Complete genome of the new bacilliform virus that causes Milky Hemolymph Syndrome in Chionoecetes bairdi (Rathbun, 1924).

The genome of a new member of the Nimaviridae family has been sequenced. The Chionoecetes bairdi bacilliform virus (CbBV) causes Milky Hemolymph Syndrome (MHS) in Chionoecetes bairdi populations of the Pacific coast of Kamchatka. The CbBV genome is represented by double-stranded DNA with a length of 245,567 nucleotides containing 120 ORFs. Of these, 85 proteins had significant matches in the NCBI database, and 57 genes encoded capsid, envelope, tegument and nonstructural proteins. Comparative analysis of the genomes of CbBV and a number of representatives of the class nuclear arthropod large DNA viruses (NALDVs) made it possible to isolate 49 evolutionarily conserved orthologue core genes. Among them, 5 were multicopy genes, and 44 were single-copy genes. There were ancestral genes characteristic of all Naldaviricetes - per os infectivity complex genes, one DNA polymerase gene and one thymidylate synthase gene. Phylogenetic analysis of representatives of the Nimaviridae family revealed that the CbBV and Chionoecetes opilio bacilliform virus (CoBV) form an independent clade within the family separate from the clade containing WSSV strains. This is supported by data on the order and arrangement of genes in the genomes of nimaviruses that were identical within each clade but differed between them. In addition, a high identity of the genomes and proteomes of CbBV and CoBV (approximately 99%) was shown, and their identity with WSSV strains was no more than 33%. The data on the structure of the genome of the new virus that causes MHS in C. bairdi indicate that it belongs to the family Nimaviridae, genus Whispovirus. Thus, the CbBV infecting the commercially important species of Tanner crab in populations of the Pacific coast of Kamchatka is the second "wild" representative of replicating nimaviruses whose genome has been characterized after the CoBV that causes MHS in C. opilio in populations of the Sea of Japan. The discovery of a new member of the family that infects decapods indicates the prevalence of nimaviruses in marine ecosystems. The information obtained is important for understanding the evolution of representatives of the class of nuclear arthropod large DNA viruses. The discovery of a new nimavirus that causes MHS in Chionoecetes crabs, in contrast to the white spot syndrome (WSS) caused by WSSV strains, makes it relevant to identify two variants and possibly species within the family, namely, WSSV and Milky Hemolymph Syndrome virus (MHSV).

Exploring Virus Diversity in the Potato leafhopper (Empoasca fabae), an Economically Important Agricultural Pest.

The potato leafhopper (Empoasca fabae, PLH) is a serious pest that feeds on a wide range of agricultural crops and is found throughout the United States but is not known to be a vector for plant-infecting viruses. We probed the diversity of virus sequences in field populations of PLH collected from four Midwestern states: Illinois, Indiana, Iowa, and Minnesota. High-throughput sequencing data from total RNAs extracted from PLH were used to assemble sequences of fifteen positive-stranded RNA viruses, two negative-stranded RNA viruses, and one DNA virus. These sequences included ten previously described plant viruses and eight putative insect-infecting viruses. All but one of the insect-specific viruses were novel and included three solemoviruses, one iflavirus, one phenuivirus, one lispivirus, and one ambidensovirus. Detailed analyses of the novel genome sequences and their evolutionary relationships with related family members were conducted. Our study revealed a diverse group of plant viruses circulating in the PLH population and discovered novel insect viruses, expanding knowledge on the untapped virus diversity in economically important crop pests. Our findings also highlight the importance of monitoring the emergence and circulation of plant-infecting viruses in agriculturally important arthropod pests.

Laboratory Replication of Ostreid Herpes Virus (OsHV-1) Using Pacific Oyster Tissue Explants.

