Endogenous Bornavirus-like Elements Research Articles

Endogenous Bornavirus-like Elements in Bats: Evolutionary Insights from the Conserved Riboviral L-Gene in Microbats and Its Antisense Transcription in Myotis daubentonii.

Bats are ecologically diverse vertebrates characterized by their ability to host a wide range of viruses without apparent illness and the presence of numerous endogenous viral elements (EVEs). EVEs are well preserved, expressed, and may affect host biology and immunity, but their role in bat immune system evolution remains unclear. Among EVEs, endogenous bornavirus-like elements (EBLs) are bornavirus sequences integrated into animal genomes. Here, we identified a novel EBL in the microbat Myotis daubentonii, EBLL-Cultervirus.10-MyoDau (short name is CV.10-MyoDau) that shows protein-level conservation with the L-protein of a Cultervirus (Wuhan sharpbelly bornavirus). Surprisingly, we discovered a transcript on the antisense strand comprising three exons, which we named AMCR-MyoDau. The active transcription in Myotis daubentonii tissues of AMCR-MyoDau, confirmed by RNA-Seq analysis and RT-PCR, highlights its potential role during viral infections. Using comparative genomics comprising 63 bat genomes, we demonstrate nucleotide-level conservation of CV.10-MyoDau and AMCR-MyoDau across various bat species and its detection in 22 Yangochiropera and 12 Yinpterochiroptera species. To the best of our knowledge, this marks the first occurrence of a conserved EVE shared among diverse bat species, which is accompanied by a conserved antisense transcript. This highlights the need for future research to explore the role of EVEs in shaping the evolution of bat immunity.

The hidden diversity ofancient bornaviral sequences from X andP genes invertebrate genomes.

Endogenous bornavirus-like elements (EBLs) are heritable sequences derived from bornaviruses in vertebrate genomes that originate from transcripts of ancient bornaviruses. EBLs have been detected using sequence similarity searches such as tBLASTn, whose technical limitations may hinder the detection of EBLs derived from small and/or rapidly evolving viral X and P genes. Indeed, no EBLs derived from the X and P genes of orthobornaviruses have been detected to date in vertebrate genomes. Here, we aimed to develop a novel strategy to detect such 'hidden' EBLs. To this aim, we focused on the 1.9-kb read-through transcript of orthobornaviruses, which encodes a well-conserved N gene and small and rapidly evolving X and P genes. We show a series of evidence supporting the existence of EBLs derived from orthobornaviral X and P genes (EBLX/Ps) in mammalian genomes. Furthermore, we found that an EBLX/P is expressed as a fusion transcript with the cellular gene, ZNF451, which potentially encodes the ZNF451/EBLP fusion protein in miniopterid bat cells. This study contributes to a deeper understanding of ancient bornaviruses and co-evolution between bornaviruses and their hosts. Furthermore, our data suggest that endogenous viral elements are more abundant than those previously appreciated using BLAST searches alone, and further studies are required to understand ancient viruses more accurately.

Viral Sequences Are Repurposed for Controlling Antiviral Responses as Non-Retroviral Endogenous Viral Elements.

Eukaryotic genomes contain numerous copies of endogenous viral elements (EVEs), most of which are considered endogenous retrovirus (ERV) sequences. Over the past decade, non-retroviral endogenous viral elements (nrEVEs) derived from ancient RNA viruses have been discovered. Several functions have been proposed for these elements, including antiviral defense. This review summarizes the current understanding of nrEVEs derived from RNA viruses, particularly endogenous bornavirus-like elements (EBLs) and endogenous filovirus-like elements (EFLs). EBLs are one of the most extensively studied nrEVEs. The EBL derived from bornavirus nucleoprotein (EBLN) is thought to function as a non-coding RNA or protein that regulates host gene expression or inhibits virus propagation. Ebolavirus and marburgvirus, which are filoviruses, induce severe hemorrhagic fever in humans and nonhuman primates. Although the ecology of filoviruses remains unclear, bats are believed to be potential reservoirs. Based on the knowledge from EBLs, it is postulated that EFLs in the bat genome help to maintain the balance between filovirus infection and the bat's defense system, which may partially explain why bats act as potential reservoirs. Further research into the functions of nrEVEs could reveal novel antiviral systems and inspire novel antiviral approaches.

