Rhabdoviral Endogenous Sequences Identified in the Leishmaniasis Vector Lutzomyia longipalpis Are Widespread in Sandflies from South America.

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.

A tale of caution: How endogenous viral elements affect virus discovery in transcriptomic data.

Large-scale metagenomic and -transcriptomic studies have revolutionized our understanding of viral diversity and abundance. In contrast, endogenous viral elements (EVEs), remnants of viral sequences integrated into host genomes, have received limited attention in the context of virus discovery, especially in RNA-Seq data. EVEs resemble their original viruses, a challenge that makes distinguishing between active infections and integrated remnants difficult, affecting virus classification and biases downstream analyses. Here, we systematically assess the effects of EVEs on a prototypical virus discovery pipeline, evaluate their impact on data integrity and classification accuracy, and provide some recommendations for better practices. We examined EVEs and exogenous viral sequences linked to Orthomyxoviridae, a diverse family of negative-sense segmented RNA viruses, in 13 genomic and 538 transcriptomic datasets of Culicinae mosquitoes. Our analysis revealed a substantial number of viral sequences in transcriptomic datasets. However, a significant portion appeared not to be exogenous viruses but transcripts derived from EVEs. Distinguishing between transcribed EVEs and exogenous virus sequences was especially difficult in samples with low viral abundance. For example, three transcribed EVEs showed full-length segments, devoid of frameshift and nonsense mutations, exhibiting sufficient mean read depths that qualify them as exogenous virus hits. Mapping reads on a host genome containing EVEs before assembly somewhat alleviated the EVE burden, but it led to a drastic reduction of viral hits and reduced quality of assemblies, especially in regions of the viral genome relatively similar to EVEs. Our study highlights that our knowledge of the genetic diversity of viruses can be altered by the underestimated presence of EVEs in transcriptomic datasets, leading to false positives and altered or missing sequence information. Thus, recognizing and addressing the influence of EVEs in virus discovery pipelines will be key in enhancing our ability to capture the full spectrum of viral diversity.

In vitro characterization of cell-fusing agent virus DNA forms in Aedes aegypti mosquitoes.

How non-retroviral endogenous viral elements (EVEs) are established is a long-standing question. Viral DNA (vDNA) forms of RNA viruses are likely to be EVE precursors. Cell-fusing agent virus (CFAV) is a major insect-specific virus (ISV) in the Aedes aegypti mosquitoes and one of the few existing non-retroviral RNA viruses found as EVEs. We characterized CFAV-derived vDNA in the cell line to understand the mechanism of why current viruses are rarely endogenized. vDNA production was affected by cell culture media independent of CFAV replication. vDNAs that correspond to different regions covering the entire viral genome were detected, implying multiple initiation sites exist. A considerable proportion of vDNAs corresponded to ssDNA. Higher vDNA copies were detected in the cytoplasm than the nucleus. Our findings provide valuable insights into the intracellular characteristics of ISV-derived vDNAs, which will aid in understanding the underlying mechanisms of non-retroviral EVE formation.

Confirmatory test of active IHHNV infection in shrimp by immunohistochemistry and IHHNV-LongAmp PCR.

The presence of endogenous viral elements (EVE) in the penaeid shrimp genome has been recently reported and suggested to be involved in the host recognition of viral invaders. Our previous report of a search for EVE of infectious hypodermal and haematopoietic necrosis virus (IHHNV-EVE) in the Thai Penaeus monodon whole genome sequence project (GenBank accession no. JABERT000000000) confirmed the presence of three clusters of EVE derived from IHHNV in the shrimp genome. This study aimed to compare an immunohistochemistry method (IHC) and a PCR method to detect infectious IHHNV infection in shrimp. First, specimens collected from farms were checked for IHHNV using three PCR methods; two methods were recommended by WOAH (309 and 389 methods), and a newly established long-range PCR for IHHNV (IHHNV-LA PCR) targeting almost the whole genome (>90%) of IHHNV. Among 29 specimens tested, 24 specimens were positive for WOAH methods (at least one method). Among 24 WOAH-positive specimens (WOAH+), there were 18 specimens with positive IHHNV-LA PCR method (WOAH+/LA+), six specimens with negative IHHNV-LA PCR method (WOAH+/LA-). Six specimens were negative for all methods (WOAH-/LA-). The positive signals detected by IHC method were found only in the specimens with WOAH+/LA+. The results suggest that the WOAH+/LA- specimens were not infected with IHHNV, and the positive WOAH method might result from the EVE-IHHNV. The study recommends combining the IHHNV-LA PCR method and IHC with positive PCR results from WOAH's recommended methods to confirm IHHNV infection.

