Endogenous Viral Elements Research Articles (Page 1)

Abstract Summary Endogenous viral elements (EVEs) offer valuable insights into virus and host evolution, but their detection remains computationally and biologically challenging. We present HI-FEVER, a user-friendly Nextflow pipeline for the discovery of EVEs in eukaryotic host genomes. HI-FEVER is highly parallelizable and customizable, ensuring computational efficiency while allowing researchers to fine-tune parameters to their specific needs. Its output provides a comprehensive analysis of discovered EVEs, including detailed annotations which can provide evolutionary insights. HI-FEVER scales seamlessly to handle millions of viral protein queries across multiple host genomes on both laptops and high-performance computing nodes. Availability and Implementation The HI-FEVER source code is available on GitHub at https://github.com/PaleovirologyLab/hi-fever. Minimal reference databases, test datasets and benchmarking results are hosted on the Open Science Framework at https://osf.io/y357r. A detailed wiki is available at https://github.com/PaleovirologyLab/hi-fever/wiki, including usage instructions, parameter descriptions, and guidance on interpreting outputs. The pipeline includes a Pixi environment compatible with Conda and Apptainer containerization, and Docker images. HI-FEVER has been tested on Linux, Windows (via WSL2), and macOS (Intel and ARM64). Supplementary Information Supplementary data are available at Bioinformatics online.

Ants, as highly eusocial insects, play vital roles in ecosystems worldwide. While numerous RNA viruses have been documented in ants, no double-stranded DNA (dsDNA) virus has previously been confirmed to infect them. Labial gland disease, reported for decades, lacks a clearly defined cause until now. Here, we identify and characterize a large filamentous dsDNA virus, Camponotus japonicus labial gland disease virus (CjLGDV), from the swollen labial gland of C. japonicus, and a closely related Anoplolepis gracilipes labial gland disease virus in A. gracilipes. Phylogenetic and genomic analyses of the two viruses support the establishment of a new viral family within the order Lefavirales, class Naldaviricetes. The discovery of endogenous viral elements related to CjLGDV in multiple ant genomes suggests the historical infection of CjLGDV-like viruses in ants. These findings broaden the known host range of naldaviricetes and shed new light on the diversity, evolution, and host interaction of large dsDNA viruses in arthropods.

A vast diversity of RNA viruses has been uncovered in agricultural pest insects over the past decade through unbiased metatranscriptomics approaches. These viruses, known as insect-specific viruses (ISVs), are restricted to insects and cannot replicate in plant hosts. The discovery of plant virus-associated ISVs, along with endogenous viral elements derived from these viruses, offers valuable insights into the evolutionary relationships between ISVs, plant viruses, and their insect hosts. Moreover, ISVs may be pathogenic or influence host biology, potentially affecting vector competence and the virulence of other pathogens in their host insects. This finding has opened new possibilities for exploring nonbaculoviral ISVs as novel biological control agents for insect pests and plant viral diseases. This review offers a concise overview of ISVs, with a focus on insect RNA viruses in agricultural pest insects, and proposes guidelines for future research in this rapidly advancing field.

