Virophages, Satellite Viruses, Virophage Replication and Its Effects and Virophage Defence Mechanisms for Giant Virus Hosts and Giant Virus Defence Systems against Virophages.

In this paper, the characteristics of 40 so far described virophages-parasites of giant viruses-are given, and the similarities and differences between virophages and satellite viruses, which also, like virophages, require helper viruses for replication, are described. The replication of virophages taking place at a specific site-the viral particle factory of giant viruses-and its consequences are presented, and the defence mechanisms of virophages for giant virus hosts, as a protective action for giant virus hosts-protozoa and algae-are approximated. The defence systems of giant viruses against virophages were also presented, which are similar to the CRISPR/Cas defence system found in bacteria and in Archea. These facts, and related to the very specific biological features of virophages (specific site of replication, specific mechanisms of their defensive effects for giant virus hosts, defence systems in giant viruses against virophages), indicate that virophages, and their host giant viruses, are biological objects, forming a 'novelty' in biology.

Isolation and Identification of a Large Green Alga Virus (Chlorella Virus XW01) of Mimiviridae and Its Virophage (Chlorella Virus Virophage SW01) by Using Unicellular Green Algal Cultures.

Virophages are a group of small double-stranded DNA viruses that infect protist hosts and parasitize the viral factory of host giant/large viruses to propagate. Here, we discover a novel cell-virus-virophage (CVv) tripartite interaction system by using unicellular micro-green algae (Chlorella sp.) as eukaryotic hosts for the first time. Viral particles, resembling known virophages and large alga viruses, are detected in culture supernatants and inside algal cells. Complete genomic sequences of the virophage (Chlorella virus virophage SW01 [CVv-SW01]; 24,744 bp) and large virus (Chlorella virus XW01 [CV-XW01]; 407,612 bp) are obtained from the cocultures. Both genomic and phylogenetic analyses show that CVv-SW01 is closely related to virophages previously found in Dishui Lake. CV-XW01 shares the greatest number of homologous genes (n = 82) with Cafeteria roenbergensis virus (CroV) and phylogenetically represents the closest relative to CroV. This is the first report of a large green alga virus being affiliated with a heterotrophic zooplankton-infecting Cafeteriavirus of the family Mimiviridae. Moreover, the codon usage preferences of CV-XW01 and CVv-SW01 are highly similar to those of CroV and its virophage Mavirus, respectively. The discovery of such a novel CVv system with the green alga Chlorella sp. as the single cellular eukaryotic host paves a way to further investigate the potential interaction mechanism of CVv and its significance in the ecology of green algae and the evolution of large/giant viruses and their parasitic viruses. IMPORTANCE Parasitic virophages are small unicellular eukaryotic dsDNA viruses that rely on the viral factories of coinfecting giant/large dsDNA viruses for propagation. Presently, the identified eukaryotic hosts of isolated virophages were restricted to a free-living amoeba, Acanthamoeba polyphaga, and a widespread marine heterotrophic flagellate, Cafeteria roenbergensis. In this study, we successfully discovered and identified a novel tripartite interaction system comprised of a micro-green alga (Chlorella sp.), Mimiviridae large green alga virus, and virophage at the coculture level, with Chlorella sp. as the eukaryotic host, based on combination analysis of infection, morphotype, genome, and phylogeny. The large green alga virus CV-XW01 represents the closest relative to the Mimiviridae giant virus Cafeteria roenbergensis virus, host virus of the virophage Mavirus, as well as a novel large virus of Mimiviridae that infects a non-protozoan protist host. The virophage CVv-SW01 highly resembles Mavirus in its codon usage frequency and preference, although they are phylogenetically distantly related. These findings give novel insights into the diversity of large/giant viruses and their virophages.

Host genome integration and giant virus-induced reactivation of the virophage mavirus.

