A Bayesian functional approach to test models of life course epidemiology over continuous time.

BACKGROUND: Life course epidemiology examines associations between repeated measures of risk and health outcomes across different phases of life. Empirical research, however, is often based on discrete-time models that assume that sporadic measurement occasions fully capture underlying long-term continuous processes of risk. METHODS: We propose (i) the functional relevant life course model (fRLM), which treats repeated, discrete measures of risk as unobserved continuous processes, and (ii) a testing procedure to assign probabilities that the data correspond to conceptual models of life course epidemiology (critical period, sensitive period and accumulation models). The performance of the fRLM is evaluated with simulations, and the approach is illustrated with empirical applications relating body mass index (BMI) to mRNA-seq signatures of chronic kidney disease, inflammation and breast cancer. RESULTS: Simulations reveal that fRLM identifies the correct life course model with three to five repeated assessments of risk and 400 subjects. The empirical examples reveal that chronic kidney disease reflects a critical period process and inflammation and breast cancer likely reflect sensitive period mechanisms. CONCLUSIONS: The proposed fRLM treats repeated measures of risk as continuous processes and, under realistic data scenarios, the method provides accurate probabilities that the data correspond to commonly studied models of life course epidemiology. fRLM is implemented with publicly-available software.

Integrated viral elements suggest the dual lifestyle of Tetraselmis spp. Polinton-like viruses.

In this study, we aimed at exploring horizontal gene transfer between viruses and Chlorodendraceae green algae (Chlorophyta) using available genomic and transcriptomic sequences for twenty algal strains. We identified a significant number of genes sharing a higher sequence similarity with viral homologues, thus signalling their possible involvement in horizontal gene transfers with viruses. Further characterization showed that many of these genes were clustered in DNA regions of several tens to hundreds of kilobases in size, originally belonging to viruses related to known Tetraselmis spp. viruses (TetV and TsV). In contrast, the remaining candidate HGT genes were randomly dispersed in the algal genomes, were more frequently transcribed, and belonged to large multigene families. The presence of homologues in Viridiplantae suggested that the latter were more likely of algal rather than viral origin. We found a remarkable diversity in polinton-like virus (PLV) elements inserted in Tetraselmis genomes, all of which were most similar to the Tetraselmis striata virus (TsV). The genes of PLV elements are transcriptionally inactive with the notable exception of the homologue of the TVSG_00024 gene of TsV whose function is unknown. We suggest that this gene may be involved in a sentinel process to trigger virus reactivation and excision in response to an environmental stimulus. Altogether, these results provide evidence that TsV-related viruses have a dual lifestyle, alternating between a free viral phase (i.e. virion) and a phase integrated into host genomes.

Diversity of Giant Viruses Infecting Vermamoeba vermiformis.

The discovery of Acanthamoeba polyphaga mimivirus in 2003 using the free-living amoeba Acanthamoeba polyphaga caused a paradigm shift in the virology field. Twelve years later, using another amoeba as a host, i.e., Vermamoeba vermiformis, novel isolates of giant viruses have been discovered. This amoeba-virus relationship led scientists to study the evolution of giant viruses and explore the origins of eukaryotes. The purpose of this article is to review all the giant viruses that have been isolated from Vermamoeba vermiformis, compare their genomic features, and report the influence of these viruses on the cell cycle of their amoebal host. To date, viruses putatively belonging to eight different viral taxa have been described: 7 are lytic and 1 is non-lytic. The comparison of giant viruses infecting Vermamoeba vermiformis has suggested three homogenous groups according to their size, the replication time inside the host cell, and the number of encoding tRNAs. This approach is an attempt at determining the evolutionary origins and trajectories of the virus; therefore, more giant viruses infecting Vermamoeba must be discovered and studied to create a comprehensive knowledge on these intriguing biological entities.

A High Rate Algal Pond Hosting a Dynamic Community of RNA Viruses.