Public databases of protein sequences, such as the National Center for Biotechnology Information (NCBI) Protein repository and UniProt, contain millions of proteins identified in samples from specific species but named as uncharacterized or hypothetical due to a lack of information about their function. Many such sequences are actually derived from RNA viruses, either due to viral infection of the original sample, contamination, or endogenous viral elements (EVEs) integrated into the genome of the sample species. Many proteins from RNA virus discovery research are also deposited into these repositories but are labelled as uncharacterized and only classified taxonomically at a superkingdom or realm level. Sequences from protein repositories not labelled specifically as being derived from the RNA-viral RNA-dependent RNA polymerase (RdRp) protein are often used as negative controls when looking to identify viral RdRp sequences, so the presence of unlabelled viruses amongst these datasets is problematic. These sequences also represent a source of information about novel viruses and EVEs. In this study, we screened uncharacterized proteins from two large public protein repositories—NCBI Protein and UniProt—to identify sequences likely to be derived from RNA viral RdRp and to perform detailed characterization of sequences of interest. We identified 3560 such sequences, many derived from EVEs. Many are previously unknown EVEs, which led to characterization of additional, related sequences. For example, a group of orbi-like viruses infecting nematodes was uncovered that appears to have both ancient endogenous and circulating exogenous members. Many integrations of mito-like viruses into plant genomes were also found. In several host taxonomic groups, the first example of an EVE, and in some cases the first example of any RNA virus, was uncovered. The large number of EVEs uncovered by this relatively small-scale search suggests that only a fraction of the true diversity of EVEs is currently known. We also provide provisional taxonomic annotations for RdRps, currently only listed as members of the Riboviria realm. A number of sequences are identified that are indistinguishable from viruses but are labelled as bacteria, seemingly as a result of mislabelling or contamination. Non-RdRp sequences that share near-significant similarity with RdRp are also characterized. Finally, recommendations are made for generating useful negative controls and sets of negative control sequences are provided.

Endogenous viral elements in termite genomes reveal extensive diversity of deltaviruses and provide insights into their origins.

Genomes of giant viruses (phylum Nucleocytoviricota) have increasingly been found integrated into the genomes of diverse eukaryotes. Here, we report eight giant endogenous viral elements (GEVEs) in the genome of the microalgae Euglena gracilis. The GEVEs in E. gracilis bear signatures of genomic erosion, including invasion of transposable elements and duplications, suggesting that they are incapable of reactivation and virion production. Most of the GEVEs exhibit high average amino acid identity and cluster near each other in phylogenies of viral marker genes, suggesting that they are derived from the same ancestral viral lineage. Phylogenetic analysis of nucleocytovirus marker proteins reveals that the viruses belong to the order Asfuvirales in the same broader lineage that includes African swine fever virus, abalone asfarvirus, and giant viral sequences recently found in the fungus Rhizophagus irregularis and the marine gastropod Elysia marginata, suggesting that widespread host range transitions have occurred in this lineage. This work expands the diversity of known endogenous giant viruses and expands the host range of the Asfuvirales to include the superkingdom Discoba.IMPORTANCEThis study expands on the rapidly growing body of literature on the impact of latent giant viruses on eukaryotic genome evolution. In this study, we describe signatures of an understudied group of giant viruses integrated in the genome of Euglena gracilis, an ecologically and industrially relevant freshwater protist. The giant viruses in the genome of Euglena are relatives of important animal pathogens, such as African swine fever virus, which infects pigs, and abalone virus, which infects abalone. The broad range of hosts infected by this lineage of viruses exemplifies the impact of giant viruses on diverse eukaryotic lineages.

BackgroundMosquitoes with aggressive biting behavior are important disease vectors threatening public health. Armigeres subalbatus, as an emerging arbovirus and filarial disease vector, exhibits aggressive host-seeking behavior and unique breeding preference for contaminated water. However, the molecular mechanisms underlying these biological characteristics remain poorly understood. This study aimed to generate a high-quality genome assembly and characterize the genetic basis of vector competence and environmental adaptation in Ar. subalbatus.MethodsWe sequenced and assembled the Ar. subalbatus genome using Oxford Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C technology. Comparative genomic analysis was performed to identify gene families related to detoxification, diapause, innate immunity, and sex determination. Gene structure analysis focused on the male-determining factor and its evolutionary relationships with other mosquito vectors.ResultsThe genome assembly consists of three chromosomes, with a total size of 1.33 Gbp and an N50 of 430.15 Mbp (GenBank assembly: GCA_024139115.2), displaying 99.4% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness. We identified the gene structure of the male-determining factor (AsuMf) and characterized its evolutionary relationship with other mosquito vectors. The analysis revealed expanded detoxification-related gene families including cytochrome P450s, which may facilitate adaptation to contaminated breeding sites. We characterized 566 putative diapause-related genes that could potentially contribute to geographical expansion, 334 innate immune genes, and 1673 endogenous viral elements, indicating complex virus-host interactions throughout evolution.ConclusionsOur study provides insights into the molecular basis of vector competence and adaptation in Ar. subalbatus. The expanded detoxification gene families may enable the species to survive in polluted environments, while the identified diapause-related genes could explain its geographical expansion capabilities. These findings establish a foundation for developing novel vector control strategies targeting this emerging disease vector.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s40249-025-01353-1.

