Structures of the Hepaci-, Pegi-, and Pestiviruses envelope proteins suggest a novel membrane fusion mechanism.

Enveloped viruses encode specialised glycoproteins that mediate fusion of viral and host membranes. Discovery and understanding of the molecular mechanisms of fusion have been achieved through structural analyses of glycoproteins from many different viruses, and yet the fusion mechanisms of some viral genera remain unknown. We have employed systematic genome annotation and AlphaFold modelling to predict the structures of the E1E2 glycoproteins from 60 viral species in the Hepacivirus, Pegivirus, and Pestivirus genera. While the predicted structure of E2 varied widely, E1 exhibited a very consistent fold across genera, despite little or no similarity at the sequence level. Critically, the structure of E1 is unlike any other known viral glycoprotein. This suggests that the Hepaci-, Pegi-, and Pestiviruses may possess a common and novel membrane fusion mechanism. Comparison of E1E2 models from various species reveals recurrent features that are likely to be mechanistically important and sheds light on the evolution of membrane fusion in these viral genera. These findings provide new fundamental understanding of viral membrane fusion and are relevant to structure-guided vaccinology.

Comparative analysis reveals the long-term coevolutionary history of parvoviruses and vertebrates.

Parvoviruses (family Parvoviridae) are small DNA viruses that cause numerous diseases of medical, veterinary, and agricultural significance and have important applications in gene and anticancer therapy. DNA sequences derived from ancient parvoviruses are common in animal genomes and analysis of these endogenous parvoviral elements (EPVs) has demonstrated that the family, which includes twelve vertebrate-specific genera, arose in the distant evolutionary past. So far, however, such "paleovirological" analysis has only provided glimpses into the biology of ancient parvoviruses and their long-term evolutionary interactions with hosts. Here, we comprehensively map EPV diversity in 752 published vertebrate genomes, revealing defining aspects of ecology and evolution within individual parvovirus genera. We identify 364 distinct EPV sequences and show these represent approximately 200 unique germline incorporation events, involving at least five distinct parvovirus genera, which took place at points throughout the Cenozoic Era. We use the spatiotemporal and host range calibrations provided by these sequences to infer defining aspects of long-term evolution within individual parvovirus genera, including mammalian vicariance for genus Protoparvovirus, and interclass transmission for genus Dependoparvovirus. Moreover, our findings support a model of virus evolution in which the long-term cocirculation of multiple parvovirus genera in vertebrates reflects the adaptation of each viral genus to fill a distinct ecological niche. Our findings show that efforts to develop parvoviruses as therapeutic tools can be approached from a rational foundation based on comparative evolutionary analysis. To support this, we published our data in the form of an open, extensible, and cross-platform database designed to facilitate the wider utilisation of evolution-related domain knowledge in parvovirus research.

Making genomic surveillance deliver: A lineage classification and nomenclature system to inform rabies elimination.

The availability of pathogen sequence data and use of genomic surveillance is rapidly increasing. Genomic tools and classification systems need updating to reflect this. Here, rabies virus is used as an example to showcase the potential value of updated genomic tools to enhance surveillance to better understand epidemiological dynamics and improve disease control. Previous studies have described the evolutionary history of rabies virus, however the resulting taxonomy lacks the definition necessary to identify incursions, lineage turnover and transmission routes at high resolution. Here we propose a lineage classification system based on the dynamic nomenclature used for SARS-CoV-2, defining a lineage by phylogenetic methods for tracking virus spread and comparing sequences across geographic areas. We demonstrate this system through application to the globally distributed Cosmopolitan clade of rabies virus, defining 96 total lineages within the clade, beyond the 22 previously reported. We further show how integration of this tool with a new rabies virus sequence data resource (RABV-GLUE) enables rapid application, for example, highlighting lineage dynamics relevant to control and elimination programmes, such as identifying importations and their sources, as well as areas of persistence and routes of virus movement, including transboundary incursions. This system and the tools developed should be useful for coordinating and targeting control programmes and monitoring progress as countries work towards eliminating dog-mediated rabies, as well as having potential for broader application to the surveillance of other viruses.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

Retroviruses drive the rapid evolution of mammalian APOBEC3 genes.