100-My history of bornavirus infections hidden in vertebrate genomes

Although viruses have threatened our ancestors for millions of years, prehistoric epidemics of viruses are largely unknown. Endogenous bornavirus-like elements (EBLs) are ancient bornavirus sequences derived from the viral messenger RNAs that were reverse transcribed and inserted into animal genomes, most likely by retrotransposons. These elements can be used as molecular fossil records to trace past bornaviral infections. In this study, we systematically identified EBLs in vertebrate genomes and revealed the history of bornavirus infections over nearly 100 My. We confirmed that ancient bornaviral infections have occurred in diverse vertebrate lineages, especially in primate ancestors. Phylogenetic analyses indicated that primate ancestors were infected with various bornaviral lineages during evolution. EBLs in primate genomes formed clades according to their integration ages, suggesting that bornavirus lineages infected with primate ancestors had changed chronologically. However, some bornaviral lineages may have coexisted with primate ancestors and underwent repeated endogenizations for tens of millions of years. Moreover, a bornaviral lineage that coexisted with primate ancestors also endogenized in the genomes of some ancestral bats. The habitats of these bat ancestors have been reported to overlap with the migration route of primate ancestors. These results suggest that long-term virus-host coexistence expanded the geographic distributions of the bornaviral lineage along with primate migration and may have spread their infections to these bat ancestors. Our findings provide insight into the history of bornavirus infections over geological timescales that cannot be deduced from research using extant viruses alone, thus broadening our perspective on virus-host coevolution.

Virus-host coevolution: Endogenous RNA viral elements as pseudogenes

RNA viruses do not need to take the form of DNAs, and RNAs alone complete their replication cycles. On the other hand, since the 1970s, it has been known that DNA fragments derived from RNA viruses can be detected in RNA virus-infected cells. Furthermore, in this decade, it has become clear that the eukaryotic genomes contain genetic sequences derived from non-retroviral RNA viruses. The DNA sequences derived from these RNA viruses are thought to be generatedby using a transposable mechanism of retrotransposon, such as LINE-1. Many endogenous RNA viral sequences are formed by the same mechanism as processed pseudogenes in eukaryotic cells, but the significance of the production of RNA viral "pseudogenes " in infected cells has not been elucidated. We have discovered endogenous bornavirus-like elements (EBLs), which derived from a negative-sense, single-stranded RNA virus, Bornaviruses, and have studied the evolution and function of EBLs in host animals. The analysis of EBLs provides us a clue to unravel the history of host-RNA virus coexistence. In this review, I overview about the function of endogenous RNA virus sequences, especially EBLs in mammalian genomes, and discuss the significance of endogenization of RNA viruses as viral pseudogenes in evolution.

Update on immunopathology of bornavirus infections in humans and animals.

Knowledge on bornaviruses has expanded tremendously during the last decade through detection of novel bornaviruses and endogenous bornavirus-like elements in many eukaryote genomes, as well as by confirmation of insectivores as reservoir species for classical Borna disease virus 1 (BoDV-1). The most intriguing finding was the demonstration of the zoonotic potential of lethal human bornavirus infections caused by a novel bornavirus of different squirrel species (variegated squirrel 1 bornavirus, VSBV-1) and by BoDV-1 known as the causative agent for the classical Borna disease in horses and sheep. Whereas a T cell-mediated immunopathology has already been confirmed as key disease mechanism for infection with BoDV-1 by experimental studies in rodents, the underlying pathomechanisms remain less clear for human bornavirus infections, infection with other bornaviruses or infection of reservoir species. Thus, an overview of current knowledge on the pathogenesis of bornavirus infections focusing on immunopathology is given.