Abstract The main purpose of this report is to provide hard evidence that the shrimp parvovirus, infectious hypodermal and hematopoietic necrosis virus (IHHNV), has not resulted “in significant consequences, for example, production losses, morbidity or mortality at a zone or country level” in Thailand since at least 2010. It also reveals that no single polymerase chain reaction (PCR) test is sufficient to identify IHHNV‐infected shrimp. It presents historical evidence and new evidence from 11 commercial ponds cultivating the giant tiger shrimp Penaeus monodon in Thailand. These ponds were selected because they were the ponds that gave positive PCR test results for IHHNV using two methods recommended for IHHNV diagnosis by World Organization for Animal Health (WOAH) (IHHNV‐309 and IHHNV‐389). However, an additional in‐house “IHHNV Long‐amp method” (IHHNV‐LA) was also used to amplify 90% of the 4‐kb IHHNV genome sequence, and it also gave false‐positive test results with 2 of the 11 ponds (IHHNV‐LA positive, but histological tests negative). Further tests using normal histopathological analysis for the presence of pathognomonic Cowdry A type inclusions (CAI), in situ hybridization (ISH) and immunohistochemistry (IHC) could confirm IHHNV infections in only two of the three ponds PCR‐positive using all three PCR methods. In addition, positive detection of CAI alone was equivalent to ISH or IHC in confirming IHHNV infection after a positive test with any of the PCR methods used. In summary, the recommended WOAH PCR methods gave false‐positive test results for IHHNV infection with 9/11 ponds (82%). All 11 ponds gave profitable harvests despite the confirmation of IHHNV in two ponds, where it was accompanied by various additional pathogens. Unfortunately, according to current practice, positive PCR test results with the WOAH methods alone sometimes leads to rejection of traded shrimp products without assurance that the test results are not false‐positive results that may arise from endogenous viral elements (EVE).

The whitefly Bemisia tabaci (Hemiptera: Aleyrodoidea) causes direct feeding damage to crop plants and transmits pathogenic plant viruses, thereby threatening global food security. Although whitefly-infecting RNA viruses are known and proposed as biocontrol agents, no insect DNA virus has been found in any member of Aleyrodoidea. Using rolling circle amplification (RCA) of viral DNA from whiteflies collected from crop fields in Morocco, followed by Illumina sequencing of the RCA products, we found a novel insect single-stranded (ss) DNA parvovirus (family Parvoviridae) in addition to plant ssDNA geminiviruses transmitted by whiteflies. Based on its genome organization with inverted terminal repeats and evolutionarily conserved proteins mediating viral DNA replication (NS1/Rep) and encapsidation (VP), encoded on the forward and reverse strands, respectively, we named this virus Bemisia tabaci ambidensovirus (BtaDV) and classified it as a founding member of a new genus within the subfamily Densovirinae. This subfamily also contains three distinct genera of ambisense densoviruses of other hemipteran insects (Aphidoidea, Coccoidea, and Psylloidea). Furthermore, we provide evidence for the genetic variants of BtaDV circulating in whitefly populations and for its partial sequences integrated into the B. tabaci genome, with one integrant locus potentially expressing a fusion protein composed of viral Rep endonuclease and host DNA-binding domains. This suggests a long-term virus-host interaction and neofunctionalization of BtaDV-derived endogenous viral elements.