Despite a surge of RNA virome sequencing in recent years, there are still many RNA viruses to uncover-as indicated by the relevance of viral dark matter to RNA virome studies (i.e., putative viruses that do not match to taxonomically identified viruses). This study explores a unique site, a high-rate algal pond (HRAP), for culturing industrially microalgae, to elucidate new RNA viruses. The importance of viral-host interactions in aquatic systems are well documented, and the ever-expanding microalgae industry is no exception. As the industry becomes a more important source of sustainable plastic manufacturing, a producer of cosmetic pigments and alternative protein sources, and a means of CO2 remediation in the face of climate change, studying microalgal viruses becomes a vital practice for proactive management of microalgae cultures at the industrial level. This study provides evidence of RNA microalgal viruses persisting in a CO2 remediation pilot project HRAP and uncovers the diversity of the RNA virosphere contained within it. Evidence shows that family Marnaviridae is cultured in the basin, alongside other potential microalgal infecting viruses (e.g., family Narnaviridae, family Totitiviridae, and family Yueviridae). Finally, we demonstrate that the RNA viral diversity of the HRAP is temporally dynamic across two successive culturing seasons.

Pacmanvirus S19, the Second Pacmanvirus Isolated from Sewage Waters in Oran, Algeria.

Acanthamoeba castellanii is an amoeba host that was used to isolate a novel strain named pacmanvirus S19. This isolate is the second strain reported and belongs to the extended Asfarviridae family. Pacmanvirus S19 harbors a 418,588-bp genome, with a GC content of 33.20%, which encodes 444 predicted proteins and a single Ile-tRNA.

Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes.

Horizontal gene transfer (HGT) is an important source of novelty in eukaryotic genomes. This is particularly true for the ochrophytes, a diverse and important group of algae. Previous studies have shown that ochrophytes possess a mosaic of genes derived from bacteria and eukaryotic algae, acquired through chloroplast endosymbiosis and from HGTs, although understanding of the time points and mechanisms underpinning these transfers has been restricted by the depth of taxonomic sampling possible. We harness an expanded set of ochrophyte sequence libraries, alongside automated and manual phylogenetic annotation, in silico modeling, and experimental techniques, to assess the frequency and functions of HGT across this lineage. Through manual annotation of thousands of single-gene trees, we identify continuous bacterial HGT as the predominant source of recently arrived genes in the model diatom Phaeodactylum tricornutum Using a large-scale automated dataset, a multigene ochrophyte reference tree, and mathematical reconciliation of gene trees, we note a probable elevation of bacterial HGTs at foundational points in diatom evolution, following their divergence from other ochrophytes. Finally, we demonstrate that throughout ochrophyte evolutionary history, bacterial HGTs have been enriched in genes encoding secreted proteins. Our study provides insights into the sources and frequency of HGTs, and functional contributions that HGT has made to algal evolution.

The Kaumoebavirus LCC10 Genome Reveals a Unique Gene Strand Bias among "Extended Asfarviridae".

Kaumoebavirus infects the amoeba Vermamoeba vermiformis and has recently been described as a distant relative of the African swine fever virus. To characterize the diversity and evolution of this novel viral genus, we report here on the isolation and genome sequencing of a second strain of Kaumoebavirus, namely LCC10. Detailed analysis of the sequencing data suggested that its 362-Kb genome is linear with covalently closed hairpin termini, so that DNA forms a single continuous polynucleotide chain. Comparative genomic analysis indicated that although the two sequenced Kaumoebavirus strains share extensive gene collinearity, 180 predicted genes were either gained or lost in only one genome. As already observed in another distant relative, i.e., Faustovirus, which infects the same host, the center and extremities of the Kaumoebavirus genome exhibited a higher rate of sequence divergence and the major capsid protein gene was colonized by type-I introns. A possible role of the Vermamoeba host in the genesis of these evolutionary traits is hypothesized. The Kaumoebavirus genome exhibited a significant gene strand bias over the two-third of genome length, a feature not seen in the other members of the "extended Asfarviridae" clade. We suggest that this gene strand bias was induced by a putative single origin of DNA replication located near the genome extremity that imparted a selective force favoring the genes positioned on the leading strand.

Long-read only assembly of Drechmeria coniospora genomes reveals widespread chromosome plasticity and illustrates the limitations of current nanopore methods.

BACKGROUND: Long-read sequencing is increasingly being used to determine eukaryotic genomes. We used nanopore technology to generate chromosome-level assemblies for 3 different strains of Drechmeria coniospora, a nematophagous fungus used extensively in the study of innate immunity in Caenorhabditis elegans. RESULTS: One natural geographical isolate demonstrated high stability over decades, whereas a second isolate not only had a profoundly altered genome structure but exhibited extensive instability. We conducted an in-depth analysis of sequence errors within the 3 genomes and established that even with state-of-the-art tools, nanopore methods alone are insufficient to generate eukaryotic genome sequences of sufficient accuracy to merit inclusion in public databases. CONCLUSIONS: Although nanopore long-read sequencing is not accurate enough to produce publishable eukaryotic genomes, in our case, it has revealed new information about genome plasticity in D. coniospora and provided a backbone that will permit future detailed study to characterize gene evolution in this important model fungal pathogen.

