Using artificial intelligence to document the hidden RNA virosphere.

Current metagenomic tools can fail to identify highly divergent RNA viruses. We developed a deep learning algorithm, termed LucaProt, to discover highly divergent RNA-dependent RNA polymerase (RdRP) sequences in 10,487 metatranscriptomes generated from diverse global ecosystems. LucaProt integrates both sequence and predicted structural information, enabling the accurate detection of RdRP sequences. Using this approach, we identified 161,979 potential RNA virus species and 180 RNA virus supergroups, including many previously poorly studied groups, as well as RNA virus genomes of exceptional length (up to 47,250 nucleotides) and genomic complexity. A subset of these novel RNA viruses was confirmed by RT-PCR and RNA/DNA sequencing. Newly discovered RNA viruses were present in diverse environments, including air, hot springs, and hydrothermal vents, with virus diversity and abundance varying substantially among ecosystems. This study advances virus discovery, highlights the scale of the virosphere, and provides computational tools to better document the global RNA virome.

Potent monoclonal antibody-mediated neutralization of a divergent Hendra virus variant.

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic Henipaviruses (HNVs) responsible for recurrent outbreaks in humans and domestic species of highly fatal (50 to 95%) disease. A HeV variant (HeV-g2) of unprecedented genetic divergence has been identified in two fatally diseased horses, and in two flying fox species in regions of Australia not previously considered at risk for HeV spillover. Given the HeV-g2 divergence from HeV while retaining equivalent pathogenicity and spillover potential, understanding receptor usage and antigenic properties is urgently required to guide One Health biosecurity. Here, we show that the HeV-g2 G glycoprotein shares a conserved receptor tropism with prototypic HeV and that a panel of monoclonal antibodies recognizing the G and F glycoproteins potently neutralizes HeV-g2­ and HeV G/F­mediated entry into cells. We determined a crystal structure of the Fab fragment of the hAH1.3 antibody bound to the HeV G head domain, revealing an antigenic site associated with potent cross-neutralization of both HeV-g2 and HeV. Structure-guided formulation of a tetravalent monoclonal antibody (mAb) mixture, targeting four distinct G head antigenic sites, results in potent neutralization of HeV and HeV-g2 and delineates a path forward for implementing multivalent mAb combinations for postexposure treatment of HNV infections.

The evolutionary history of vertebrate RNA viruses.

Our understanding of the diversity and evolution of vertebrate RNA viruses is largely limited to those found in mammalian and avian hosts and associated with overt disease. Here, using a large-scale meta-transcriptomic approach, we discover 214 vertebrate-associated viruses in reptiles, amphibians, lungfish, ray-finned fish, cartilaginous fish and jawless fish. The newly discovered viruses appear in every family or genus of RNA virus associated with vertebrate infection, including those containing human pathogens such as influenza virus, the Arenaviridae and Filoviridae families, and have branching orders that broadly reflected the phylogenetic history of their hosts. We establish a long evolutionary history for most groups of vertebrate RNA virus, and support this by evaluating evolutionary timescales using dated orthologous endogenous virus elements. We also identify new vertebrate-specific RNA viruses and genome architectures, and re-evaluate the evolution of vector-borne RNA viruses. In summary, this study reveals diverse virus-host associations across the entire evolutionary history of the vertebrates.

Author Correction: The evolutionary history of vertebrate RNA viruses.

Change history: In this Article, author Li Liu should be associated with affiliation number 5 (College of Marine Sciences, South China Agricultural University, Guangzhou, Guangdong, China), rather than affiliation number 4 (Wenzhou Center for Disease Control and Prevention, Wenzhou, Zhejiang, China). This has been corrected online.

New Detection of Locally Acquired Japanese Encephalitis Virus Using Clinical Metagenomics, New South Wales, Australia.

In the context of an emerging Japanese encephalitis outbreak within Australia, we describe a novel locally acquired case in New South Wales. A man in his 70s had rapidly progressive, fatal meningoencephalitis, diagnosed as caused by Japanese encephalitis virus by RNA-based metagenomic next-generation sequencing performed on postmortem brain tissue.

Fatal Human Neurologic Infection Caused by Pigeon Avian Paramyxovirus-1, Australia.

Avian paramyxovirus type 1 (APMV-1) is a virus of birds that results in a range of outcomes, from asymptomatic infections to outbreaks of systemic respiratory and neurologic disease, depending on the virus strain and the avian species affected. Humans are rarely affected; those who are predominantly experience mild conjunctivitis. We report a fatal case of neurologic disease in a 2-year-old immunocompromised child in Australia. Metagenomic sequencing and histopathology identified the causative agent as the pigeon variant of APMV-1. This diagnosis should be considered in neurologic conditions of undefined etiologies. Agnostic metagenomic sequencing methods are useful in such settings to direct diagnostic and therapeutic efforts.

Plasma-Derived HIV-1 Virions Contain Considerable Levels of Defective Genomes.

