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.

Emerging Viral Pathogens in Sturgeon Aquaculture in Poland: Focus on Herpesviruses and Mimivirus Detection.

Recently, Poland has become a leading producer of sturgeon meat and caviar in Europe and is one of the largest in the world. The growing importance of this branch of aquaculture means that diseases of these fish, especially viral ones, are becoming the object of interest for ichthyopathologists. In recent years, there have been increasing reports of health problems in the dynamically developing sturgeon farming. The greatest risk appears to be emerging infectious diseases that are caused by viruses and that can become a serious threat to the development of the aquaculture industry and the success of sturgeon restitution programs undertaken in many European countries, including Poland. In this paper, an attempt was made to determine the spread of the two most important groups of viruses in Polish sturgeon farming: These include the herpesviruses and sturgeon nucleocytoplasmic large DNA viruses (sNCLDV), in particular, mimiviruses. In the years 2016-2020, 136 samples from nine farms were collected and tested by using the WSSK-1 cell line, PCR and Real Time PCR methods. All results were negative for herpesviruses. Out of the samples, 26% of the samples have been tested positive for mimiviruses. Sanger sequencing of mimiviruses demonstrated their affiliation with AciV-E. The sequence characterization confirmed the presence of both V1 and V2 lineages in Polish fish facilities, but variant V2 seems to be more widespread, as is observed in other European countries.

Revealing the Viral Community in the Hadal Sediment of the New Britain Trench.

Marine viruses are widely distributed and influence matter and energy transformation in ecosystems by modulating hosts' metabolism. The hadal trenches represent the deepest marine habitat on Earth, for which the viral communities and related biogeochemical functions are least explored and poorly understood. Here, using the sediment samples (8720 m below sea level) collected from the New Britain Trench (NBT), we investigated the viral community, diversity, and genetic potentials in the hadal sediment habitat for the first time by deep shotgun metagenomic sequencing. We found the NBT sediment viral community was dominated by Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, and Phycodnaviridae, which belong to the dsDNA viruses. However, the large majority of them remained uncharacterized. We found the hadal sediment virome had some common components by comparing the hadal sediment viruses with those of hadal aquatic habitats and those of bathypelagic and terrestrial habitats. It was also distinctive in community structure and had many novel viral clusters not associated with the other habitual virome included in our analyses. Further phylogenetic analysis on its Caudovirales showed novel diversities, including new clades specially evolved in the hadal sediment habitat. Annotation of the NBT sediment viruses indicated the viruses might influence microbial hydrocarbon biodegradation and carbon and sulfur cycling via metabolic augmentation through auxiliary metabolic genes (AMGs). Our study filled in the knowledge gaps on the virome of the hadal sediment habitats and provided insight into the evolution and the potential metabolic functions of the hadal sediment virome.

Morphological and Taxonomic Properties of the Newly Isolated Cotonvirus japonicus, a New Lineage of the Subfamily Megavirinae.

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.

Isolation and genomic characterization of a new mimivirus of lineage B from a Brazilian river.

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.

Lack of Marseillevirus DNA in immunocompetent and immunocompromised Italian patients.

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.

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.

Co-Isolation and Characterization of Two Pandoraviruses and a Mimivirus from a Riverbank in Japan.

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.

Seasonal Dynamics of Algae-Infecting Viruses and Their Inferred Interactions with Protists.

Viruses are a highly abundant, dynamic, and diverse component of planktonic communities that have key roles in marine ecosystems. We aimed to reveal the diversity and dynamics of marine large dsDNA viruses infecting algae in the Northern Skagerrak, South Norway through the year by metabarcoding, targeting the major capsid protein (MCP) and its correlation to protist diversity and dynamics. Metabarcoding results demonstrated a high diversity of algal viruses compared to previous metabarcoding surveys in Norwegian coastal waters. We obtained 313 putative algal virus operational taxonomic units (vOTUs), all classified by phylogenetic analyses to either the Phycodnaviridae or Mimiviridae families, most of them in clades without any cultured or environmental reference sequences. The viral community showed a clear temporal variation, with some vOTUs persisting for several months. The results indicate co-occurrences between abundant viruses and potential hosts during long periods. This study gives new insights into the virus-algal host dynamics and provides a baseline for future studies of algal virus diversity and temporal dynamics.

The Complex Nature of Tupanviruses.

The discovery of giant viruses revealed a new level of complexity in the virosphere, raising important questions about the diversity, ecology, and evolution of these viruses. The family Mimiviridae was the first group of amoebal giant viruses to be discovered (by Bernard La Scola and Didier Raoult team), containing viruses with structural and genetic features that challenged many concepts of classic virology. The tupanviruses are among the newest members of this family and exhibit structural, biological, and genetic features never previously observed in other giant viruses. The complexity of these viruses has put us one step forward toward the comprehension of giant virus biology and evolution, but also has raised important questions that still need to be addressed. In this chapter, we tell the history behind the discovery of one of the most complex viruses isolated to date, highlighting the unique features exhibited by tupanviruses, and discuss how these giant viruses have contributed to redefining limits for the virosphere.