Endogenous viral elements are increasingly found in eukaryotic genomes, yet little is known about their origins, dynamics, or function. Here we provide a compelling example of a DNA virus that readily integrates into a eukaryotic genome where it acts as an inducible antiviral defence system. We found that the virophage mavirus, a parasite of the giant Cafeteria roenbergensis virus (CroV), integrates at multiple sites within the nuclear genome of the marine protozoan Cafeteria roenbergensis. The endogenous mavirus is structurally and genetically similar to eukaryotic DNA transposons and endogenous viruses of the Maverick/Polinton family. Provirophage genes are not constitutively expressed, but are specifically activated by superinfection with CroV, which induces the production of infectious mavirus particles. Virophages can inhibit the replication of mimivirus-like giant viruses and an anti-viral protective effect of provirophages on their hosts has been hypothesized. We find that provirophage-carrying cells are not directly protected from CroV; however, lysis of these cells releases infectious mavirus particles that are then able to suppress CroV replication and enhance host survival during subsequent rounds of infection. The microbial host-parasite interaction described here involves an altruistic aspect and suggests that giant-virus-induced activation of provirophages might be ecologically relevant in natural protist populations.

The Virophage Family Lavidaviridae.

Double-stranded (ds) DNA viruses of the family Lavidaviridae, commonly known as virophages, are a fascinating group of eukaryotic viruses that depend on a coinfecting giant dsDNA virus of the Mimiviridae for their propagation. Instead of replicating in the nucleus, virophages multiply in the cytoplasmic virion factory of a coinfecting giant virus inside a phototrophic or heterotrophic protistal host cell. Virophages are parasites of giant viruses and can inhibit their replication, which may lead to increased survival rates of the infected host cell population. The genomes of virophages are 17-33 kilobase pairs (kbp) long and encode 16-34 proteins. Genetic signatures of virophages can be found in metagenomic datasets from various saltwater and freshwater environments around the planet. Most virophages share a set of conserved genes that code for a major and a minor capsid protein, a cysteine protease, a genome-packaging ATPase, and a superfamily 3 helicase, although the genomes are otherwise diverse and variable. Lavidaviruses share genes with other mobile genetic elements, suggesting that horizontal gene transfer and recombination have been major forces in shaping these viral genomes. Integrases are occasionally found in virophage genomes and enable these DNA viruses to persist as provirophages in the chromosomes of their viral and cellular hosts. As we watch the genetic diversity of this new viral family unfold through metagenomics, additional isolates are still lacking and critical questions regarding their infection cycle, host range, and ecology remain to be answered.

Novel Cell-Virus-Virophage Tripartite Infection Systems Discovered in the Freshwater Lake Dishui Lake in Shanghai, China.

Virophages are small parasitic double-stranded DNA (dsDNA) viruses of giant dsDNA viruses infecting unicellular eukaryotes. Except for a few isolated virophages characterized by parasitization mechanisms, features of virophages discovered in metagenomic data sets remain largely unknown. Here, the complete genomes of seven virophages (26.6 to 31.5 kbp) and four large DNA viruses (190.4 to 392.5 kbp) that coexist in the freshwater lake Dishui Lake, Shanghai, China, have been identified based on environmental metagenomic investigation. Both genomic and phylogenetic analyses indicate that Dishui Lake virophages (DSLVs) are closely related to each other and to other lake virophages, and Dishui Lake large DNA viruses are affiliated with the micro-green alga-infecting Prasinovirus of the Phycodnaviridae (named Dishui Lake phycodnaviruses [DSLPVs]) and protist (protozoan and alga)-infecting Mimiviridae (named Dishui Lake large alga virus [DSLLAV]). The DSLVs possess more genes with closer homology to that of large alga viruses than to that of giant protozoan viruses. Furthermore, the DSLVs are strongly associated with large green alga viruses, including DSLPV4 and DSLLAV1, based on codon usage as well as oligonucleotide frequency and correlation analyses. Surprisingly, a nonhomologous CRISPR-Cas like system is found in DSLLAV1, which appears to protect DSLLAV1 from the parasitization of DSLV5 and DSLV8. These results suggest that novel cell-virus-virophage (CVv) tripartite infection systems of green algae, large green alga virus (Phycodnaviridae- and Mimiviridae-related), and virophage exist in Dishui Lake, which will contribute to further deep investigations of the evolutionary interaction of virophages and large alga viruses as well as of the essential roles that the CVv plays in the ecology of algae.IMPORTANCE Virophages are small parasitizing viruses of large/giant viruses. To our knowledge, the few isolated virophages all parasitize giant protozoan viruses (Mimiviridae) for propagation and form a tripartite infection system with hosts, here named the cell-virus-virophage (CVv) system. However, the CVv system remains largely unknown in environmental metagenomic data sets. In this study, we systematically investigated the metagenomic data set from the freshwater lake Dishui Lake, Shanghai, China. Consequently, four novel large alga viruses and seven virophages were discovered to coexist in Dishui Lake. Surprisingly, a novel CVv tripartite infection system comprising green algae, large green alga viruses (Phycodnaviridae- and Mimiviridae-related), and virophages was identified based on genetic link, genomic signature, and CRISPR system analyses. Meanwhile, a nonhomologous CRISPR-like system was found in Dishui Lake large alga viruses, which appears to protect the virus host from the infection of Dishui Lake virophages (DSLVs). These findings are critical to give insight into the potential significance of CVv in global evolution and ecology.