Endogenous viral elements (EVEs) serve as molecular fossils that record the ancient co-evolutionary arms race between viruses and their hosts. In this study, by analyzing 105 host crustacean genomes, we identified 252 infectious hypodermal and haematopoietic necrosis virus-derived EVEs (IHHNV-EVEs), which include 183 ancient and 6 recently inserted EVEs. These IHHNV-EVEs are widely distributed among Decapoda, Thoracica, and Isopoda, with some of them exhibiting a syntenic distribution relative toneighboringhost sequences, suggesting that the IHHNV or its ancestor are potential pathogens of these species with a long-time dynamic interaction during the evolutionary history. An expansion of IHHNV-EVEs was observed indecapodagenomes, reflecting a reinforced arm race betweendecapodaand IHHNV. Notably, we found that nearly all recent IHHNV-EVEs were laboratory contaminants, except for a single authentic integration in Penaeus monodon that persists intact across 16 samples from the 2 populations. These temporal dynamics-ancient genomic stabilization versus modern colonization activity-highlight that EVEs serve as dual archives: historical records of past conflicts and active participants in current evolutionary battles. Our findings redefine viral genomic colonization as a continuum, where ancient EVE fixation coexists with persistent integration processes, providing new insights into host-virus co-evolutionary trajectories.

The online version contains supplementary material available at 10.1007/s13205-025-04412-4.

Endogenous viral elements (EVEs) are widespread in the genomes of various organisms and have played a crucial role in evolution. Historically, research on EVEs primarily focused on those derived from retroviruses; however, the significance of non-retroviral EVEs (nrEVEs) has gradually gained recognition. In this study, we employ an approach that combines protein structure prediction with sequence analysis to identify a large group of previously unrecognized nrEVEs across spider genomes. Additionally, we identify nrEVE-related messenger RNAs, small interfering RNAs, and PIWI-interacting RNAs in spiders, suggesting that these nrEVEs may be functionally active. We also experimentally confirm the presence of spider nrEVEs and their transcripts in individual spiders. Evolutionary analysis suggests that these spider nrEVEs derived from unidentified nuclear arthropod large DNA viruses belonging to the order Lefavirales, class Naldaviricetes. Multiple integration events must have occurred both anciently and recently during the evolutionary history of spiders to explain these nrEVEs. Our findings reveal a novel group of nrEVEs and provide valuable insights into their evolutionary relationship with arthropods.

Virus discovery in mass-reared insects is a growing topic of interest due to outbreak risks and for insect welfare concerns. In the case of black soldier flies (Hermetia illucens, BSF), pioneering bioinformatic studies have uncovered exogenous viruses from the orders Ghabrivirales and Bunyavirales, as well as endogenous viral elements from five virus families. This prompted further virome investigation of BSF metagenomes and metatranscriptomes, including from BSF individuals displaying signs and symptoms of disease. A high-throughput pipeline allowed the simultaneous investigation of 203 next generation sequencing datasets. This revealed the presence of seven viruses belonging to the families Dicistroviridae, Iflaviridae, Rhabdoviridae, Solinviviridae, Inseviridae, Lebotiviridae, and an unclassified Bunyavirales. Here we describe five viruses, which were detected in BSF from multiple origins, outlining the diversity of naturally occurring viruses associated with BSF colonies. As this viral community may also include BSF pathogens, we developed molecular detection tools which could be used for viral surveillance, both in mass-reared and wild populations of BSF.

Isolation of an infectious mammalian chu-like virus from tumor cells of the endangered Tasmanian devil (Sarcophilus harrisii).