Pacific oysters (Crassostrea or Magallana gigas) are one of the most economically important aquaculture species globally. Over the past two decades, ostreid herpesvirus (OsHV-1) has become a major pathogen of cultured Pacific oysters, resulting in widespread mortality with a global distribution. Experimental use of OsHV-1 is challenging for many reasons, including both complexity of host-pathogen dynamics and a lack of functioning model systems. The goal of this study was to improve the tools available for working with OsHV-1 in both whole animals and in tissue explants established from oysters maintained in controlled laboratory conditions. Tissue explants were taken from oysters originating from two different sources that have different levels of mortality in experimental OsHV-1 infections and were exposed to OsHV-1. A whole-animal infection experiment was run concurrently as a comparison. Quantitative PCR and electron microscopy were used to confirm that the explants were capable of replicating OsHV-1. Furthermore, the quantitative PCR results suggest that the source of the oysters was significant in determining the outcome of infection in the explants, supporting the validity of the explant model for OsHV-1 infection. This tissue explant approach for studying OsHV-1 allows for the control of confounding factors in the disease outcome that is not possible in whole-animal experiments, providing a new tool for the study of OsHV-1 in Pacific oysters.

Complex Genomes of Early Nucleocytoviruses Revealed by Ancient Origins of Viral Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (aaRSs), also known as tRNA ligases, are essential enzymes in translation. Owing to their functional essentiality, these enzymes are conserved in all domains of life and used as informative markers to trace the evolutionary history of cellular organisms. Unlike cellular organisms, viruses generally lack aaRSs because of their obligate parasitic nature, but several large and giant DNA viruses in the phylum Nucleocytoviricota encode aaRSs in their genomes. The discovery of viral aaRSs led to the idea that the phylogenetic analysis of aaRSs can shed light on ancient viral evolution. However, conflicting results have been reported from previous phylogenetic studies: one posited that nucleocytoviruses recently acquired their aaRSs from their host eukaryotes, while another hypothesized that the viral aaRSs have ancient origins. Here, we investigated 4,168 nucleocytovirus genomes, including metagenome-assembled genomes (MAGs) derived from large-scale metagenomic studies. In total, we identified 780 viral aaRS sequences in 273 viral genomes. We generated and examined phylogenetic trees of these aaRSs with a large set of cellular sequences to trace evolutionary relationships between viral and cellular aaRSs. The analyses suggest that the origins of some viral aaRSs predate the last common eukaryotic ancestor. Inside viral aaRS clades, we identify intricate evolutionary trajectories of viral aaRSs with horizontal transfers, losses, and displacements. Overall, these results suggest that ancestral nucleocytoviruses already developed complex genomes with an expanded set of aaRSs in the proto-eukaryotic era.

A systematic review on current approaches in bat virus discovered between 2018 and 2022.

Zoonotic viruses are widely seen as the primary threat for future pandemics. Bats are the most diverse group of mammals, with more than 1400 species distributed across most habitats on Earth. So far, 31 known virus families were associated with bats, although the understanding of most viruses were insufficient. Continuous efforts to discover, understand and monitor these bats viruses, is thereby an area of public health interest. This systematic review was designed to catalogue publications reporting novel bat virus discoveries within PubMed, SCOPUS, and Web of Science databases, within a 5-year period from 2018 to 2022. Various experimental parameters, including sampling locations, methodology, bat species diversity, similarity to known viruses, species demarcation of new viruses, and genomic sequencing strategies, were extracted from 41 publications and analyzed. In total, 72 novel viruses from 19 virus families were identified between 2018 and 2022, particularly from Genomoviridae (DNA viruses) and Coronaviridae (RNA viruses). That said, only a limited number of bat families featured extensively despite noticeable shift towards next generation sequencing methods and metagenomics pipeline for virus identification across different sampling methods. This review aims to provide a comprehensive analysis of the global efforts made over the past five years to identify and characterize emerging viruses in bat species, and to provide a detailed overview of the current technologies and methodologies used in these studies.

The Study of Metalloproteome of DNA Viruses: Identification, Functional Annotation, and Diversity Analysis of Viral Metal-Binding Proteins.