Comparative Genomics Unveils Regionalized Evolution of the Faustovirus Genomes.

Faustovirus is a recently discovered genus of large DNA virus infecting the amoeba Vermamoeba vermiformis, which is phylogenetically related to Asfarviridae. To better understand the diversity and evolution of this viral group, we sequenced six novel Faustovirus strains, mined published metagenomic datasets and performed a comparative genomic analysis. Genomic sequences revealed three consistent phylogenetic groups, within which genetic diversity was moderate. The comparison of the major capsid protein (MCP) genes unveiled between 13 and 18 type-I introns that likely evolved through a still-active birth and death process mediated by intron-encoded homing endonucleases that began before the Faustovirus radiation. Genome-wide alignments indicated that despite genomes retaining high levels of gene collinearity, the central region containing the MCP gene together with the extremities of the chromosomes evolved at a faster rate due to increased indel accumulation and local rearrangements. The fluctuation of the nucleotide composition along the Faustovirus (FV) genomes is mostly imprinted by the consistent nucleotide bias of coding sequences and provided no evidence for a single DNA replication origin like in circular bacterial genomes.

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata.

BACKGROUND: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. RESULTS: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. CONCLUSIONS: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

Exploring the microbiome of the "star" freshwater diatom Asterionella formosa in a laboratory context.

Most of our knowledge on the mechanisms underlying diatom-bacterial interactions has been acquired through studies involving isolation of culturable partners. Here, we established a laboratory model of intermediate complexity between complex natural communities and laboratory pure culture models. We investigated the whole community formed by the freshwater diatom Asterionella formosa and its associated bacteria in a laboratory context, including both culturable and unculturable bacteria. Combining cellular and molecular approaches, we showed that in laboratory cultures, A. formosa microbiome was dynamic and comprised of numerous bacterial species (mainly Proteobacteria and Bacteroidetes). Using metagenomics, we explored several metabolic potentials present within the bacterial community. Our analyses suggested that bacteria were heterotrophic although a third of them (Alpha- and Beta-proteobacteria) could also be phototrophic. About 60% of the bacteria, phylogenetically diverse, could metabolize glycolate. The capacity to synthesize molecules such as B vitamins appeared unevenly distributed among bacteria. Altogether, our results brought insights into the bacterial diversity found in diatom-bacterial communities and hinted at metabolic interdependencies within the community that could result in diatom-bacterial and bacterial-bacterial interactions. The present work allowed us to explore the functional architecture of the bacterial community associated with A. formosa in culture and is complementary to field studies.

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.

Comparative Genomics of Chrysochromulina Ericina Virus and Other Microalga-Infecting Large DNA Viruses Highlights Their Intricate Evolutionary Relationship with the Established Mimiviridae Family.

Chrysochromulina ericina virus CeV-01B (CeV) was isolated from Norwegian coastal waters in 1998. Its icosahedral particle is 160 nm in diameter and encloses a 474-kb double-stranded DNA (dsDNA) genome. This virus, although infecting a microalga (the haptophyceae Haptolina ericina, formerly Chrysochromulina ericina), is phylogenetically related to members of the Mimiviridae family, initially established with the acanthamoeba-infecting mimivirus and megavirus as prototypes. This family was later split into two genera (Mimivirus and Cafeteriavirus) following the characterization of a virus infecting the heterotrophic stramenopile Cafeteria roenbergensis (CroV). CeV, as well as two of its close relatives, which infect the unicellular photosynthetic eukaryotes Phaeocystis globosa (Phaeocystis globosa virus [PgV]) and Aureococcus anophagefferens (Aureococcus anophagefferens virus [AaV]), are currently unclassified by the International Committee on Viral Taxonomy (ICTV). The detailed comparative analysis of the CeV genome presented here confirms the phylogenetic affinity of this emerging group of microalga-infecting viruses with the Mimiviridae but argues in favor of their classification inside a distinct clade within the family. Although CeV, PgV, and AaV share more common features among them than with the larger Mimiviridae, they also exhibit a large complement of unique genes, attesting to their complex evolutionary history. We identified several gene fusion events and cases of convergent evolution involving independent lateral gene acquisitions. Finally, CeV possesses an unusual number of inteins, some of which are closely related despite being inserted in nonhomologous genes. This appears to contradict the paradigm of allele-specific inteins and suggests that the Mimiviridae are especially efficient in spreading inteins while enlarging their repertoire of homing genes.IMPORTANCE Although it infects the microalga Chrysochromulina ericina, CeV is more closely related to acanthamoeba-infecting viruses of the Mimiviridae family than to any member of the Phycodnaviridae, the ICTV-approved family historically including all alga-infecting large dsDNA viruses. CeV, as well as its relatives that infect the microalgae Phaeocystic globosa (PgV) and Aureococcus anophagefferens (AaV), remains officially unclassified and a source of confusion in the literature. Our comparative analysis of the CeV genome in the context of this emerging group of alga-infecting viruses suggests that they belong to a distinct clade within the established Mimiviridae family. The presence of a large number of unique genes as well as specific gene fusion events, evolutionary convergences, and inteins integrated at unusual locations document the complex evolutionary history of the CeV lineage.