Hidden diversity of soil giant viruses.

Known giant virus diversity is currently skewed towards viruses isolated from aquatic environments and cultivated in the laboratory. Here, we employ cultivation-independent metagenomics and mini-metagenomics on soils from the Harvard Forest, leading to the discovery of 16 novel giant viruses, chiefly recovered by mini-metagenomics. The candidate viruses greatly expand phylogenetic diversity of known giant viruses and either represented novel lineages or are affiliated with klosneuviruses, Cafeteria roenbergensis virus or tupanviruses. One assembled genome with a size of 2.4 Mb represents the largest currently known viral genome in the Mimiviridae, and others encode up to 80% orphan genes. In addition, we find more than 240 major capsid proteins encoded on unbinned metagenome fragments, further indicating that giant viruses are underexplored in soil ecosystems. The fact that most of these novel viruses evaded detection in bulk metagenomes suggests that mini-metagenomics could be a valuable approach to unearth viral giants.

Mimiviridae: An Expanding Family of Highly Diverse Large dsDNA Viruses Infecting a Wide Phylogenetic Range of Aquatic Eukaryotes.

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.

Isolation and Quantification of Mimivirus-Like and Marseillevirus-Like Viruses from Soil Samples in An Aboriginal (Orang asli) Village in Peninsular Malaysia.

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 kinetoplastid-infecting Bodo saltans virus (BsV), a window into the most abundant giant viruses in the sea.

Giant viruses are ecologically important players in aquatic ecosystems that have challenged concepts of what constitutes a virus. Herein, we present the giant Bodo saltans virus (BsV), the first characterized representative of the most abundant group of giant viruses in ocean metagenomes, and the first isolate of a klosneuvirus, a subgroup of the Mimiviridae proposed from metagenomic data. BsV infects an ecologically important microzooplankton, the kinetoplastid Bodo saltans. Its 1.39 Mb genome encodes 1227 predicted ORFs, including a complex replication machinery. Yet, much of its translational apparatus has been lost, including all tRNAs. Essential genes are invaded by homing endonuclease-encoding self-splicing introns that may defend against competing viruses. Putative anti-host factors show extensive gene duplication via a genomic accordion indicating an ongoing evolutionary arms race and highlighting the rapid evolution and genomic plasticity that has led to genome gigantism and the enigma that is giant viruses.

Analyses of the Kroon Virus Major Capsid Gene and Its Transcript Highlight a Distinct Pattern of Gene Evolution and Splicing among Mimiviruses.

The inclusion of Mimiviridae members in the putative monophyletic nucleocytoplasmic large DNA virus (NCLDV) group is based on genomic and phylogenomic patterns. This shows that, along with other viral families, they share a set of genes known as core or "hallmark genes," including the gene for the major capsid protein (MCP). Although previous studies have suggested that the maturation of mimivirus MCP transcripts is dependent on splicing, there is little information about the processing of this transcript in other mimivirus isolates. Here we report the characterization of a new mimivirus isolate, called Kroon virus (KV) mimivirus. Analysis of the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates revealed a remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. In addition, sequencing of KV and Acanthamoeba polyphaga mimivirus (APMV) MCP transcripts has shown that inside the family, even related giant viruses may present different ways to process the MCP mRNA. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.IMPORTANCE Mimivirus isolates have been obtained by prospecting studies since 2003. Based on genomic and phylogenomic studies of conserved genes, these viruses have been clustered together with members of six other viral families. Although the major capsid protein (MCP) gene is an important member of the so-called "hallmark genes," there is little information about the processing and structure of this gene in many mimivirus isolates. In this work, we have analyzed the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates; these genes showed remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.

Viruses in cancers among the immunosuppressed.

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.

Detection of mimivirus genome and neutralizing antibodies in humans from Brazil.

In recent years, giant viruses belonging to the family Mimiviridae have been proposed to be infectious agents in humans. In this work we provide evidence of mimivirus genome and neutralizing antibodies detection in humans.

The "Giant Virus Finder" discovers an abundance of giant viruses in the Antarctic dry valleys.

Mimivirus was identified in 2003 from a biofilm of an industrial water-cooling tower in England. Later, numerous new giant viruses were found in oceans and freshwater habitats, some of them having 2,500 genes. We have demonstrated their likely presence in four soil samples taken from the Kutch Desert (Gujarat, India). Here we describe a bioinformatics work-flow, called the "Giant Virus Finder" that is capable of discovering the likely presence of the genomes of giant viruses in metagenomic shotgun-sequenced datasets. The new workflow is applied to numerous hot and cold desert soil samples as well as some tundra- and forest soils. We show that most of these samples contain giant viruses, especially in the Antarctic dry valleys. The results imply that giant viruses could be frequent not only in aqueous habitats, but in a wide spectrum of soils on our planet.