Metalloproteins are fundamental to diverse biological processes but still lack extensive investigation in viral contexts. This study reveals the prevalence and functional diversity of metal-binding proteins in DNA viruses. Among a subset of 1432 metalloproteins, zinc and magnesium-binding proteins are notably abundant, indicating their importance in viral biology. Furthermore, significant numbers of proteins binding to iron, manganese, copper, nickel, mercury, and cadmium were also detected. Human-infecting viral proteins displayed a rich landscape of metalloproteins, with MeBiPred (964 proteins) and Pfam (666) yielding the highest numbers. Interestingly, many essential viral proteins exhibited metal-binding capabilities, including polymerases, DNA binding proteins, helicases, dUPTase, thymidine kinase, and various structural and accessory proteins. This study sheds light on the ubiquitous presence of metalloproteins, their functional signatures, subcellular placements, and metal-utilization patterns, providing valuable insights into viral biology. A similar metal utilization pattern was observed in similar functional proteins across the various DNA viruses. Furthermore, these findings provide a foundation for identifying potential drug targets for combating viral infections.

2024 Smacoviridae family update: 59 new species in seven genera.

Family Smacoviridae (order Cremevirales, class Arfiviricetes, phylum Cressdnaviricota) comprises viruses with small circular genomes of ~2300-3000 nt in length that encode at least two proteins, the rolling-circle replication associated protein (Rep) and the capsid protein (CP). Smacovirids have been discovered in fecal samples of various animals and display remarkable sequence diversity. Here, we provide an overview of the genomic properties of classified smacovirids and report on the latest taxonomy update in the family Smacoviridae. The family has been expanded by 59 new species in the genera Porprismacovirus (n = 25), Inpeasmacovirus (n = 1), Felismacovirus (n = 22), Drosmacovirus (n = 4), Dragsmacovirus (n = 2), Bovismacovirus (n = 4), and Bonzesmacovirus (n = 1) and currently includes 12 genera with 143 species officially recognized by the International Committee on Taxonomy of Viruses (ICTV).

Temperature modulates dominance of a superinfecting Arctic virus in its unicellular algal host.

Complex virus-virus interactions can arise when multiple viruses coinfect the same host, impacting infection outcomes with broader ecological and evolutionary significance for viruses and host. Yet, our knowledge regarding virus competition is still limited, especially for single-celled eukaryotic host-virus systems. Here, we report on mutual interference of two dsDNA viruses, MpoV-45T and MpoV-46T, competing for their Arctic algal host Micromonas polaris. Both viruses affected each other's gene expression and displayed reduced genome replication during coinfection. MpoV-45T was the dominant virus, likely due to interference in the DNA replication of is competitor. Even when its coinfection was delayed, the dominant virus still prevailed while genome production of the other virus was strongly suppressed. This contrasts with typical superinfection exclusion, where the primary infection prevents secondary infection by other viruses. Higher temperature made the suppressed virus a stronger competitor, signifying that global warming is likely to alter virus-virus interactions in Arctic waters.

Characterization of CRESS-DNA viruses in human vaginal secretions: An exploratory metagenomic investigation.

The Phylum Cressdnaviricota consists of a large number of circular Rep-encoding single-stranded (CRESS)-DNA viruses. Recently, metagenomic analyzes revealed their ubiquitous distribution in a diverse range of eukaryotes. Data relating to CRESS-DNA viruses in humans remains scarce. Our study investigated the presence and genetic diversity of CRESS-DNA viruses in human vaginal secretions. Vaginal swabs were collected from 28 women between 29 and 43 years old attending a fertility clinic in New York City. An exploratory metagenomic analysis was performed and detection of CRESS-DNA viruses was confirmed through analysis of near full-length sequences of the viral isolates. A phylogenetic tree was based on the REP open reading frame sequences of the CRESS-DNA virus genome. Eleven nearly complete CRESS-DNA viral genomes were identified in 16 (57.1%) women. There were no associations between the presence of these viruses and any demographic or clinical parameters. Phylogenetic analysis indicated that one of the sequences belonged to the genus Gemycircularvirus within the Genomoviridae family, while ten sequences represented previously unclassified species of CRESS-DNA viruses. Novel species of CRESS-DNA viruses are present in the vaginal tract of adult women. Although they be transient commensal agents, the potential clinical implications for their presence at this site cannot be dismissed.