Genomic Signatures Among Acanthamoeba polyphaga Entoorganisms Unveil Evidence of Coevolution.

The definition of a genomic signature (GS) is "the total net response to selective pressure". Recent isolation and sequencing of naturally occurring organisms, hereby named entoorganisms, within Acanthamoeba polyphaga, raised the hypothesis of a common genomic signature despite their diverse and unrelated evolutionary origin. Widely accepted and implemented tests for GS detection are oligonucleotide relative frequencies (OnRF) and relative codon usage (RCU) surveys. A common pattern and strong correlations were unveiled from OnRFs among A. polyphaga's Mimivirus and virophage Sputnik. RCU showed a common A-T bias at third codon position. We expanded tests to the amoebal mitochondrial genome and amoeba-resistant bacteria, achieving strikingly coherent results to the aforementioned viral analyses. The GSs in these entoorganisms of diverse evolutionary origin are coevolutionarily conserved within an intracellular environment that provides sanctuary for species of ecological and biomedical relevance.

Complex transcriptional regulations of a hyperparasitic quadripartite system in giant viruses infecting protists.

Hyperparasitism is a common pattern in nature that is not limited to cellular organisms. Giant viruses infecting protists can be hyperparasitized by smaller ones named virophages. In addition, both may carry episomal DNA molecules known as transpovirons in their particles. They all share transcriptional regulatory elements that dictate the expression of their genes within viral factories built by giant viruses in the host cytoplasm. This suggests the existence of interactions between their respective transcriptional networks. Here we investigated Acanthamoeba castellanii cells infected by a giant virus (megavirus chilensis), and coinfected with a virophage (zamilon vitis) and/or a transpoviron (megavirus vitis transpoviron). Infectious cycles were monitored through time-course RNA sequencing to decipher the transcriptional program of each partner and its impact on the gene expression of the others. We found highly diverse transcriptional responses. While the giant virus drastically reshaped the host cell transcriptome, the transpoviron had no effect on the gene expression of any of the players. In contrast, the virophage strongly modified the giant virus gene expression, albeit transiently, without altering the protein composition of mature viral particles. The virophage also induced the overexpression of transpoviron genes, likely through the indirect upregulation of giant virus-encoded transcription factors. Together, these analyses document the intricated transcriptionally regulated networks taking place in the infected cell.

The polinton-like supergroup of viruses: evolution, molecular biology, and taxonomy.

SUMMARYPolintons are 15-20 kb-long self-synthesizing transposons that are widespread in eukaryotic, and in particular protist, genomes. Apart from a transposase and a protein-primed DNA polymerase, polintons encode homologs of major and minor jelly-roll capsid proteins, DNA-packaging ATPases, and proteases involved in capsid maturation of diverse eukaryotic viruses of kingdom Bamfordvirae. Given the conservation of these structural and morphogenetic proteins among polintons, these elements are predicted to alternate between transposon and viral lifestyles and, although virions have thus far not been detected, are classified as viruses (class Polintoviricetes) in the phylum Preplasmiviricota. Related to polintoviricetes are vertebrate adenovirids; unclassified polinton-like viruses (PLVs) identified in various environments or integrated into diverse protist genomes; virophages (Maveriviricetes), which are part of tripartite hyperparasitic systems including protist hosts and giant viruses; and capsid-less derivatives, such as cytoplasmic linear DNA plasmids of fungi and transpovirons. Phylogenomic analysis indicates that the polinton-like supergroup of viruses bridges bacterial tectivirids (preplasmiviricot class Tectiliviricetes) to the phylum Nucleocytoviricota that includes large and giant eukaryotic DNA viruses. Comparative structural analysis of proteins encoded by polinton-like viruses led to the discovery of previously undetected functional domains, such as terminal proteins and distinct proteases implicated in DNA polymerase processing, and clarified the evolutionary relationships within Polintoviricetes. Here, we leverage these insights into the evolution of the polinton-like supergroup to develop an amended megataxonomy that groups Polintoviricetes, PLVs (new class 'Aquintoviricetes'), and virophages (renamed class 'Virophaviricetes') together with Adenoviridae (new class 'Pharingeaviricetes') in a preplasmiviricot subphylum 'Polisuviricotina' sister to a subphylum including Tectiliviricetes ('Prepoliviricotina').