APOBEC3 (A3) genes are members of the AID/APOBEC gene family that are found exclusively in mammals. A3 genes encode antiviral proteins that restrict the replication of retroviruses by inducing G-to-A mutations in their genomes and have undergone extensive amplification and diversification during mammalian evolution. Endogenous retroviruses (ERVs) are sequences derived from ancient retroviruses that are widespread mammalian genomes. In this study we characterize the A3 repertoire and use the ERV fossil record to explore the long-term history of coevolutionary interaction between A3s and retroviruses. We examine the genomes of 160 mammalian species and identify 1,420 AID/APOBEC-related genes, including representatives of previously uncharacterized lineages. We show that A3 genes have been amplified in mammals and that amplification is positively correlated with the extent of germline colonization by ERVs. Moreover, we demonstrate that the signatures of A3-mediated mutation can be detected in ERVs found throughout mammalian genomes and show that in mammalian species with expanded A3 repertoires, ERVs are significantly enriched for G-to-A mutations. Finally, we show that A3 amplification occurred concurrently with prominent ERV invasions in primates. Our findings establish that conflict with retroviruses is a major driving force for the rapid evolution of mammalian A3 genes.

An endogenous lentivirus in the germline of a rodent.

Lentiviruses (genus Lentivirus) are complex retroviruses that infect a broad range of mammals, including humans. Unlike many other retrovirus genera, lentiviruses have only rarely been incorporated into the mammalian germline. However, a small number of endogenous retrovirus (ERV) lineages have been identified, and these rare genomic "fossils" can provide crucial insights into the long-term history of lentivirus evolution. Here, we describe a previously unreported endogenous lentivirus lineage in the genome of the South African springhare (Pedetes capensis), demonstrating that the host range of lentiviruses has historically extended to rodents (order Rodentia). Furthermore, through comparative and phylogenetic analysis of lentivirus and ERV genomes, considering the biogeographic and ecological characteristics of host species, we reveal broader insights into the long-term evolutionary history of the genus.

Derivation of simian tropic HIV-1 infectious clone reveals virus adaptation to a new host.

To replicate in a new host, lentiviruses must adapt to exploit required host factors and evade species-specific antiviral proteins. Understanding how host protein variation drives lentivirus adaptation allowed us to expand the host range of HIV-1 to pigtail macaques. We have previously derived a viral swarm (in the blood of infected animals) that can cause AIDS in this new host. To further exploit this reagent, we generated infectious molecular clones (IMCs) from the viral swarm. We identified clones with high replicative capacity in pigtail peripheral blood mononuclear cells (PBMC) in vitro and used in vivo replication to select an individual IMC, named stHIV-A19 (for simian tropic HIV-1 clone A19), which recapitulated the phenotype obtained with the viral swarm. Adaptation of HIV-1 in macaques led to the acquisition of amino acid changes in viral proteins, such as capsid (CA), that are rarely seen in HIV-1-infected humans. Using stHIV-A19, we show that these CA changes confer a partial resistance to the host cell inhibitor Mx2 from pigtail macaques, but that complete resistance is associated with a fitness defect. Adaptation of HIV-1 to a new host will lead to a more accurate animal model and a better understanding of virus-host interactions.

Molecular Properties and Evolutionary Origins of a Parvovirus-Derived Myosin Fusion Gene in Guinea Pigs.

Sequences derived from parvoviruses (family Parvoviridae) are relatively common in animal genomes, but the functional significance of these endogenous parvoviral element (EPV) sequences remains unclear. In this study, we used a combination of in silico and molecular biological approaches to investigate a fusion gene carried by guinea pigs (genus Cavia) that is partially derived from an EPV. This gene, named enRep-M9l, encodes a predicted polypeptide gene product comprising a partial myosin9-like (M9l) gene fused to a 3' truncated, EPV-encoded replicase. We examined the genomic and phylogenetic characteristics of the EPV locus (enRep) that encodes the viral portions of enRep-M9l, revealing that it derives from an ancient dependoparvovirus (genus Dependoparvovirus) that was incorporated into the guinea pig germ line between approximately 22 and 35 million years ago (MYA). Despite these ancient origins, the regions of the enRep locus that are expressed in the enRep-M9l gene are conserved across multiple species in the family Caviidae (guinea pigs and cavies), consistent with a potential function at the amino acid level. Using molecular biological approaches, we further demonstrated that (i) enRep-M9l mRNA is broadly transcribed in guinea pig cells, (ii) the cloned enRep-M9l transcript can express a protein of the expected size in guinea pig cells in vitro, and (iii) the expressed protein localizes to the cytosol. Our findings demonstrate that, consistent with a functional role, the enRep-M9l fusion gene is evolutionarily conserved, broadly transcribed, and capable of expressing protein.IMPORTANCE DNA from viruses has been "horizontally transferred" to mammalian genomes during evolution, but the impact of this process on mammalian biology remains poorly understood. The findings of our study indicate that a novel gene has evolved in guinea pigs through fusion of host and virus genes.