Cutavirus Infection in Large-Plaque Parapsoriasis, a Premalignant Condition of Mycosis Fungoides.

BACKGROUND: Cutavirus (CuV) is associated with mycosis fungoides; however, the CuV status in parapsoriasis en plaques (PP), a premalignant inflammatory condition of mycosis fungoides, has not been fully delineated. METHODS: Fifty-five Japanese patients with chronic inflammatory skin diseases, including 13 patients with PP, were studied. RESULTS: CuV DNA was detected significantly more frequently in biopsies of the lesional skin from patients with PP (38%; 4 of 13) than in those from patients with other inflammatory skin diseases (2%; 1 of 42; P = .009). All CuV-positive PP cases were of the large-plaque parapsoriasis (LPP) subtype. The viral loads ranged from 83 450 to 2 164 170 copies/103 cells. We recovered near-full-length CuV sequences from the CuV-positive LPP biopsies, all of which were of the Japanese/Asian genotype. The CuV genome appeared to be present within lymphoid cells infiltrating the epidermis and dermis. CuV NS1 and VP1 gene transcripts were also detected in the affected tissues. CONCLUSIONS: The detection of high levels of CuV DNA with the expression of viral mRNA suggests a potential role for CuV in the pathogenesis of LPP, making it necessary to study further the impact of CuV, especially regarding the viral genotype, on the outcomes of patients with CuV-positive LPP.

Transient, context-dependent fitness costs accompanying viral resistance in isolates of the marine microalga Micromonas sp. (class Mamiellophyceae).

Marine microbes are important in biogeochemical cycling, but the nature and magnitude of their contributions are influenced by their associated viruses. In the presence of a lytic virus, cells that have evolved resistance to infection have an obvious fitness advantage over relatives that remain susceptible. However, susceptible cells remain extant in the wild, implying that the evolution of a fitness advantage in one dimension (virus resistance) must be accompanied by a fitness cost in another dimension. Identifying costs of resistance is challenging because fitness is context-dependent. We examined the context dependence of fitness costs in isolates of the picophytoplankton genus Micromonas and their co-occurring dsDNA viruses using experimental evolution. After generating 88 resistant lineages from two ancestral Micromonas strains, each challenged with one of four distinct viral strains, we found resistance led to a 46% decrease in mean growth rate under high irradiance and a 19% decrease under low. After a year in culture, the experimentally selected lines remained resistant, but fitness costs had attenuated. Our results suggest that the cost of resistance in Micromonas is dependent on environmental conditions and the duration of population adaptation, illustrating the dynamic nature of fitness costs of viral resistance among marine protists.

DNA Virome in Cardiac Tissue from Green Sea Turtles (Chelonia mydas) with Myocarditis.

As part of a sea turtle health monitoring program on the central east coast of Queensland, Australia, stranded and sick green sea turtles (Chelonia mydas) were subjected to necropsy and histopathology. A subset of these turtles had myocarditis of varying severity, which could not be attributed to parasitism by spirorchid flukes or bacterial infections. We, therefore, undertook an investigation to determine whether virus infections might be part of the pathogenesis. Deep sequencing revealed abundant DNA virus contigs in the heart tissue, of which CRESS and circoviruses appeared to be the most consistently present. Further analysis revealed the homology of some of the circoviruses to the beak and feather disease virus. While a causative link to myocarditis could not be established, the presence of these viruses may play a contributing role by affecting the immune system and overall health of animals exposed to pollutants, higher water temperatures, and decreasing nutrition.

[Comparative analysis of the taxonomic classification criteria for a number of groups of pathogenic DNA and RNA viruses based on genomic data].