Comparison of Polymerase Chain Reaction (PCR) assay performance in detecting Decapod penstylhamaparvovirus 1 in penaeid shrimp.

Decapod Penstylhamaparvovirus 1, commonly known as infectious hypodermal and hematopoietic necrosis virus (IHHNV), remains an economically important viral pathogen for penaeid shrimp aquaculture due to its effects on growth performance. The World Organization for Animal Health (WOAH, Paris, France) recommended methods for the detection of IHHNV include both conventional and real-time PCR. However, published reports and anecdotal evidence suggest the occurrence of non-specific amplifications when testing for IHHNV using the WOAH protocols. Studies were designed to develop a sensitive, robust TaqMan PCR method for detection of IHHNV in the three commercially important penaeid shrimp: Penaeus vannamei, P. monodon and P. stylirostris. We compared the performance of the WOAH-recommended real-time PCR method to several published as well as in-house designed primer/probe sets spanning the entire genome of IHHNV. Our results show that (1) more than one primer/ probe set is needed when testing for the infectious form of IHHNV in all three species of shrimp and (2) primer pairs qIH-Fw/qIH-Rv and 3144F/ 3232R have diagnostic characteristics that would enable IHHNV detection in all three shrimp species. These findings are valuable for a large-scale screening of shrimp using a TaqMan real-time PCR assay.

Large-scale reference-free analysis of flavivirus sequences in Aedes aegypti whole genome DNA sequencing data.

Flaviviruses are a diverse group of RNA viruses, which include the etiological agents of Zika, dengue and yellow fever that are transmitted by mosquitoes. Flaviviruses do not encode reverse transcriptase and cannot reverse transcribe into DNA, yet DNA sequences of flaviviruses are found both integrated in the chromosomes of Aedes aegypti mosquitoes and as extrachromosomal sequences. We have previously examined the Ae. aegypti reference genome to identify flavivirus integrations and analyzed conservation of these sequences among whole-genome data of 464 Ae. aegypti collected across 10 countries globally. Here, we extended this analysis by identifying flavivirus sequences in these samples independently of the Ae. aegypti reference assembly. Our aim was to identify the complete set of viral sequences, including those absent in the reference genome, and their geographical distribution. We compared the identified sequences using BLASTn and applied machine learning methods to identify clusters of similar sequences. Apart from clusters of sequences that correspond to the four viral integration events that we had previously described, we identified 19 smaller clusters. The only cluster with a strong geographic association consisted of Cell-fusing agent virus-like sequences specific to Thailand. The remaining clusters did not have a geographic association and mostly consisted of near identical short sequences without strong similarity to any known flaviviral genomes. The short read sequencing data did not permit us to determine whether identified sequences were extrachromosomal or integrated into Ae. aegypti chromosomes. Our results suggest that Liverpool strain and field Ae. aegypti mosquitoes have a similar variety of conserved flaviviral DNA, whose functional role should be investigated in follow-up studies.

Teratorn and Its Related Elements - a Novel Group of Herpesviruses Widespread in Teleost Genomes.