A Glimpse of Nucleo-Cytoplasmic Large DNA Virus Biodiversity through the Eukaryotic Genomics Window.

The nucleocytoplasmic large DNA viruses (NCLDV) are a group of extremely complex double-stranded DNA viruses, which are major parasites of a variety of eukaryotes. Recent studies showed that certain eukaryotes contain fragments of NCLDV DNA integrated in their genome, when surprisingly many of these organisms were not previously shown to be infected by NCLDVs. We performed an update survey of NCLDV genes hidden in eukaryotic sequences to measure the incidence of this phenomenon in common public sequence databases. A total of 66 eukaryotic genomic or transcriptomic datasets-many of which are from algae and aquatic protists-contained at least one of the five most consistently conserved NCLDV core genes. Phylogenetic study of the eukaryotic NCLDV-like sequences identified putative new members of already recognized viral families, as well as members of as yet unknown viral clades. Genomic evidence suggested that most of these sequences resulted from viral DNA integrations rather than contaminating viruses. Furthermore, the nature of the inserted viral genes helped predicting original functional capacities of the donor viruses. These insights confirm that genomic insertions of NCLDV DNA are common in eukaryotes and can be exploited to delineate the contours of NCLDV biodiversity.

Complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa.

We report the complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa. The large 61.9 kb circular sequence encodes 34 proteins and 25 tRNAs that are universally conserved in other sequenced diatoms. We fully resolved a unique 24 kb region containing highly conserved repeated sequence units, possibly collocating with an origin of replication.

Study of Gene Trafficking between Acanthamoeba and Giant Viruses Suggests an Undiscovered Family of Amoeba-Infecting Viruses.

The nucleocytoplasmic large DNA viruses (NCLDV) are a group of extremely complex double-stranded DNA viruses, which are major parasites of a variety of eukaryotes. Recent studies showed that certain unicellular eukaryotes contain fragments of NCLDV DNA integrated in their genome, when surprisingly many of these organisms were not previously shown to be infected by NCLDVs. These findings prompted us to search the genome of Acanthamoeba castellanii strain Neff (Neff), one of the most prolific hosts in the discovery of giant NCLDVs, for possible DNA inserts of viral origin. We report the identification of 267 markers of lateral gene transfer with viruses, approximately half of which are clustered in Neff genome regions of viral origins, transcriptionally inactive or exhibit nucleotide-composition signatures suggestive of a foreign origin. The integrated viral genes had diverse origin among relatives of viruses that infect Neff, including Mollivirus, Pandoravirus, Marseillevirus, Pithovirus, and Mimivirus However, phylogenetic analysis suggests the existence of a yet-undiscovered family of amoeba-infecting NCLDV in addition to the five already characterized. The active transcription of some apparently anciently integrated virus-like genes suggests that some viral genes might have been domesticated during the amoeba evolution. These insights confirm that genomic insertion of NCLDV DNA is a common theme in eukaryotes. This gene flow contributed fertilizing the eukaryotic gene repertoire and participated in the occurrence of orphan genes, a long standing issue in genomics. Search for viral inserts in eukaryotic genomes followed by environmental screening of the original viruses should be used to isolate radically new NCLDVs.

Giant viruses at the core of microscopic wars with global impacts.