The basis for criteria of the taxonomic classification of DNA and RNA viruses based on data of the genomic sequencing are viewed in this review. The genomic sequences of viruses, which have genome represented by double-stranded DNA (orthopoxviruses as example), positive-sense single-stranded RNA (alphaviruses and flaviviruses as example), non-segmented negative-sense single-stranded RNA (filoviruses as example), segmented negative-sense single-stranded RNA (arenaviruses and phleboviruses as example) are analyzed. The levels of genetic variability that determine the assignment of compared viruses to taxa of various orders are established for each group of viruses.

The plasma viral communities associate with clinical profiles in a large-scale haematological patients cohort.

BACKGROUND: Haematological patients exhibit immune system abnormalities that make them susceptible to viral infections. Understanding the relationship between the virome in the blood plasma of haematological patients and their clinical characteristic is crucial for disease management. We aimed to explore the presence of viral pathogens and identify close associations between viral infections and various clinical features. RESULTS: A total of 21 DNA viruses and 6 RNA viruses from 12 virus families were identified from 1383 patients. Patients with haematological diseases exhibited significantly higher diversity, prevalence, and co-detection rates of viral pathogens. During fever episodes, pathogen detection was notably higher, with Epstein-Barr virus (EBV) and Mucorales infections being the most probable culprits for fever symptoms in non-haematological patients. The detection rate of torque teno virus (TTV) significantly increases in haematological patients after transplantation and during primary lung infections. Additionally, TTV-positive patients demonstrate significantly higher absolute neutrophil counts, while C-reactive protein and procalcitonin levels are notably lower. Furthermore, TTV, cytomegalovirus, and parvovirus B19 (B19V) were found to be more prevalent in non-neutropenic patients, while non-viral pathogenic infections, such as Gram-negative bacteria and Mucorales, were more common in neutropenic patients. Pegivirus C (HPgV-C) infection often occurred post-transplantation, regardless of neutropenia. Additionally, some viruses such as TTV, B19V, EBV, and HPgV-C showed preferences for age and seasonal infections. CONCLUSIONS: Analysis of the plasma virome revealed the susceptibility of haematological patients to plasma viral infections at specific disease stages, along with the occurrence of mixed infections with non-viral pathogens. Close associations were observed between the plasma virome and various clinical characteristics, as well as clinical detection parameters. Understanding plasma virome aids in auxiliary clinical diagnosis and treatment, enabling early prevention to reduce infection rates in patients and improve their quality of life. Video Abstract.

ICTV Virus Taxonomy Profile: Turriviridae 2024.

The family Turriviridae includes viruses with a dsDNA genome of 16-17 kbp. Virions are spherical with a diameter of approximately 75 nm and comprise a host-derived internal lipid membrane surrounded by a proteinaceous capsid shell. Members of the family Turriviridae infect extremophilic archaea of the genera Sulfolobus and Saccharolobus. Viral infection results in cell lysis for Sulfolobus turreted icosahedral virus 1 infection but other members of the family can be temperate. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Turriviridae, which is available at ictv.global/report/turriviridae.

Full-genome sequencing of dozens of new DNA viruses found in Spanish bat feces.

Bats are natural hosts of multiple viruses, many of which have clear zoonotic potential. The search for emerging viruses has been aided by the implementation of metagenomic tools, which have also enabled the detection of unprecedented viral diversity. Currently, this search is mainly focused on RNA viruses, which are largely over-represented in databases. To compensate for this research bias, we analyzed fecal samples from 189 Spanish bats belonging to 22 different species using viral metagenomics. This allowed us to identify 52 complete or near-complete viral genomes belonging to the families Adenoviridae, Circoviridae, Genomoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae and Smacoviridae. Of these, 30 could constitute new species, doubling the number of viruses currently described in Europe. These findings open the door to a more thorough analysis of bat DNA viruses and their zoonotic potential. IMPORTANCE: Metagenomics has become a fundamental tool to characterize the global virosphere, allowing us not only to understand the existing viral diversity and its ecological implications but also to identify new and emerging viruses. RNA viruses have a higher zoonotic potential, but this risk is also present for some DNA virus families. In our study, we analyzed the DNA fraction of fecal samples from 22 Spanish bat species, identifying 52 complete or near-complete genomes of different viral families with zoonotic potential. This doubles the number of genomes currently described in Europe. Metagenomic data often produce partial genomes that can be difficult to analyze. Our work, however, has characterized a large number of complete genomes, thus facilitating their taxonomic classification and enabling different analyses to be carried out to evaluate their zoonotic potential. For example, recombination studies are relevant since this phenomenon could play a major role in cross-species transmission.