Herpesviruses are a large family of DNA viruses infecting vertebrates and invertebrates, and are important pathogens in the field of aquaculture. In general, herpesviruses do not have the ability to integrate into the host genomes since they do not have a chromosomal integration step in their life cycles. Recently, we identified a novel group of herpesviruses, "Teratorn" and its related elements, in the genomes of various teleost fish species. At least some of the Teratorn-like herpesviruses are fused with a piggyBac-like DNA transposon, suggesting that they have acquired the transposon-like intragenomic lifestyle by hijacking the transposon system. In this review, we describe the sequence characteristics of Teratorn-like herpesviruses and phylogenetic relationships with other herpesviruses. Then we discuss the process of transposon-herpesvirus fusion, their life cycle, and the generality of transposon-virus fusion. Teratorn-like herpesviruses provide a piece of concrete evidence that even non-retroviral elements can become intragenomic parasites retaining replication capacity, by acquiring transposition machinery from other sources.

Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA biogenesis in mosquitoes.

The piRNA pathway in mosquitoes differs substantially from other model organisms, with an expanded PIWI gene family and functions in antiviral defense. Here, we define core piRNA clusters as genomic loci that show ubiquitous piRNA expression in both somatic and germline tissues. These core piRNA clusters are enriched for non-retroviral endogenous viral elements (nrEVEs) in antisense orientation and depend on key biogenesis factors, Veneno, Tejas, Yb, and Shutdown. Combined transcriptome and chromatin state analyses identify transcriptional readthrough as a conserved mechanism for cluster-derived piRNA biogenesis in the vector mosquitoes Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae. Comparative analyses between the two Aedes species suggest that piRNA clusters function as traps for nrEVEs, allowing adaptation to environmental challenges such as virus infection. Our systematic transcriptome and chromatin state analyses lay the foundation for studies of gene regulation, genome evolution, and piRNA function in these important vector species.

Endogenous Viral Elements in Shrew Genomes Provide Insights into Pestivirus Ancient History.

As viral genomic imprints in host genomes, endogenous viral elements (EVEs) shed light on the deep evolutionary history of viruses, ancestral host ranges, and ancient viral-host interactions. In addition, they may provide crucial information for calibrating viral evolutionary timescales. In this study, we conducted a comprehensive in silico screening of a large data set of available mammalian genomes for EVEs deriving from members of the viral family Flaviviridae, an important group of viruses including well-known human pathogens, such as Zika, dengue, or hepatitis C viruses. We identified two novel pestivirus-like EVEs in the reference genome of the Indochinese shrew (Crocidura indochinensis). Homologs of these novel EVEs were subsequently detected in vivo by molecular detection and sequencing in 27 shrew species, including 26 species representing a wide distribution within the Crocidurinae subfamily and one in the Soricinae subfamily on different continents. Based on this wide distribution, we estimate that the integration event occurred before the last common ancestor of the subfamily, about 10.8 million years ago, attesting to an ancient origin of pestiviruses and Flaviviridae in general. Moreover, we provide the first description of Flaviviridae-derived EVEs in mammals even though the family encompasses numerous mammal-infecting members. This also suggests that shrews were past and perhaps also current natural reservoirs of pestiviruses. Taken together, our results expand the current known Pestivirus host range and provide novel insight into the ancient evolutionary history of pestiviruses and the Flaviviridae family in general.

Shrimp genome sequence contains independent clusters of ancient and current Endogenous Viral Elements (EVE) of the parvovirus IHHNV.

BACKGROUND: Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). RESULTS: The shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called "non-infectious IHHNV Type A" (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV. CONCLUSIONS: Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

Virophages and retrotransposons colonize the genomes of a heterotrophic flagellate.