The unicellular eukaryotes (also called protists) that inhabit the contemporary oceans have large impacts on major biogeochemical cycles. Populations of oceanic protists are to a large extent regulated by their viral parasites, especially nucleocytoplasmic large DNA viruses (NCLDVs). NCLDVs can themselves be the prey of smaller viruses called virophages and can also be infected by transposable elements termed transpovirons. These entangled parasitisms have fostered the emergence of sophisticated infection and defence strategies. In addition persistent contact has facilitated the exchange of genes between different parties. Recent advances shed light on the strategies that govern such microbial wars. Endogenous virophage-like elements found in the genome of a marine alga could for instance provide the host acquired immunity against NCLDVs. In return, it was recently speculated that virophage sequences can be hijacked by NCLDVs and used as genetic weapons against virophages.

Provirophages in the Bigelowiella genome bear testimony to past encounters with giant viruses.

Virophages are recently discovered double-stranded DNA virus satellites that prey on giant viruses (nucleocytoplasmic large DNA viruses; NCLDVs), which are themselves parasites of unicellular eukaryotes. This coupled parasitism can result in the indirect control of eukaryotic cell mortality by virophages. However, the details of such tripartite relationships remain largely unexplored. We have discovered ∼300 predicted genes of putative virophage origin in the nuclear genome of the unicellular alga Bigelowiella natans. Physical clustering of these genes indicates that virophage genomes are integrated into the B. natans genome. Virophage inserts show high levels of similarity and synteny between each other, indicating that they are closely related. Virophage genes are transcribed not only in the sequenced B. natans strain but also in other Bigelowiella isolates, suggesting that transcriptionally active virophage inserts are widespread in Bigelowiella populations. Evidence that B. natans is also a host to NCLDV members is provided by the identification of NCLDV inserts in its genome. These putative large DNA viruses may be infected by B. natans virophages. We also identify four repeated elements sharing structural and genetic similarities with transpovirons--a class of mobile elements first discovered in giant viruses--that were probably independently inserted in the B. natans genome. We argue that endogenized provirophages may be beneficial to both the virophage and B. natans by (i) increasing the chances for the virophage to coinfect the host cell with an NCLDV prey and (ii) defending the host cell against fatal NCLDV infections.

Yersinia ruckeri Biotypes 1 and 2 in France: presence and antibiotic susceptibility.

Yersinia ruckeri is the causative agent of yersiniosis, a disease reported in a number of fish species, especially rainbow trout. This study was undertaken to describe the phenotypes of Y. ruckeri on French rainbow trout farms. More than 100 isolates, collected during recent outbreaks on trout farms, were characterized by phenotypic tests, namely using biochemical tests of the API 20E system, serotyping, biotyping (tests for motility and lipase activity) and by describing the pattern of susceptibility to several antibiotics. The isolates showed a low phenotypic diversity with a prevalent serotype (O1) and API 20E profile 5 1(3)07 100. As in other European countries, Biotype 2 (BT2), which lacks both motility and secreted lipase activity, was found to be present in France. The emergence of 'French' BT2 was different than that observed for other European countries (Finland, Spain, Denmark and the UK). The antibiotic pattern was uniform for all isolates, regardless of the geographical area studied. The results indicate that no resistance has yet emerged, and the efficacy of the antibiotic generally used against yersiniosis in France, trimethoprim/sulfamethoxasol, is not compromised (minimum inhibitory concentrations [MIC] of between 0.016 and 0.128 µg ml-1). Enrofloxacin and doxycycline, not used as a first-line treatment in fish diseases, have reasonably good efficacies (with MICs ≤0.128 and 0.256, respectively).

Plant genomes enclose footprints of past infections by giant virus relatives.

Nucleocytoplasmic large DNA viruses (NCLDVs) are eukaryotic viruses with large genomes (100 kb-2.5 Mb), which include giant Mimivirus, Megavirus and Pandoravirus. NCLDVs are known to infect animals, protists and phytoplankton but were never described as pathogens of land plants. Here, we show that the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii have open reading frames (ORFs) with high phylogenetic affinities to NCLDV homologues. The P. patens genes are clustered in DNA stretches (up to 13 kb) containing up to 16 NCLDV-like ORFs. Molecular evolution analysis suggests that the NCLDV-like regions were acquired by horizontal gene transfer from distinct but closely related viruses that possibly define a new family of NCLDVs. Transcriptomics and DNA methylation data indicate that the NCLDV-like regions are transcriptionally inactive and are highly cytosine methylated through a mechanism not relying on small RNAs. Altogether, our data show that members of NCLDV have infected land plants.