Divergent bornaviruses from Australian carpet pythons with neurological disease date the origin of extant Bornaviridae prior to the end-Cretaceous extinction.

Tissue samples from Australian carpet pythons (Morelia spilota) with neurological disease were screened for viruses using next-generation sequencing. Coding complete genomes of two bornaviruses were identified with the gene order 3’-N-X-P-G-M-L, representing a transposition of the G and M genes compared to other bornaviruses and most mononegaviruses. Use of these viruses to search available vertebrate genomes enabled recognition of further endogenous bornavirus-like elements (EBLs) in diverse placental mammals, including humans. Codivergence patterns and shared integration sites revealed an ancestral laurasiatherian EBLG integration (77 million years ago [MYA]) and a previously identified afrotherian EBLG integration (83 MYA). The novel python bornaviruses clustered more closely with these EBLs than with other exogenous bornaviruses, suggesting that these viruses diverged from previously known bornaviruses prior to the end-Cretaceous (K-Pg) extinction, 66 MYA. It is possible that EBLs protected mammals from ancient bornaviral disease, providing a selective advantage in the recovery from the K-Pg extinction. A degenerate PCR primer set was developed to detect a highly conserved region of the bornaviral polymerase gene. It was used to detect 15 more genetically distinct bornaviruses from Australian pythons that represent a group that is likely to contain a number of novel species.

Systematic estimation of insertion dates of endogenous bornavirus-like elements in vesper bats.

Endogenous bornavirus-like elements (EBLs) are sequences derived from bornaviruses (the family Bornaviridae) that are integrated into animal genomes. They are formed through germline insertions of segments of bornaviral transcripts into animal genomes. Because EBLs are molecular fossils of bornaviruses, they serve as precious sources of information to understand the evolutionary history of bornaviruses. Previous studies revealed the presence of many EBLs in bat genomes, especially in vesper bats, and suggested the long-term association between bats and bornaviruses. However, insertion dates of EBLs are largely unknown because of the limitations of available bat genome sequences in the public database. In this study, through a combination of database searches, PCR, and sequencing approaches, we systematically determined the gene orthologies of 13 lineages of EBLs in bats of the genus Myotis and Eptesicus and family Vespertilionidae. Using the above data, we estimated their insertion dates: the EBLs in vesper bats were inserted approximately 14.2 to 53 million years ago. These results suggest that vesper bats have been repeatedly infected by bornaviruses at different points in time during evolution. This study provides novel insights into the evolutionary history of bornaviruses and demonstrates the robustness of combining database searches, PCR, and sequencing approaches to estimate insertion dates of bornaviruses.

Potential Links between Hepadnavirus and Bornavirus Sequences in the Host Genome and Cancer.

Various viruses leave their sequences in the host genomes during infection. Such events occur mainly in retrovirus infection but also sometimes in DNA and non-retroviral RNA virus infections. If viral sequences are integrated into the genomes of germ line cells, the sequences can become inherited as endogenous viral elements (EVEs). The integration events of viral sequences may have oncogenic potential. Because proviral integrations of some retroviruses and/or reactivation of endogenous retroviruses are closely linked to cancers, viral insertions related to non-retroviral viruses also possibly contribute to cancer development. This article focuses on genomic viral sequences derived from two non-retroviral viruses, whose endogenization is already reported, and discusses their possible contributions to cancer. Viral insertions of hepatitis B virus play roles in the development of hepatocellular carcinoma. Endogenous bornavirus-like elements, the only non-retroviral RNA virus-related EVEs found in the human genome, may also be involved in cancer formation. In addition, the possible contribution of the interactions between viruses and retrotransposons, which seem to be a major driving force for generating EVEs related to non-retroviral RNA viruses, to cancers will be discussed. Future studies regarding the possible links described here may open a new avenue for the development of novel therapeutics for tumor virus-related cancers and/or provide novel insights into EVE functions.

Possible roles of endogenous RNA virus elements in RNA virus infection.