Virophages can parasitize giant DNA viruses and may provide adaptive anti-giant virus defense in unicellular eukaryotes. Under laboratory conditions, the virophage mavirus integrates into the nuclear genome of the marine flagellate Cafeteria burkhardae and reactivates upon superinfection with the giant virus CroV. In natural systems, however, the prevalence and diversity of host-virophage associations has not been systematically explored. Here, we report dozens of integrated virophages in four globally sampled C. burkhardae strains that constitute up to 2% of their host genomes. These endogenous mavirus-like elements (EMALEs) separated into eight types based on GC-content, nucleotide similarity, and coding potential and carried diverse promoter motifs implicating interactions with different giant viruses. Between host strains, some EMALE insertion loci were conserved indicating ancient integration events, whereas the majority of insertion sites were unique to a given host strain suggesting that EMALEs are active and mobile. Furthermore, we uncovered a unique association between EMALEs and a group of tyrosine recombinase retrotransposons, revealing yet another layer of parasitism in this nested microbial system. Our findings show that virophages are widespread and dynamic in wild Cafeteria populations, supporting their potential role in antiviral defense in protists.

Viruses exist in all ecosystems in vast numbers and infect many organisms. Some of them are harmful but others can protect the organisms they infect. For example, a group of viruses called virophages protect microscopic sea creatures called plankton from deadly infections by so-called giant viruses. In fact, virophages need plankton infected with giant viruses to survive because they use enzymes from the giant viruses to turn on their own genes. A virophage called mavirus integrates its genes into the DNA of a type of plankton called Cafeteria. It lays dormant in the DNA until a giant virus called CroV infects the plankton. This suggests that the mavirus may be a built-in defense against CroV infections and laboratory studies seem to confirm this. But whether wild Cafeteria also use these defenses is unknown. Hackl et al. show that virophages are common in the DNA of wild Cafeteria and that the two appear to have a mutually beneficial relationship. In the experiments, the researchers sequenced the genomes of four Cafeteria populations from the Atlantic and Pacific Oceans and looked for virophages in their DNA. Each of the four Cafeteria genomes contained dozens of virophages, which suggests that virophages are important to these plankton. This included several relatives of the mavirus and seven new virophages. Virophage genes were often interrupted by so called jumping genes, which may take advantage of the virophages the way the virophages use giant viruses to meet their own needs. The experiments show that virophages often co-exist with marine plankton from around the world and these relationships are likely beneficial. In fact, the experiments suggest that the virophages may have played an important role in the evolution of these plankton. Further studies may help scientists learn more about virus ecology and how viruses have shaped the evolution of other creatures.

Ancient viral integrations in marsupials: a potential antiviral defence.

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed 'endogenous viral elements' (EVEs), could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing data sets (n = 35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae, or Parvoviridae EVEs. The other twelve transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n = 188) were mapped to marsupial genomes (where available, n = 5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum, and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n = 71). Every marsupial has Bornaviridae, Filoviridae, and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American-Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence data sets, we detected Bornaviridae replicase and Filoviridae nucleoprotein produced small RNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

The discovery, distribution, and diversity of DNA viruses associated with Drosophila melanogaster in Europe.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Flavivirus integrations in Aedes aegypti are limited and highly conserved across samples from different geographic regions unlike integrations in Aedes albopictus.

Mosquitoes of the genus Aedes are the main vectors of many viruses, e.g. dengue and Zika, which affect millions of people each year and for which there are limited treatment options. Understanding how Aedes mosquitoes tolerate high viral loads may lead to better disease control strategies. Elucidating endogenous viral elements (EVEs) within vector genomes may give exploitable biological insights. Previous studies have reported the presence of a large number of EVEs in Aedes genomes. Here we investigated if flavivirus EVEs are conserved across populations and different Aedes species by using ~ 500 whole genome sequence libraries from Aedes aegypti and Aedes albopictus, sourced from colonies and field mosquitoes across continents. We found that nearly all flavivirus EVEs in the Ae. aegypti reference genome originate from four separate putative viral integration events, and that they are highly conserved across geographically diverse samples. By contrast, flavivirus EVEs in the Ae. albopictus reference genome originate from up to nine distinct integration events and show low levels of conservation, even within samples from narrow geographical ranges. Our analysis suggests that flaviviruses integrated as long sequences and were subsequently fragmented and shuffled by transposable elements. Given that EVEs of Ae. aegypti and Ae. albopictus belong to different phylogenetic clades and have very differing levels of conservation, they may have different evolutionary origins and potentially different functional roles.

