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HUH endonucleases of the Rep (replication protein) class mediate the replication of highly diverse plasmids and viral genomes across all domains of life. HUH transposases have independently evolved from Reps, giving rise to three major transposable element groups: the prokaryotic insertion sequences IS200/IS605 and IS91/ISCR, and the eukaryotic Helitrons. Here, I present Replitrons, a second group of eukaryotic transposons encoding Rep HUH endonuclease. Replitron transposases feature a Rep domain with one catalytic Tyr (Y1) and an adjacent domain that may function in oligomerization, contrasting with Helitron transposases that feature Rep with two Tyr (Y2) and a fused helicase domain (i.e., RepHel). Protein clustering found no link between Replitron transposases and described HUH transposases, and instead recovered a weak association with Reps of circular Rep-encoding single-stranded (CRESS) DNA viruses and their related plasmids (pCRESS). The predicted tertiary structure of the transposase of Replitron-1, the founding member of the group that is active in the green alga Chlamydomonas reinhardtii, closely resembles that of CRESS- DNA viruses and other HUH endonucleases. Replitrons are present in at least three eukaryotic supergroups and reach high copy numbers in nonseed plant genomes. Replitron DNA sequences feature short direct repeats at, or potentially near, their termini. Finally, I characterize copy-and-paste de novo insertions of Replitron-1 using long-read sequencing of C. reinhardtii experimental lines. These results support an ancient and evolutionarily independent origin of Replitrons, in line with other major groups of eukaryotic transposons. This work expands the known diversity of both transposons and HUH endonucleases in eukaryotes.

Viruses that infect single-celled algae strongly regulate microalgae growth and community composition through cell lysis, enable nutrient recycling in marine ecosystems, and offer valuable insights into early stages of viral evolution. One major group, the Bacilladnaviridae family of single-stranded DNA viruses, infects diatoms in marine environments. Here, we present the capsid structure of Chaetoceros lorenzianus DNA virus (ClorDNAV, Protobacilladnavirus chaelor ) determined at 2.2 Å resolution, thereby expanding the known structural diversity within the Cressdnaviricota phylum. The ClorDNAV capsid protein (CP) contains a conserved jelly-roll fold and a surface-exposed projection domain, with both N- and C-termini oriented toward the capsid interior. A low-resolution reconstruction of the genome revealed a spooled arrangement of the outer DNA layer, similar to that observed in Chaetoceros tenuissimus DNA virus type II (CtenDNAV-II). Structural comparison with CtenDNAV-II revealed five key CP differences: the absence of surface-exposed C-terminal tails in ClorDNAV, the presence of a helical domain, differences in the projection domain conformation, variation in the number of β-strands in the jelly-roll fold, and the lack of ion-attributed densities at subunit interfaces. Together with the genome reconstruction, these findings underscore the importance of experimentally determined structures for understanding viral architecture and evolution. To complement these results, we analyzed AlphaFold3-predicted CPs from all classified Bacilladnaviridae genera. These models confirmed the conservation of the jelly-roll fold across the family while revealing variability in the surface-exposed and terminal regions, likely reflecting host-specific adaptations and genome packaging strategies. Together, the experimental and predicted structures provide a comprehensive view of structural conservation and divergence in Bacilladnaviridae . Furthermore, the results provide additional structural evidence for the evolution of ssDNA Bacilladnaviridae from a noda-like ssRNA virus ancestor and suggest a shared genome organization resembling that of double-stranded viruses.

Circoviruses (CVs) infect a wide range of avian hosts, including domestic, ornamental and wild birds. The immunosuppressive effect of these viruses could make the host more susceptible to other pathogens. This study aimed to assess the potential hosts and genetic features of CVs or other related viruses in wild birds. Cloacal swab samples of 588 birds were processed for screening of circoviral sequences using nested PCR. Altogether, 19 complete genome and 21 partial sequences of small, circular, rep-encoding DNA viruses were determined, 32 of which belonged to CVs. Along with some newly established CV species (virus name long-eared owl-associated CV1 and barn owl-associated CV1), genomic sequences of previously characterized avian CVs (pigeon CV, duck CV, goose CV, gull CV, swan CV and little bittern CV) were identified. Pathogenic CVs and CVs of unknown aetiology occur in wild birds taxonomically distant from the originally described host species, such as the duck CV and pigeon CV in white stork ( Ciconia ciconia ), as well as pigeon CV in peregrine falcon ( Falco peregrinus ) and black-headed gull ( Chroicocephalus ridibundus ). The results draw attention to the widespread distribution of these viruses among wild birds which, by hiding in the host and reducing defensibility, could pose a threat to both poultry farming and efforts to wild bird conservation.