Examination and Reconstruction of Three Ancient Endogenous Parvovirus Capsid Protein Gene Remnants Found in Rodent Genomes.

Parvovirus-derived endogenous viral elements (EVEs) have been found in the genomes of many different animal species, resulting from integration events that may have occurred from more than 50 million years ago to much more recently. Here, we further investigate the properties of autonomous parvovirus EVEs and describe their relationships to contemporary viruses. While we did not find any intact capsid protein open reading frames in the integrated viral sequences, we examined three EVEs that were repaired to form full-length sequences with relatively few changes. These sequences were found in the genomes of Rattus norvegicus (brown rat), Mus spretus (Algerian mouse), and Apodemus sylvaticus (wood mouse). The R. norvegicus sequence was not present in the genomes of the closely related species R. rattus, R. tanezumi, R. exulans, and R. everetti, indicating that it was less than 2 million years old, and the M. spretus and A. sylvaticus sequences were not found in the published genomes of other mouse species, also indicating relatively recent insertions. The M. spretus VP2 sequence assembled into capsids, which had high thermal stability, bound the sialic acid N-acetylneuraminic acid, and entered murine L cells. The 3.89-Å structure of the M. spretus virus-like particles (VLPs), determined using cryo-electron microscopy, showed similarities to rodent and porcine parvovirus capsids. The repaired VP2 sequences from R. norvegicus and A. sylvaticus did not assemble as first prepared, but chimeras combining capsid surface loops from R. norvegicus with canine parvovirus assembled, allowing some of that capsid's structures and functions to be examined.IMPORTANCE Parvovirus endogenous viral elements (EVEs) that have been incorporated into the genomes of different animals represent remnants of the DNA sequences of ancient viruses that infected the ancestors of those animals millions of years ago, but we know little about their properties or how they differ from currently circulating parvoviruses. By expressing the capsid proteins of different parvovirus EVEs that were found integrated into the genomes of three different rodents, we can examine their structures and functions. A VP2 (major capsid protein) EVE sequence from a mouse genome assembled into capsids that had a similar structure and biophysical properties to extant parvoviruses and also bound sialic acids and entered rodent cells. Chimeras formed from combinations of canine parvovirus and portions of the parvovirus sequences from the brown rat genome allowed us to examine the structures and functions of the surface loops of that EVE capsid.

Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses.

The host innate immune response mediated by type I interferon (IFN) and the resulting up-regulation of hundreds of interferon-stimulated genes (ISGs) provide an immediate barrier to virus infection. Studies of the type I 'interferome' have mainly been carried out at a single species level, often lacking the power necessary to understand key evolutionary features of this pathway. Here, using a single experimental platform, we determined the properties of the interferomes of multiple vertebrate species and developed a webserver to mine the dataset. This approach revealed a conserved 'core' of 62 ISGs, including genes not previously associated with IFN, underscoring the ancestral functions associated with this antiviral host response. We show that gene expansion contributes to the evolution of the IFN system and that interferomes are shaped by lineage-specific pressures. Consequently, each mammal possesses a unique repertoire of ISGs, including genes common to all mammals and others unique to their specific species or phylogenetic lineages. An analysis of genes commonly down-regulated by IFN suggests that epigenetic regulation of transcription is a fundamental aspect of the IFN response. Our study provides a resource for the scientific community highlighting key paradigms of the type I IFN response.

Deltaretroviruses have circulated since at least the Paleogene and infected a broad range of mammalian species.