Microsatellite signature analysis of twenty-one virophage genomes of the family Lavidaviridae.

Microsatellites or Simple Sequence Repeats (SSRs) are short motif repeat sequences constituting the most hypervariable regions of genomes. Present study extracts and analyzes the SSRs from genomes of 21 virophages. Genomic sequences were retrieved from NCBI and the microsatellite data was extracted through MISA web server. Phylogenetic analysis was performed by using MAFFT and MEGAX as per standardized protocols. The virophages have a circular/linear ds DNA genome of ~17-30 kb size. The GC% of genomes ranged from 26.8 (PSAV13) to 51.1 (PSAV12). A total of 3664 SSRs and 488 cSSR were observed with an average incidence of 174 and 23 respectively. The total SSR incidence in a genome ranged from 120 (PSAV19) to 264 (PSAV14). The cSSR (compound SSR) incidence ranged from 8 (PSAV12) to 47 (PSAV14). Mono-nucleotide repeats are the most incident microsatellites (1129 SSRs) followed by di-nucleotide (1036 SSRs) and tri-nucleotide repeats (368 SSRs). However, the same is not true for individual genomes. There are 14, 16 and 17 genomes which have no incidence of tetra-, penta- and hexa-nucleotide repeats respectively. Mono 'A' repeats having the maximum representation (average ~33 per genome) in mono-nucleotide repeats. For the di-nucleotide repeats, AT/TA motif had the highest frequency (average ~30) distantly followed by AG/GA; and CT/TC (average 5.6 & 5.5 respectively). A total of 1946 SSRs (76%) were found in the coding region. All genomes had a higher SSR density in non-coding as compared to the coding region. There are fifteen genomes which have at least one gene with no SSR. A total of 41 cSSRs with incidence across minimum of two virophages was observed. There were 12 cSSRs which had multiple presence within the same genome. The heat map of the genomes on one hand corroborates the phylogenetic tree with similar sequences (PSAV2, PSAV5, PSAV6, PSAV17 and PSAV18) being positioned together in the phylogenetic analysis while on the other hand it also highlights the diversity of the studied sequences. The conservation of cSSRs across multiple virophages highlights their potential as biomarkers.

Updated Virophage Taxonomy and Distinction from Polinton-like Viruses.

Virophages are small dsDNA viruses that hijack the machinery of giant viruses during the co-infection of a protist (i.e., microeukaryotic) host and represent an exceptional case of "hyperparasitism" in the viral world. While only a handful of virophages have been isolated, a vast diversity of virophage-like sequences have been uncovered from diverse metagenomes. Their wide ecological distribution, idiosyncratic infection and replication strategy, ability to integrate into protist and giant virus genomes and potential role in antiviral defense have made virophages a topic of broad interest. However, one limitation for further studies is the lack of clarity regarding the nomenclature and taxonomy of this group of viruses. Specifically, virophages have been linked in the literature to other "virophage-like" mobile genetic elements and viruses, including polinton-like viruses (PLVs), but there are no formal demarcation criteria and proper nomenclature for either group, i.e., virophage or PLVs. Here, as part of the ICTV Virophage Study Group, we leverage a large set of genomes gathered from published datasets as well as newly generated protist genomes to propose delineation criteria and classification methods at multiple taxonomic ranks for virophages 'sensu stricto', i.e., genomes related to the prototype isolates Sputnik and mavirus. Based on a combination of comparative genomics and phylogenetic analyses, we show that this group of virophages forms a cohesive taxon that we propose to establish at the class level and suggest a subdivision into four orders and seven families with distinctive ecogenomic features. Finally, to illustrate how the proposed delineation criteria and classification method would be used, we apply these to two recently published datasets, which we show include both virophages and other virophage-related elements. Overall, we see this proposed classification as a necessary first step to provide a robust taxonomic framework in this area of the virosphere, which will need to be expanded in the future to cover other virophage-related viruses such as PLVs.

