virus, Insectomime virus, Tunisvirus, Melbournevirus, Port-Miou virus, and Brazilian Marseillevirus, which have been isolated from Europe, Africa, Australia, and South America. The morphological and genomic properties of a new Marseilleviridae family member, Tokyovirus, discovered in a water/soil sample from a Japanese river in Tokyo, were described in the present study. Tokyovirus possesses icosahedral particles of up to 200 nm in diameter, as revealed by a transmission electron microscopy (TEM) analysis, which form a giant virion factory in Acanthamoeba cells. A preliminary genome analysis predicted 487 coding sequences. A dot plot analysis and phylogenetic analysis using family B DNA polymerase, proliferating cell nuclear antigen (PCNA), and DNA-directed RNA polymerase alpha subunit genes revealed that Tokyovirus shares similarities with Marseillevirus, Melbournevirus, and Cannes 8 virus (Marseilleviridae subclade A), but not with Lausannevirus and Port-Miou virus (subclade B), Tunisvirus and Insectomime virus (subclade C), or Brazilian Marseillevirus (subclade D), suggesting that Tokyovirus has evolved separately from the previously described Marseilleviridae members.

Background While the genomes of eukaryotes and Archaea both encode the histone-fold domain, only eukaryotes encode the core histone paralogs H2A, H2B, H3, and H4. With DNA, these core histones assemble into the nucleosomal octamer underlying eukaryotic chromatin. Importantly, core histones for H2A and H3 are maintained as neofunctionalized paralogs adapted for general bulk chromatin (canonical H2 and H3) or specialized chromatin (H2A.Z enriched at gene promoters and cenH3s enriched at centromeres). In this context, the identification of core histone-like “doublets” in the cytoplasmic replication factories of the Marseilleviridae (MV) is a novel finding with possible relevance to understanding the origin of eukaryotic chromatin. Here, we analyze and compare the core histone doublet genes from all known MV genomes as well as other MV genes relevant to the origin of the eukaryotic replisome. Results Using different phylogenetic approaches, we show that MV histone domains encode obligate H2B-H2A and H4-H3 dimers of possible proto-eukaryotic origin. MV core histone moieties form sister clades to each of the four eukaryotic clades of canonical and variant core histones. This suggests that MV core histone moieties diverged prior to eukaryotic neofunctionalizations associated with paired linear chromosomes and variant histone octamer assembly. We also show that MV genomes encode a proto-eukaryotic DNA topoisomerase II enzyme that forms a sister clade to eukaryotes. This is a relevant finding given that DNA topo II influences histone deposition and chromatin compaction and is the second most abundant nuclear protein after histones. Conclusions The combined domain architecture and phylogenomic analyses presented here suggest that a primitive origin for MV histone genes is a more parsimonious explanation than horizontal gene transfers + gene fusions + sufficient divergence to eliminate relatedness to eukaryotic neofunctionalizations within the H2A and H3 clades without loss of relatedness to each of the four core histone clades. We thus suggest MV histone doublet genes and their DNA topo II gene possibly were acquired from an organism with a chromatinized replisome that diverged prior to the origin of eukaryotic core histone variants for H2/H2A.Z and H3/cenH3. These results also imply that core histones were utilized ancestrally in viral DNA compaction and/or protection from host endonucleases. Electronic supplementary material The online version of this article (10.1186/s13072-017-0162-0) contains supplementary material, which is available to authorized users.

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.

Giant viruses are protist-associated viruses belonging to the proposed order Megavirales ; almost all have been isolated from Acanthamoeba spp. Their isolation in humans suggests that they are part of the human virome. Using a high-throughput strategy to isolate new giant viruses from their original protozoan hosts, we obtained eight isolates of a new giant viral lineage from Vermamoeba vermiformis , the most common free-living protist found in human environments. This new lineage was proposed to be the faustovirus lineage. The prototype member, faustovirus E12, forms icosahedral virions of ≈200 nm that are devoid of fibrils and that encapsidate a 466-kbp genome encoding 451 predicted proteins. Of these, 164 are found in the virion. Phylogenetic analysis of the core viral genes showed that faustovirus is distantly related to the mammalian pathogen African swine fever virus, but it encodes ≈3 times more mosaic gene complements. About two-thirds of these genes do not show significant similarity to genes encoding any known proteins. These findings show that expanding the panel of protists to discover new giant viruses is a fruitful strategy. IMPORTANCE By using Vermamoeba , a protist living in humans and their environment, we isolated eight strains of a new giant virus that we named faustovirus. The genomes of these strains were sequenced, and their sequences showed that faustoviruses are related to but different from the vertebrate pathogen African swine fever virus (ASFV), which belongs to the family Asfarviridae . Moreover, the faustovirus gene repertoire is ≈3 times larger than that of ASFV and comprises approximately two-thirds ORFans (open reading frames [ORFs] with no detectable homology to other ORFs in a database).

BACKGROUND: A new Marseilleviridae virus family member, giant blood Marseille-like (GBM) virus, was recently reported in persons from France in the serum of an infant with adenitis, in the blood of 4% of healthy blood donors, and in 9% of multiply transfused thalassemia patients. These results suggested the presence of a nucleocytoplasmic large DNA virus potentially transmissible by blood product transfusion. STUDY DESIGN AND METHODS: To investigate this possibility we tested the plasma from 113 US blood donors and 74 multiply transfused Cameroon patients for GBM viral DNA using highly sensitive polymerase chain reaction (PCR) assays. RESULTS: GBM DNA was not detected by nested PCR in any of these 187 human specimens. CONCLUSIONS: Further testing is required to confirm the occurrence of human GBM virus infections.

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.