The Deltaretrovirus genus of retroviruses (family Retroviridae) includes the human T cell leukemia viruses and bovine leukemia virus (BLV). Relatively little is known about the biology and evolution of these viruses, because only a few species have been identified and the genomic 'fossil record' is relatively sparse. Here, we report the discovery of multiple novel endogenous retroviruses (ERVs) derived from ancestral deltaretroviruses. These sequences-two of which contain complete or near complete internal coding regions-reside in genomes of several distinct mammalian orders, including bats, carnivores, cetaceans, and insectivores. We demonstrate that two of these ERVs contain unambiguous homologs of the tax gene, indicating that complex gene regulation has ancient origins within the Deltaretrovirus genus. ERVs demonstrate that the host range of the deltaretrovirus genus is much more extensive than suggested by the relatively small number of exogenous deltaretroviruses described so far, and allow the evolutionary timeline of deltaretrovirus-mammal interaction to be more accurately calibrated.

Origin and recent expansion of an endogenous gammaretroviral lineage in domestic and wild canids.

BACKGROUND: Vertebrate genomes contain a record of retroviruses that invaded the germlines of ancestral hosts and are passed to offspring as endogenous retroviruses (ERVs). ERVs can impact host function since they contain the necessary sequences for expression within the host. Dogs are an important system for the study of disease and evolution, yet no substantiated reports of infectious retroviruses in dogs exist. Here, we utilized Illumina whole genome sequence data to assess the origin and evolution of a recently active gammaretroviral lineage in domestic and wild canids. RESULTS: We identified numerous recently integrated loci of a canid-specific ERV-Fc sublineage within Canis, including 58 insertions that were absent from the reference assembly. Insertions were found throughout the dog genome including within and near gene models. By comparison of orthologous occupied sites, we characterized element prevalence across 332 genomes including all nine extant canid species, revealing evolutionary patterns of ERV-Fc segregation among species as well as subpopulations. CONCLUSIONS: Sequence analysis revealed common disruptive mutations, suggesting a predominant form of ERV-Fc spread by trans complementation of defective proviruses. ERV-Fc activity included multiple circulating variants that infected canid ancestors from the last 20 million to within 1.6 million years, with recent bursts of germline invasion in the sublineage leading to wolves and dogs.

Insights into Circovirus Host Range from the Genomic Fossil Record.

A diverse range of DNA sequences derived from circoviruses (family Circoviridae) has been identified in samples obtained from humans and domestic animals, often in association with pathological conditions. In the majority of cases, however, little is known about the natural biology of the viruses from which these sequences are derived. Endogenous circoviral elements (CVe) are DNA sequences derived from circoviruses that occur in animal genomes and provide a useful source of information about circovirus-host relationships. In this study, we screened genome assemblies of 675 animal species and identified numerous circovirus-related sequences, including the first examples of CVe derived from cycloviruses. We confirmed the presence of these CVe in the germ line of the elongate twig ant (Pseudomyrmex gracilis), thereby establishing that cycloviruses infect insects. We examined the evolutionary relationships between CVe and contemporary circoviruses, showing that CVe from ants and mites group relatively closely with cycloviruses in phylogenies. Furthermore, the relatively random interspersion of CVe from insect genomes with cyclovirus sequences recovered from vertebrate samples suggested that contamination might be an important consideration in studies reporting these viruses. Our study demonstrates how endogenous viral sequences can inform metagenomics-based virus discovery. In addition, it raises doubts about the role of cycloviruses as pathogens of humans and other vertebrates.IMPORTANCE Advances in DNA sequencing have dramatically increased the rate at which new viruses are being identified. However, the host species associations of most virus sequences identified in metagenomic samples are difficult to determine. Our analysis indicates that viruses proposed to infect vertebrates (in some cases being linked to human disease) may in fact be restricted to arthropod hosts. The detection of these sequences in vertebrate samples may reflect their widespread presence in the environment as viruses of parasitic arthropods.

GLUE: a flexible software system for virus sequence data.

BACKGROUND: Virus genome sequences, generated in ever-higher volumes, can provide new scientific insights and inform our responses to epidemics and outbreaks. To facilitate interpretation, such data must be organised and processed within scalable computing resources that encapsulate virology expertise. GLUE (Genes Linked by Underlying Evolution) is a data-centric bioinformatics environment for building such resources. The GLUE core data schema organises sequence data along evolutionary lines, capturing not only nucleotide data but associated items such as alignments, genotype definitions, genome annotations and motifs. Its flexible design emphasises applicability to different viruses and to diverse needs within research, clinical or public health contexts. RESULTS: HCV-GLUE is a case study GLUE resource for hepatitis C virus (HCV). It includes an interactive public web application providing sequence analysis in the form of a maximum-likelihood-based genotyping method, antiviral resistance detection and graphical sequence visualisation. HCV sequence data from GenBank is categorised and stored in a large-scale sequence alignment which is accessible via web-based queries. Whereas this web resource provides a range of basic functionality, the underlying GLUE project can also be downloaded and extended by bioinformaticians addressing more advanced questions. CONCLUSION: GLUE can be used to rapidly develop virus sequence data resources with public health, research and clinical applications. This streamlined approach, with its focus on reuse, will help realise the full value of virus sequence data.