Endogenous bornavirus-like elements (EBLs) are ancient bornavirus-derived sequence in the genomes of eukaryotes. Expression profile of EBLs suggests that, although most of the EBLs in mammalian genomes have lost their coding potential, many of them are transcribed in a cell-type specific or ubiquitous manner. This observation leads us to speculate that EBLs may have functions in their host cells. Here we describe possible functions of EBLs and their evolutionary significance. Our recent studies revealed that EBLs in some mammals, including humans, play critical roles in viral infection as either RNAs or proteins in previously undescribed mechanisms. Considering that species having EBLs in their genomes appear to be relatively resistance to BDV-mediated pathogenesis, endogenization of RNA viruses might be an evolutionarily inevitable event in the adaptation of hosts to the viruses.

Taxonomic reorganization of the family Bornaviridae.

Knowledge of bornaviruses has expanded considerably during the last decade. A possible reservoir of mammalian Borna disease virus has been identified, divergent bornaviruses have been detected in birds and reptiles, and endogenous bornavirus-like elements have been discovered in the genomes of vertebrates of several species. Previous sequence comparisons and alignments have indicated that the members of the current family Bornaviridae are phylogenetically diverse and are not adequately classified in the existing bornavirus taxonomy supported by the International Committee on Taxonomy of Viruses (ICTV). We provide an update of these analyses and describe their implications for taxonomy. We propose retaining the family name Bornaviridae and the genus Bornavirus but reorganizing species classification. PAirwise Sequence Comparison (PASC) of bornavirus genomes and Basic Local Alignment Search Tool (BLAST) comparison of genomic and protein sequences, in combination with other already published phylogenetic analyses and known biological characteristics of bornaviruses, indicate that this genus should include at least five species: Mammalian 1 bornavirus (classical Borna disease virus and divergent Borna disease virus isolate No/98), Psittaciform 1 bornavirus (avian/psittacine bornaviruses 1, 2, 3, 4, 7), Passeriform 1 bornavirus (avian/canary bornaviruses C1, C2, C3, LS), Passeriform 2 bornavirus (estrildid finch bornavirus EF), and Waterbird 1 bornavirus (avian bornavirus 062CG). This classification is also in line with biological characteristics of these viruses and their vertebrate hosts. A snake bornavirus, proposed to be named Loveridge's garter snake virus 1, should be classified as a member of an additional species (Elapid 1 bornavirus), unassigned to a genus, in the family Bornaviridae. Avian bornaviruses 5, 6, MALL, and another "reptile bornavirus" ("Gaboon viper virus") should stay unclassified until further information becomes available. Finally, we propose new virus names and abbreviations when necessary to achieve clear differentiation and unique identification.

Inhibition of Borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome.

Animal genomes contain endogenous viral sequences, such as endogenous retroviruses and retrotransposons. Recently, we and others discovered that nonretroviral viruses also have been endogenized in many vertebrate genomes. Bornaviruses belong to the Mononegavirales and have left endogenous fragments, called "endogenous bornavirus-like elements" (EBLs), in the genomes of many mammals. The striking features of EBLs are that they contain relatively long ORFs which have high sequence homology to the extant bornavirus proteins. Furthermore, some EBLs derived from bornavirus nucleoprotein (EBLNs) have been shown to be transcribed as mRNA and probably are translated into proteins. These features lead us to speculate that EBLs may function as cellular coopted genes. An EBLN element in the genome of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), itEBLN, encodes an ORF with 77% amino acid sequence identity to the current bornavirus nucleoprotein. In this study, we cloned itEBLN from the ground squirrel genome and investigated its involvement in Borna disease virus (BDV) replication. Interestingly, itEBLN, but not a human EBLN, colocalized with the viral factory in the nucleus and appeared to affect BDV polymerase activity by being incorporated into the viral ribonucleoprotein. Our data show that, as do certain endogenous retroviruses, itEBLN potentially may inhibit infection by related exogenous viruses in vivo.