Genetically-characterizing full-length HIV-1 RNA is critical for identifying genetically-intact genomes and for comparing these RNA genomes to proviral DNA. We have developed a method for sequencing plasma-derived RNA using long-range sequencing (PRLS assay; ∼8.3 kb from gag to the 3' end or ∼5 kb from integrase to the 3' end). We employed the gag-3' PRLS assay to sequence HIV-1 RNA genomes from ART-naive participants during acute/early infection (n = 6) or chronic infection (n = 2). On average, only 65% of plasma-derived genomes were genetically-intact. Defects were found in all genomic regions but were concentrated in env and pol. We compared these genomes to near-full-length proviral sequences from paired peripheral blood mononuclear cell (PBMC) samples for the acute/early group and found that near-identical (>99.98% identical) sequences were identified only during acute infection. For three participants who initiated therapy during acute infection, we used the int-3' PRLS assay to sequence plasma-derived genomes from an analytical treatment interruption and identified 100% identical genomes between pretherapy and rebound time points. The PRLS assay provides a new level of sensitivity for understanding the genetic composition of plasma-derived HIV-1 RNA from viremic individuals either pretherapy or after treatment interruption, which will be invaluable in assessing possible HIV-1 curative strategies. IMPORTANCE We developed novel plasma-derived RNA using long-range sequencing assays (PRLS assay; 8.3 kb, gag-3', and 5.0 kb, int-3'). Employing the gag-3' PRLS assay, we found that 26% to 51% of plasma-derived genomes are genetically-defective, largely as a result of frameshift mutations and deletions. These genetic defects were concentrated in the env region compared to gag and pol, likely a reflection of viral immune escape in env during untreated HIV-1 infection. Employing the int-3' PRLS assay, we found that analytical treatment interruption (ATI) plasma-derived sequences were identical and genetically-intact. Several sequences from the ATI plasma samples were identical to viral sequences from pretherapy plasma and PBMC samples, indicating that HIV-1 reservoirs established prior to therapy contribute to viral rebound during an ATI. Therefore, near-full-length sequencing of HIV-1 particles is required to gain an accurate picture of the genetic landscape of plasma HIV-1 virions in studies of HIV-1 replication and persistence.

Cellular Activation, Differentiation, and Proliferation Influence the Dynamics of Genetically Intact Proviruses Over Time.

Human immunodeficiency virus (HIV) persists in cells despite antiretroviral therapy; however, the influence of cellular mechanisms such as activation, differentiation, and proliferation upon the distribution of proviruses over time is unclear. To address this, we used full-length sequencing to examine proviruses within memory CD4+ T-cell subsets longitudinally in 8 participants. Over time, the odds of identifying a provirus increased in effector and decreased in transitional memory cells. In all subsets, more activated (HLA-DR-expressing) cells contained a higher frequency of intact provirus, as did more differentiated cells such as transitional and effector memory subsets. The proportion of genetically identical proviruses increased over time, indicating that cellular proliferation was maintaining the persistent reservoir; however, the number of genetically identical proviral clusters in each subset was stable. As such, key biological processes of activation, differentiation, and proliferation influence the dynamics of the HIV reservoir and must be considered during the development of any immune intervention.

Novel Hendra Virus Variant Circulating in Black Flying Foxes and Grey-Headed Flying Foxes, Australia.

A novel Hendra virus variant, genotype 2, was recently discovered in a horse that died after acute illness and in Pteropus flying fox tissues in Australia. We detected the variant in flying fox urine, the pathway relevant for spillover, supporting an expanded geographic range of Hendra virus risk to horses and humans.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Identification of SARS-CoV-2 Nucleocapsid and Spike T-Cell Epitopes for Assessing T-Cell Immunity.

Developing optimal T-cell response assays to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is critical for measuring the duration of immunity to this disease and assessing the efficacy of vaccine candidates. These assays need to target conserved regions of SARS-CoV-2 global variants and avoid cross-reactivity to seasonal human coronaviruses. To contribute to this effort, we employed an in silico immunoinformatics analysis pipeline to identify immunogenic peptides resulting from conserved and highly networked regions with topological importance from the SARS-CoV-2 nucleocapsid and spike proteins. A total of 57 highly networked T-cell epitopes that are conserved across geographic viral variants were identified from these viral proteins, with a binding potential to diverse HLA alleles and 80 to 100% global population coverage. Importantly, 18 of these T-cell epitope derived peptides had limited homology to seasonal human coronaviruses making them promising candidates for SARS-CoV-2-specific T-cell immunity assays. Moreover, two of the NC-derived peptides elicited effector/polyfunctional responses of CD8+ T cells derived from SARS-CoV-2 convalescent patients.IMPORTANCE The development of specific and validated immunologic tools is critical for understanding the level and duration of the cellular response induced by SARS-CoV-2 infection and/or vaccines against this novel coronavirus disease. To contribute to this effort, we employed an immunoinformatics analysis pipeline to define 57 SARS-CoV-2 immunogenic peptides within topologically important regions of the nucleocapsid (NC) and spike (S) proteins that will be effective for detecting cellular immune responses in 80 to 100% of the global population. Our immunoinformatics analysis revealed that 18 of these peptides had limited homology to circulating seasonal human coronaviruses and therefore are promising candidates for distinguishing SARS-CoV-2-specific immune responses from pre-existing coronavirus immunity. Importantly, CD8+ T cells derived from SARS-CoV-2 survivors exhibited polyfunctional effector responses to two novel NC-derived peptides identified as HLA-binders. These studies provide a proof of concept that our immunoinformatics analysis pipeline identifies novel immunogens which can elicit polyfunctional SARS-CoV-2-specific T-cell responses.