An Intact Retroviral Gene Conserved in Spiny-Rayed Fishes for over 100 My.

We have identified a retroviral envelope gene with a complete, intact open reading frame (ORF) in 20 species of spiny-rayed fishes (Acanthomorpha). The taxonomic distribution of the gene, "percomORF", indicates insertion into the ancestral lineage >110 Ma, making it the oldest known conserved gene of viral origin in a vertebrate genome. Underscoring its ancient provenence, percomORF exists as an isolated ORF within the intron of a widely conserved host gene, with no discernible proviral sequence nearby. Despite its remarkable age, percomORF retains canonical features of a retroviral glycoprotein, and tests for selection strongly suggest cooption for a host function. Retroviral envelope genes have been coopted for a role in placentogenesis by numerous lineages of mammals, including eutherians and marsupials, representing a variety of placental structures. Therefore percomORF's presence within the group Percomorpha-unique among spiny-finned fishes in having evolved placentation and live birth-is especially intriguing.

Reconstruction of a replication-competent ancestral murine endogenous retrovirus-L.

BACKGROUND: About 10% of the mouse genome is composed of endogenous retroviruses (ERVs) that represent a molecular fossil record of past retroviral infections. One such retrovirus, murine ERV-L (MuERV-L) is an env-deficient ERV that has undergone episodic proliferation, with the most recent amplification occurring ~ 2 million years ago. MuERV-L related sequences have been co-opted by mice for antiretroviral defense, and possibly as promoters for some genes that regulate totipotency in early mouse embryos. However, MuERV-L sequences present in modern mouse genomes have not been observed to replicate. RESULTS: Here, we describe the reconstruction of an ancestral MuERV-L (ancML) sequence through paleovirological analyses of MuERV-L elements in the modern mouse genome. The resulting MuERV-L (ancML) sequence was synthesized and a reporter gene embedded. The reconstructed MuERV-L (ancML) could replicate in a manner that is dependent on reverse transcription and generated de novo integrants. Notably, MuERV-L (ancML) exhibited a narrow host range. Interferon-α could reduce MuERV-L (ancML) replication, suggesting the existence of interferon-inducible genes that could inhibit MuERV-L replication. While mouse APOBEC3 was able to restrict the replication of MuERV-L (ancML), inspection of endogenous MuERV-L sequences suggested that the impact of APOBEC3 mediated hypermutation on MuERV-L has been minimal. CONCLUSION: The reconstruction of an ancestral MuERV-L sequence highlights the potential for the retroviral fossil record to illuminate ancient events and enable studies of the impact of retroviral elements on animal evolution.

Nomenclature for endogenous retrovirus (ERV) loci.

Retroviral integration into germline DNA can result in the formation of a vertically inherited proviral sequence called an endogenous retrovirus (ERV). Over the course of their evolution, vertebrate genomes have accumulated many thousands of ERV loci. These sequences provide useful retrospective information about ancient retroviruses, and have also played an important role in shaping the evolution of vertebrate genomes. There is an immediate need for a unified system of nomenclature for ERV loci, not only to assist genome annotation, but also to facilitate research on ERVs and their impact on genome biology and evolution. In this review, we examine how ERV nomenclatures have developed, and consider the possibilities for the implementation of a systematic approach for naming ERV loci. We propose that such a nomenclature should not only provide unique identifiers for individual loci, but also denote orthologous relationships between ERVs in different species. In addition, we propose that-where possible-mnemonic links to previous, well-established names for ERV loci and groups should be retained. We show how this approach can be applied and integrated into existing taxonomic and nomenclature schemes for retroviruses, ERVs and transposable elements.

Late Ebola virus relapse causing meningoencephalitis: a case report.

BACKGROUND: There are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13.2). METHODS: A 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach. FINDINGS: On admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23.7) than plasma (31.3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus. INTERPRETATION: Our report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern. FUNDING: Royal Free London NHS Foundation Trust.