Oysters that filter feed can accumulate numerous pathogens, including viruses, which can serve as a valuable viral repository. As oyster farming becomes more prevalent, concerns are mounting about diseases that can harm both cultivated and wild oysters. Unfortunately, there is a lack of research on the viruses and other factors that can cause illness in shellfish. This means that it is harder to find ways to prevent these diseases and protect the oysters. This is part of a previously started project, the Dataset of Oyster Virome, in which we further study 30 almost complete genomes of oyster-associated CRESS DNA viruses. The replication-associated proteins and capsid proteins found in CRESS DNA viruses display varying evolutionary rates and frequently undergo recombination. Additionally, some CRESS DNA viruses have the capability for cross-species transmission. A plethora of unclassified CRESS DNA viruses are detectable in transcriptome libraries, exhibiting higher levels of transcriptional activity than those found in metagenome libraries. The study significantly enhances our understanding of the diversity of oyster-associated CRESS DNA viruses, emphasizing the widespread presence of CRESS DNA viruses in the natural environment and the substantial portion of CRESS DNA viruses that remain unidentified. This study’s findings provide a basis for further research on the biological and ecological roles of viruses in oysters and their environment.

Numerous metagenomic studies have uncovered a remarkable diversity of circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses, the majority of which are uncultured and unclassified. Unlike capsid proteins, the Reps show significant similarity across different groups of CRESS DNA viruses and have conserved domain organization with the N-terminal nuclease and the C-terminal helicase domain. Consequently, Rep is widely used as a marker for identification, classification and assessment of the diversity of CRESS DNA viruses. However, it has been shown that in certain viruses the Rep nuclease and helicase domains display incongruent evolutionary histories. Here, we systematically evaluated the co-evolutionary patterns of the two Rep domains across classified and unclassified CRESS DNA viruses. Our analysis indicates that the Reps encoded by members of the families Bacilladnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Nanoviridae and Smacoviridae display largely congruent evolutionary patterns in the two domains. By contrast, among the unclassified CRESS DNA viruses, 71% appear to have chimeric Reps. Such massive chimerism suggests that unclassified CRESS DNA viruses represent a dynamic population in which exchange of gene fragments encoding the nuclease and helicase domains is extremely common. Furthermore, purging of the chimeric sequences uncovered six monophyletic Rep groups that may represent new families of CRESS DNA viruses.

Background Musk deer can produce musk which has high medicinal value and is closely related to human health. Viruses in forest musk deer both threaten the health of forest musk deer and human beings. Methods Using viral metagenomics we investigated the virome in 85 faeces samples collected from forest musk deer. Results In this article, eight novel CRESS-DNA viruses were characterized, whole genomes were 2148 nt–3852 nt in length. Phylogenetic analysis indicated that some viral genomes were part of four different groups of CRESS-DNA virus belonging in the unclassified CRESS-DNA virus, Smacoviridae , pCPa-like virus and pPAPh2-like virus. UJSL001 (MN621482), UJSL003 (MN621469) and UJSL017 (MN621476) fall into the branch of unclassified CRESS-DNA virus (CRESSV1–2), UJSL002 (MN621468), UJSL004 (MN621481) and UJSL007 (MN621470) belong to the cluster of Smacoviridae , UJSL005 (MN604398) showing close relationship with pCPa-like (pCRESS4–8) clusters and UJSL006 (MN621480) clustered into the branch of pPAPh2-like (pCRESS9) virus, respectively. Conclusion The virome in faeces samples of forest musk deer from Chengdu, Sichuan province, China was revealed, which further characterized the diversity of viruses in forest musk deer intestinal tract.