BackgroundPolinton-like viruses (PLVs) are diverse eukaryotic DNA viral elements (14–40 kb) that often undergo significant expansion within protist genomes through repeated insertion events. Emerging evidence indicates they function as antiviral defense systems in protists, reducing the progeny yield of their infecting giant viruses (phylum Nucleocytoviricota) and influencing the population dynamics and evolution of both viruses and their hosts. While many PLVs have been identified within the genomes of sequenced protists, most were recovered from metagenomic data. Even with the large number of PLVs identified from metagenomic data, their host-virus linkages remain unknown owing to the scarcity of ecologically relevant protist genomes. Additionally, the extent of PLV diversification within abundant freshwater taxa remains undetermined. In order to tackle these questions, high-quality genomes of abundant and representative taxa that bridge genomic and metagenomic PLVs are necessary. In this regard, cryptophytes, which are among the most widely distributed, abundant organisms in freshwaters and have remained largely out of bounds of genomic and metagenomic approaches, are ideal candidates for investigating the diversification of such viral elements both in cellular and environmental context.ResultsWe leveraged long-read sequencing to recover large (200–600 Mb), high-quality, and highly repetitive (> 60%) genomes of representative freshwater and marine photosynthetic cryptophytes. We uncovered over a thousand complete PLVs within these genomes, revealing vast lineage-specific expansions, particularly in the common freshwater cryptophyte Rhodomonas lacustris. By combining deep sequence homology annotation with biological network analyses, we discern well-defined PLV groups defined by characteristic gene-sharing patterns and the use of distinct strategies for replication and integration within host genomes. Finally, the PLVs recovered from these cryptophyte genomes also allow us to assign host-virus linkages in environmental sequencing data.ConclusionsOur findings provide a primer for understanding the evolutionary history, gene content, modes of replication and infection strategies of cryptophyte PLVs, with special emphasis on their expansion as endogenous viral elements (EVEs) in freshwater bloom-forming R. lacustris.5wVn_eVLoqFBm1YzScaWETVideo

Tick-borne viruses are primarily transmitted to vertebrates by infected ticks during blood feeding and cause various diseases in humans and animals. Haemaphysalis longicornis is one of the main tick species responsible for human tick bites and is thought to be the primary vector of severe fever with thrombocytopenia syndrome, an important tick-borne viral disease in Japan. Although H. longicornis ticks pose a potential risk to humans in Japan, studies on tick-borne viral prevalence of this tick species in Japan are limited. In this study, we conducted RNA virome analysis of H. longicornis ticks collected in Toyooka City, Hyogo Prefecture, Japan. Two known viruses, Dabieshan tick virus and Hubei sobemo-like virus 15, and putative novel quaranjavirus-like sequences were detected. Additionally, assessments of endogenous viral elements (EVEs) related to the virus and virus-like sequences suggested putative novel quaranjavirus-like sequences existed in both RNA and DNA forms. However, we could not determine whether this quaranjavirus-like sequence was of viral origin and could not conclude whether the DNA forms of the quaranjavirus-like sequence existed as EVEs in ticks. This study provides new insights into the prevalence of tick-associated viruses in ixodid ticks and serves as a reference for future approaches to prevent tick-borne diseases.

Endogenous viral elements (EVEs) are genomic sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express co-opted proteins in their host. Furthermore, some EVEs that are expressed as proteins have become part of cellular genes that are fusions of hosts and EVE sequences. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and some of them contain ORFs and can be expressed as proteins. We have shown that an EPV containing an ORF is part of the guinea pig gene enRep-M9l. This gene is broadly transcribed in vivo, indicating that it can be translated into a protein. By generating antibodies against the enRep coding sequence of the enRep-M9l ORF, we showed that the protein enRep-M9l is expressed in vivo and in the guinea pig-derived cell line JH4. By immunofluorescence and in situ proximity ligation assays, we observed that enRep-M9l protein has a cytoplasmic localization near microtubules. The results of this study suggest that the guinea pig EPV-derived protein enRep-M9l is a microtubule-associated protein. To our knowledge, this is the second demonstration that an EPV-derived protein is expressed in vivo.