Virophages Found in Viromes from Lake Baikal.

In this study, a previously little-studied group of viruses-virophages-was searched for and identified in the viromes of the ancient oligotrophic Lake Baikal. Virophages are small dsDNA viruses that parasitize giant viruses (e.g., Mimiviridae), which in turn affect unicellular eukaryotes. We analyzed eight viromes obtained from the deep-water areas of three basins of Lake Baikal and the shallow-water strait Maloye More in different seasons. The sequences of virophages were revealed in all viromes and were dominant after bacteriophages and algal viruses. Sixteen putative complete genomes of virophages were assembled, all of which contained four conserved genes encoding major capsid protein (MCP), minor capsid protein (mCP), maturation cysteine protease (PRO), and FtsK-HerA family DNA-packaging ATPase (ATPase). The MCP-based cluster analysis showed a sequence separation according to seasons, and a dependence on the geographical localization was not detected.

Isolation and infection cycle of a polinton-like virus virophage in an abundant marine alga.

Virophages are small double stranded DNA (dsDNA) viruses that can only replicate in a host by co-infecting with another virus. Marine algae are commonly associated with virophage-like elements such as Polinton-like viruses (PLVs) that remain largely uncharacterized. Here we isolated a PLV that co-infects the alga Phaeocystis globosa with the Phaeocystis globosa virus-14T (PgV-14T), a close relative of the "Phaeocystis globosa virus-virophage" genomic sequence. We name this PLV 'Gezel-14T. Gezel is phylogenetically distinct from the Lavidaviridae family where all known virophages belong. Gezel-14T co-infection decreases the fitness of its viral host by reducing burst sizes of PgV-14T, yet insufficiently to spare the cellular host population. Genomic screens show Gezel-14T-like PLVs integrated into Phaeocystis genomes, suggesting that these widespread viruses are capable of integration into cellular host genomes. This system presents an opportunity to better understand the evolution of eukaryotic dsDNA viruses as well as the complex dynamics and implications of viral parasitism.

Simultaneous Giant Virus and Virophage Quantification Using Droplet Digital PCR.

Viruses are an abundant component of aquatic systems, but their detection and quantification remain a challenge. Virophages co-replicate with giant viruses in the shared host cell, and can inhibit the production of new giant virus particles, thereby increasing the survival of the infected host population. Here, we present a protocol for Droplet Digital PCR (ddPCR) to quantify simultaneously giant virus and virophage in a mixed sample, enabling the rapid, culture-free and high throughput detection of virus and virophage. As virophage can be present as free virus particles or integrated into the virus host's genome as well as associated with organic particles, we developed a simple method that enables discrimination between free and particle-associated virophages. The latter include aggregated virophage particles as well as virophage integrated into the host genome. We used, for our experiments, a host-virus-virophage system consisting of Cafeteria burkhardae, CroV and mavirus. Our results show that ddPCR can be an efficient method to quantify virus and virophage, and we discuss potential applications of the method for studying ecological and evolutionary processes of virus and virophages.

The Discovery of a New Mimivirus Isolate in Association with Virophage-Transpoviron Elements in Brazil Highlights the Main Genomic and Evolutionary Features of This Tripartite System.

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.

Polinton-like viruses are abundant in aquatic ecosystems.