Identifying Protein Interactions with Viral DNA Genomes during Virus Infection.

Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.

Reconstructing Prehistoric Viral Genomes from Neanderthal Sequencing Data.

DNA viruses that produce persistent infections have been proposed as potential causes for the extinction of Neanderthals, and, therefore, the identification of viral genome remnants in Neanderthal sequence reads is an initial step to address this hypothesis. Here, as proof of concept, we searched for viral remnants in sequence reads of Neanderthal genome data by mapping to adenovirus, herpesvirus and papillomavirus, which are double-stranded DNA viruses that may establish lifelong latency and can produce persistent infections. The reconstructed ancient viral genomes of adenovirus, herpesvirus and papillomavirus revealed conserved segments, with nucleotide identity to extant viral genomes and variable regions in coding regions with substantial divergence to extant close relatives. Sequence reads mapped to extant viral genomes showed deamination patterns of ancient DNA, and these ancient viral genomes showed divergence consistent with the age of these samples (≈50,000 years) and viral evolutionary rates (10-5 to 10-8 substitutions/site/year). Analysis of random effects showed that the Neanderthal mapping to genomes of extant persistent viruses is above what is expected by random similarities of short reads. Also, negative control with a nonpersistent DNA virus does not yield statistically significant assemblies. This work demonstrates the feasibility of identifying viral genome remnants in archaeological samples with signal-to-noise assessment.

Mastering Death: The Roles of Viral Bcl-2 in dsDNA Viruses.

Proteins of the Bcl-2 family regulate cellular fate via multiple mechanisms including apoptosis, autophagy, senescence, metabolism, inflammation, redox homeostasis, and calcium flux. There are several regulated cell death (RCD) pathways, including apoptosis and autophagy, that use distinct molecular mechanisms to elicit the death response. However, the same proteins/genes may be deployed in multiple biochemical pathways. In apoptosis, Bcl-2 proteins control the integrity of the mitochondrial outer membrane (MOM) by regulating the formation of pores in the MOM and apoptotic cell death. A number of prosurvival genes populate the genomes of viruses including those of the pro-survival Bcl-2 family. Viral Bcl-2 proteins are sequence and structural homologs of their cellular counterparts and interact with cellular proteins in apoptotic and autophagic pathways, potentially allowing them to modulate these pathways and determine cellular fate.

Mriyaviruses: small relatives of giant viruses.

The phylum Nucleocytoviricota consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35-45 kb that appear to be related to the Nucleocytoviricota. In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCPs) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the Nucleocytoviricota and accordingly would comprise a class which we propose to name "Mriyaviricetes" (after Ukrainian "mriya," dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the Nucleocytoviricota. In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family Yaraviridae and proposed new family "Gamadviridae." The previously characterized members of these families, yaravirus and Pleurochrysis sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the Nucleocytoviricota, and RNA polymerase subunits encoded by all cytoplasmic viruses among the Nucleocytoviricota, suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism. IMPORTANCE: The origin of giant viruses of eukaryotes that belong to the phylum Nucleocytoviricota is not thoroughly understood and remains a matter of major interest and debate. Here, we combine metagenome database searches with extensive protein sequence and structure analysis to describe a distinct group of viruses with comparatively small genomes of 35-45 kilobases that appear to comprise a distinct class within the phylum Nucleocytoviricota that we provisionally named "Mriyaviricetes." Mriyaviruses appear to be the closest identified relatives of the ancestors of the Nucleocytoviricota. Analysis of proteins encoded in mriyavirus genomes suggests that they replicate their genome via the rolling circle mechanism that is unusual among viruses with double-stranded DNA genomes and so far not described for members of Nucleocytoviricota.