This study identified and characterised three Genomoviruses during a circular DNA-enriched sequencing project aimed at assessing the evolution of Cassava mosaic begomoviruses in Nigeria. Using a combination of rolling circle amplification, Oxford Nanopore Sequencing and targeted amplicon sequencing, three full-length Genomovirus genomes were recovered. The recovered genomes ranged from 2090 to 2188 nucleotides in length, contained two open reading frames (Rep and CP) in an ambisense orientation and shared between 84.81 and 95.37% nucleotide similarity with isolates in the NCBI GenBank repository. Motif analyses confirmed the presence of conserved rolling circle replication (RCR) and helicase motifs in all three isolates; however, one isolate lacked the RCR II motif. Phylogenetic inference using Rep and CP nucleotide sequences suggested that the isolates belonged to a divergent lineage within the Genomovirus family. These findings expand current knowledge of Genomovirus diversity and highlight the potential of cassava as a source for identifying novel CRESS-DNA viruses.

Viruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and they appear to have multiple origins from prokaryotic plasmids. A subset of CRESS-DNA viruses, the cruciviruses, have homologues of capsid proteins encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability. The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viruses that encode capsid proteins that are most closely related to those encoded by RNA viruses in the family Tombusviridae . The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of capsid genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and 10 capsid-encoding circular genetic elements through de novo assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication-associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins and that the exchange of Rep protein domains between cruciviruses is rarer than intergenic recombination. Additionally, we suggest members of the stramenopiles/alveolates/Rhizaria supergroup as possible crucivirus hosts. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses. IMPORTANCE Viruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and they appear to have multiple origins from prokaryotic plasmids. A subset of CRESS-DNA viruses, the cruciviruses, have homologues of capsid proteins encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.

Capybaras (Hydrochoerus hydrochaeris), the world’s largest rodents, are distributed throughout South America. These wild herbivores are commonly found near water bodies and are well adapted to rural and urban areas. There is limited information on the viruses circulating through capybaras. This study aimed to expand the knowledge on the viral diversity associated with capybaras by sampling their faeces. Using a viral metagenomics approach, we identified diverse single-stranded DNA viruses in the capybara faeces sampled in the Distrito Federal, Brazil. A total of 148 complete genomes of viruses in the Microviridae family were identified. In addition, 14 genomoviruses (family Genomoviridae), a novel cyclovirus (family Circoviridae), and a smacovirus (family Smacoviridae) were identified. Also, 37 diverse viruses that cannot be assigned to known families and more broadly referred to as unclassified circular replication associated protein encoding single-stranded (CRESS) DNA viruses were identified. This study provides a snapshot of the viral diversity associated with capybaras that may be infectious to these animals or associated with their microbiota or diet.

Background Faced with the recrudescence of viral CRESS-DNA plant diseases, the availability of efficient and cost-effective tools for routine diagnosis and genomic characterisation is vital. As these viruses possess circular single-strand DNA genomes, they have been routinely characterised using rolling circle amplification (RCA) coupled with Sanger sequencing. However, while providing the basis of our knowledge of the diverse CRESS-DNA viruses, this approach is laboratory-intensive, time-consuming and ultimately ineffective faced with co-infection or viruses with multiple genomic components, two common characteristics of these viruses. Whereas alternatives have proved effective in some applications, there is a strong need for next-generation sequencing methods suitable for small-scale projects that can routinely produce high quality sequences comparable to the gold standard Sanger sequencing. Results Here, we present an RCA sequencing diagnostic technique using the latest Oxford Nanopore Technology flongle flow cells. Originally, using the tandem-repeat nature of RCA products, we were able to improve the quality of each viral read and assemble high-quality genomic components. The effectiveness of the method was demonstrated on two plant samples, one infected with the bipartite begomovirus African cassava mosaic virus (ACMV) and the other infected with the nanovirus faba bean necrotic stunt virus (FBNSV), a virus with eight genomic segments. This method allow us to recover all genomic components of both viruses. The assembled genomes of ACMV and FBNSV shared 100% nucleotide identity with those obtained with Sanger sequencing. Additionally, our experiments demonstrated that for similar-sized components, the number of reads was proportional to the segment frequencies measured using qPCR. Conclusion In this study, we demonstrated an accessible and effective Nanopore-based method for high-quality genomic characterisation of CRESS-DNA viruses, comparable to Sanger sequencing. Face with of increasing challenges posed by viral CRESS-DNA plant diseases, integrating this approach into routine workflows could pave the way for more proactive responses to viral epidemics.