Horseshoe crabs (Xiphosura: Limulidae) are the sole surviving species of the class Merostomata, with only four extant species remaining today. Recent advances in metagenomic next-generation sequencing have unveiled a vast diversity of RNA viruses and non-retroviral endogenous RNA viral elements (nrEVEs) in invertebrates. This raises intriguing questions about the RNA virome and nrEVEs in horseshoe crabs as "living fossils," potentially offering insights into the evolutionary relationships between RNA viruses and these ancient organisms. In this study, 22 novel RNA viruses were identified across the four horseshoe crab species by screening 117 data sets, including picornaviruses, totiviruses, a flavivirus, a rhabdovirus, as well as a plant-associated tombusvirus and a fungi-associated narnavirus. Additionally, 20 nrEVEs were identified in the genomes of the four horseshoe crab species (hcEVEs), with most sharing homology with the viral family Chuviridae (N = 11), supporting the hypothesis that modern negative-sense RNA viruses may trace their origins to ancient oceanic chuviruses. A time-scaled phylogenetic tree based on hcEVEs suggests that at least two independent ancient chuvirus infections and genome integration events occurred in the common ancestor of horseshoe crab species. Interestingly, transcriptional analyses indicated that hcEVE-containing transcripts display typical exon-intron structures in the three Asian horseshoe crab species, suggesting that these hcEVEs may have been co-opted by horseshoe crabs during coevolution. These findings advance our understanding of the RNA viruses associated with horseshoe crabs and shed light on the potential role of RNA viruses in shaping the evolutionary history of this "living fossil" arthropod host.IMPORTANCERecent studies have discovered abundant RNA viruses in invertebrates, revealing that viral genomes may integrate into host genomes, creating a genetic record of past infections. In this study, we explored the evolutionary relationship between RNA viruses and the four extant horseshoe crab species-the last representatives of the class Merostomata, often termed "living fossils"-by analyzing viral sequences embedded in their genomes. The presence of chuvirus-like sequences in the genomes of these horseshoe crabs suggests that modern negative-sense RNA viruses may trace their origins back to ancient chuviruses from the ocean. Furthermore, we identified at least two independent ancient integrations of chuviruses in the evolutionary history of horseshoe crabs, with one orthologous gene containing a chuvirus-derived G protein gene/coding sequence potentially inherited from a common ancestor of the three Asian species before their divergence. Our findings contribute to a deeper understanding of the long-term coevolution between RNA viruses and their arthropod hosts.

BackgroundAlthough advances in metagenomics, viral diversity and non-retroviral endogenous viral elements (EVEs) in wheat aphids remain underexplored. By analyzing 470 publicly available datasets and one laboratory-generated transcriptome, the RNA virome and EVEs in the genomes of Sitobion avenae, Schizaphis graminum, and Rhopalosiphum padi were systematically investigated.ResultsWe identified 43 RNA viruses, including 12 novel and 31 known RNA viruses. These viruses were widely distributed and abundant in different geographic populations of three wheat aphid species. +ssRNA viruses were the dominant type of aphid viruses. Besides, 90 EVEs were discovered in the genomes of three aphid species. In addition, the EVEs exhibit potential domestication and novel functional roles within aphid genomes.ConclusionsThis study expands the understanding of RNA virus diversity in aphids and provides valuable insights into the potential functions of EVEs in virus-host coevolution.

Bangladesh has a warm climate and landscapes favourable for the proliferation of mosquitoes. Mosquito-borne pathogens including malaria and arthropod-borne viruses (arboviruses) remain a serious threat to the public health requiring constant vector control and disease surveillance. From November 2018 to April 2019, Anopheles mosquitoes were collected in three unions in the Ramu Upazila (sub-district) of Cox's Bazar District, Bangladesh. The mosquito specimens were combined into pools based on date of collection, household ID, and sex. Metagenome next-generation sequencing was conducted to elucidate diversity of virus sequences in each pool. Homology-based taxonomic classification and phylogenetic analyses identified a broad diversity of putative viruses from 12 known families, with additional unclassified viruses also likely present. Analysis of male mosquitoes showed some of these viruses are likely capable of being vertically transmitted. Moreover, many of the assembled virus sequences share homology and phylogenetic affinity with segments in sequenced Anopheles genomes, and may represent endogenous viral elements derived from a past evolutionary relationship between these putative viruses and their mosquito hosts.