A Human Endogenous Bornavirus-Like Nucleoprotein Encodes a Mitochondrial Protein Associated with Cell Viability.

Endogenous retroviruses (ERVs) are sequences in animal genomes that originated from ancient retrovirus infections; they provide genetic novelty in hosts by being coopted as functional genes or elements during evolution. Recently, we demonstrated that endogenous elements from not only from retroviruses but also nonretroviral RNA viruses are a possible source of functional genes in host animals. The remnants of ancient bornavirus infections, called endogenous bornavirus-like elements (EBLs), are present in the genomes of a wide variety of vertebrate species, and some express functional products in host cells. Previous studies have predicted that the human EBL locus derived from bornavirus nucleoprotein, termed hsEBLN-2, expresses mRNA encoding a protein, suggesting that hsEBLN-2 has acquired a cellular function during evolution. However, the detailed function of the hsEBLN-2-derived product remains to be elucidated. In this study, we show that the hsEBLN-2-derived protein E2 acts as a mitochondrial protein that interacts with mitochondrial host factors associated with apoptosis, such as HAX-1. We also demonstrate that knockdown of hsEBLN-2-derived RNA increased the levels of PARP and caspase-3 cleavage and markedly decreased cell viability. In contrast, overexpression of E2 enhanced cell viability, as well as the intracellular stability of HAX-1, under stress conditions. Our results suggest that hsEBLN-2 has been coopted as a host gene, the product of which is involved in cell viability by interacting with mitochondrial proteins. IMPORTANCE Our genomes contain molecular fossils of ancient viruses, called endogenous virus elements (EVEs). Mounting evidence suggests that EVEs derived from nonretroviral RNA viruses have acquired functions in host cells during evolution. Previous studies have revealed that a locus encoding a bornavirus-derived EVE, hsEBLN-2, which was generated approximately 43 million years ago in a human ancestor, may be linked to the development of some tumors. However, the function of hsEBLN-2 has not been determined. In this study, we found that the E2 protein, an expression product of hsEBLN-2, interacts with apoptosis-related host proteins as a mitochondrial protein and affects cell viability. This study suggests that nonretroviral RNA viral EVEs have been coopted by hosts with more diverse functions than previously thought, showing a pivotal role for RNA virus infection in evolution.

Identification of African swine fever virus-like elements in the soft tick genome provides insights into the virus' evolution.

BACKGROUND: African swine fever virus (ASFV) is a most devastating pathogen affecting swine. In 2007, ASFV was introduced into Eastern Europe where it continuously circulates and recently reached Western Europe and Asia, leading to a socio-economic crisis of global proportion. In Africa, where ASFV was first described in 1921, it is transmitted between warthogs and soft ticks of the genus Ornithodoros in a so-called sylvatic cycle. However, analyses into this virus' evolution are aggravated by the absence of any closely related viruses. Even ancient endogenous viral elements, viral sequences integrated into a host's genome many thousand years ago that have proven extremely valuable to analyse virus evolution, remain to be identified. Therefore, the evolution of ASFV, the only known DNA virus transmitted by arthropods, remains a mystery. RESULTS: For the identification of ASFV-like sequences, we sequenced DNA from different recent Ornithodoros tick species, e.g. O. moubata and O. porcinus, O. moubata tick cells and also 100-year-old O. moubata and O. porcinus ticks using high-throughput sequencing. We used BLAST analyses for the identification of ASFV-like sequences and further analysed the data through phylogenetic reconstruction and molecular clock analyses. In addition, we performed tick infection experiments as well as additional small RNA sequencing of O. moubata and O. porcinus soft ticks. CONCLUSION: Here, we show that soft ticks of the Ornithodoros moubata group, the natural arthropod vector of ASFV, harbour African swine fever virus-like integrated (ASFLI) elements corresponding to up to 10% (over 20 kb) of the ASFV genome. Through orthologous dating and molecular clock analyses, we provide data suggesting that integration could have occurred over 1.47 million years ago. Furthermore, we provide data showing ASFLI-element specific siRNA and piRNA in ticks and tick cells allowing for speculations on a possible role of ASFLI-elements in RNA interference-based protection against ASFV in ticks. We suggest that these elements, shaped through many years of co-evolution, could be part of an evolutionary virus-vector 'arms race', a finding that has not only high impact on our understanding of the co-evolution of viruses with their hosts but also provides a glimpse into the evolution of ASFV.