BACKGROUND: Polintons are large mobile genetic elements found in the genomes of eukaryotic organisms that are considered the ancient ancestors of most eukaryotic dsDNA viruses. Originally considered as transposons, they have been found to encode virus capsid genes, suggesting they may actually be integrated viruses; however, an extracellular form has yet to be detected. Recently, circa 25 Polinton-like viruses have been discovered in environmental metagenomes and algal genomes, which shared distantly related genes to both Polintons and virophages (Lavidaviridae). These entities could be the first members of a major class of ancient eukaryotic viruses; however, owing to the lack of available genomes for analysis, information on their global diversity, evolutionary relationships, eukaryotic hosts, and status as free virus particles is limited. RESULTS: Here, we analysed the metaviromes of an alpine lake to show that Polinton-like virus genome sequences are abundant in the water column. We identify major capsid protein genes belonging to 82 new Polinton-like viruses and use these to interrogate publicly available metagenomic datasets, identifying 543 genomes and a further 16 integrated into eukaryotic genomes. Using an analysis of shared gene content and major capsid protein phylogeny, we define large groups of Polinton-like viruses and link them to diverse eukaryotic hosts, including a new group of viruses, which possess all the core genes of virophages and infect oomycetes and Chrysophyceae. CONCLUSIONS: Our study increased the number of known Polinton-like viruses by 25-fold, identifying five major new groups of eukaryotic viruses, which until now have been hidden in metagenomic datasets. The large enrichment (> 100-fold) of Polinton-like virus sequences in the virus-sized fraction of this alpine lake and the fact that their viral major capsid proteins are found in eukaryotic host transcriptomes support the hypothesis that Polintons in unicellular eukaryotes are viruses. In summary, our data reveals a diverse assemblage of globally distributed viruses, associated with a wide range of unicellular eukaryotic hosts. We anticipate that the methods we have developed for Polinton-like virus detection and the database of over 20,000 genes we present will allow for continued discovery and analysis of these new viral groups. Video abstract.

Exploration of the propagation of transpovirons within Mimiviridae reveals a unique example of commensalism in the viral world.

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.

The dual lifestyle of genome-integrating virophages in protists.

DNA viruses with efficient host genome integration capability were unknown in eukaryotes until recently. The discovery of virophages, satellite-like DNA viruses that depend on lytic giant viruses that infect protists, revealed a genetically diverse group of viruses with high genome mobility. Virophages can act as strong inhibitors of their associated giant viruses, and the resulting beneficial effects on their unicellular hosts resemble a population-based antiviral defense mechanism. By comparing various aspects of genome-integrating virophages, in particular the virophage mavirus, with other mobile genetic elements and parasite-derived defense mechanisms in eukaryotes and prokaryotes, we show that virophages share many features with other host-parasite systems. Yet, the dual lifestyle exhibited by mavirus remains unprecedented among eukaryotic DNA viruses, with potentially far-reaching ecological and evolutionary consequences for the host.

Diversity, evolution, and classification of virophages uncovered through global metagenomics.

BACKGROUND: Virophages are small viruses with double-stranded DNA genomes that replicate along with giant viruses and co-infect eukaryotic cells. Due to the paucity of virophage reference genomes, a collective understanding of the global virophage diversity, distribution, and evolution is lacking. RESULTS: Here we screened a public collection of over 14,000 metagenomes using the virophage-specific major capsid protein (MCP) as "bait." We identified 44,221 assembled virophage sequences, of which 328 represent high-quality (complete or near-complete) genomes from diverse habitats including the human gut, plant rhizosphere, and terrestrial subsurface. Comparative genomic analysis confirmed the presence of four core genes in a conserved block. We used these genes to establish a revised virophage classification including 27 clades with consistent genome length, gene content, and habitat distribution. Moreover, for eight high-quality virophage genomes, we computationally predicted putative eukaryotic virus hosts. CONCLUSION: Overall, our approach has increased the number of known virophage genomes by 10-fold and revealed patterns of genome evolution and global virophage distribution. We anticipate that the expanded diversity presented here will provide the backbone for further virophage studies.

Deciphering the genomes of 16 Acanthamoeba species does not provide evidence of integration of known giant virus-associated mobile genetic elements.

Giant viruses infect protozoa, especially amoebae of the genus Acanthamoeba. These viruses possess genetic elements named Mobilome. So far, this mobilome comprises provirophages which are integrated into the genome of their hosts, transpovirons, and Maverick/Polintons. Virophages replicate inside virus factories within Acanthamoeba and can decrease the infectivity of giant viruses. The virophage infecting CroV was found to be integrated in the host of CroV, Cafeteria roenbergensis, thus protecting C. roenbergensis by reduction of CroV multiplication. Because of this unique property, assessment of the mechanisms of replication of virophages and their relationship with giant viruses is a key element of this investigation. This work aimed at evaluating the presence and the dynamic of these mobile elements in sixteen Acanthamoeba genomes. No significant traces of the integration of genomes or sequences from known virophages were identified in all the available Acanthamoeba genomes. These results brought us to hypothesize that the interactions between mimiviruses and their virophages might occur through different mechanisms, or at low frequency. An additional explanation could be that our knowledge of the diversity of virophages is still very limited.

Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles.

Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.