Evolution of dependoparvoviruses across geological timescales-implications for design of AAV-based gene therapy vectors.

Endogenous viral elements (EVEs) are genetic remnants of viruses that have integrated into host genomes millions of years ago and retained as heritable elements passed on to offspring until present-day. As a result, EVEs provide an opportunity to analyse the genomes of extinct viruses utilizing these genomic viral fossils to study evolution of viruses over large timescales. Analysis of sequences from near full-length EVEs of dependoparvoviral origin identified within three mammalian taxa, Whippomorpha (whales and hippos), Vespertilionidae (smooth-nosed bats), and Lagomorpha (rabbits, hares, and pikas), indicates that distinct ancestral dependoparvovirus species integrated into these host genomes approximately 77 to 23 million years ago. These ancestral viruses are unique relative to modern adeno-associated viruses (AAVs), and distinct from extant species of genus Dependoparvovirus. These EVE sequences show characteristics previously unseen in modern, mammalian AAVs, but instead appear more similar to the more primitive, autonomously replicating and pathogenic waterfowl dependoparvoviruses. Phylogeny reconstruction suggests that the whippomorph EVE orthologue derives from exogenous ancestors of autonomous and highly pathogenic dependoparvovirus lineages, believed to have uniquely co-evolved with waterfowl birds to present date. In contrast, ancestors of the two other mammalian orthologues (Lagomorpha and Vespertilionidae) likely shared the same lineage as all other known mammalian exogenous AAVs. Comparative in silico analysis of the EVE genomes revealed remarkable overall conservation of AAV rep and cap genes, despite millions of years of integration within the host germline. Modelling these proteins identified unexpected variety, even between orthologues, in previously defined capsid viral protein (VP) variable regions, especially in those related to the three- and fivefold symmetry axes of the capsid. Moreover, the normally well-conserved phospholipase A2 domain of the predicted minor VP1 also exhibited a high degree of sequence variance. These findings may indicate unique biological properties for these virus 'fossils' relative to extant dependoparvoviruses and suggest key regions to explore within capsid sequences that may confer novel properties for engineered gene therapy vectors based on paleovirology data.

Non-retroviral Endogenous Viral Element Limits Cognate Virus Replication in Aedes aegypti Ovaries.

Endogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR-Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived P-element-induced wimpy testis-interacting RNAs (piRNAs). Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction.

Endogenous viral elements integrated into the genome of the soybean aphid, Aphis glycines.

Sequence analysis of the genomic DNA isolated from four biotypes of the soybean aphid, Aphis glycines (AG), revealed that in addition to the commonly observed retrovirus-related retrotransposons, viral sequences derived from multiple RNA and DNA viruses have integrated into the genome. Notably, sequences of more than 60 nudiviral genes were identified from de novo assembled DNA contigs, and mapped to assembled genomic scaffolds of AG, indicating that an ancient nudivirus, named Aphis glycines endogenous nudivirus (AgENV), had integrated into the AG genome. Furthermore, sequences derived from a similar endogenous nudivirus, Melanaphis sacchari endogenous nudivirus (MsENV), were identified from the genomic scaffolds of the sugarcane aphid, Melanaphis sacchari. Analysis of transcriptome and small RNA sequence data derived from AG did not provide evidence for transcription of the integrated AgENV genes. Hence, the genes of AgENV may be present as pseudogenes. Phylogenetic analysis based on nudivirus core genes indicated that these aphid ENVs belong to the genus Alphanudivirus.