Redefining the invertebrate RNA virosphere.

Current knowledge of RNA virus biodiversity is both biased and fragmentary, reflecting a focus on culturable or disease-causing agents. Here we profile the transcriptomes of over 220 invertebrate species sampled across nine animal phyla and report the discovery of 1,445 RNA viruses, including some that are sufficiently divergent to comprise new families. The identified viruses fill major gaps in the RNA virus phylogeny and reveal an evolutionary history that is characterized by both host switching and co-divergence. The invertebrate virome also reveals remarkable genomic flexibility that includes frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Together, these data present a view of the RNA virosphere that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.

Metatranscriptomic Analysis of Virus Diversity in Urban Wild Birds with Paretic Disease.

Wild birds are major natural reservoirs and potential dispersers of a variety of infectious diseases. As such, it is important to determine the diversity of viruses they carry and use this information to help understand the potential risks of spillover to humans, domestic animals, and other wildlife. We investigated the potential viral causes of paresis in long-standing, but undiagnosed, disease syndromes in wild Australian birds. RNA from diseased birds was extracted and pooled based on tissue type, host species, and clinical manifestation for metagenomic sequencing. Using a bulk and unbiased metatranscriptomic approach, combined with clinical investigation and histopathology, we identified a number of novel viruses from the families Astroviridae, Adenoviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, and Circoviridae in common urban wild birds, including Australian magpies, magpie larks, pied currawongs, Australian ravens, and rainbow lorikeets. In each case, the presence of the virus was confirmed by reverse transcription (RT)-PCR. These data revealed a number of candidate viral pathogens that may contribute to coronary, skeletal muscle, vascular, and neuropathology in birds of the Corvidae and Artamidae families and neuropathology in members of the Psittaculidae The existence of such a diverse virome in urban avian species highlights the importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban environments. This information will be increasingly important for managing disease risks and conducting surveillance for potential viral threats to wildlife, livestock, and human health.IMPORTANCE Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of novel diseases in domestic animals and human populations. Using unbiased RNA sequencing, we identified highly diverse viruses in native birds from Australian urban environments presenting with paresis. This research included the clinical investigation and description of poorly understood recurring syndromes of unknown etiology: clenched claw syndrome and black and white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, this study describes methods that can effectively and efficiently characterize emergent disease syndromes in free-ranging wildlife and promotes further surveillance for specific pathogens of potential conservation and zoonotic concern.

Novel RNA viruses associated with Plasmodium vivax in human malaria and Leucocytozoon parasites in avian disease.

Eukaryotes of the genus Plasmodium cause malaria, a parasitic disease responsible for substantial morbidity and mortality in humans. Yet, the nature and abundance of any viruses carried by these divergent eukaryotic parasites is unknown. We investigated the Plasmodium virome by performing a meta-transcriptomic analysis of blood samples taken from patients suffering from malaria and infected with P. vivax, P. falciparum or P. knowlesi. This resulted in the identification of a narnavirus-like sequence, encoding an RNA polymerase and restricted to P. vivax samples, as well as an associated viral segment of unknown function. These data, confirmed by PCR, are indicative of a novel RNA virus that we term Matryoshka RNA virus 1 (MaRNAV-1) to reflect its analogy to a "Russian doll": a virus, infecting a parasite, infecting an animal. Additional screening revealed that MaRNAV-1 was abundant in geographically diverse P. vivax derived from humans and mosquitoes, strongly supporting its association with this parasite, and not in any of the other Plasmodium samples analyzed here nor Anopheles mosquitoes in the absence of Plasmodium. Notably, related bi-segmented narnavirus-like sequences (MaRNAV-2) were retrieved from Australian birds infected with a Leucocytozoon-a genus of eukaryotic parasites that group with Plasmodium in the Apicomplexa subclass hematozoa. Together, these data support the establishment of two new phylogenetically divergent and genomically distinct viral species associated with protists, including the first virus likely infecting Plasmodium parasites. As well as broadening our understanding of the diversity and evolutionary history of the eukaryotic virosphere, the restriction to P. vivax may be of importance in understanding P. vivax-specific biology in humans and mosquitoes, and how viral co-infection might alter host responses at each stage of the P. vivax life-cycle.

Punctuated Evolution of Myxoma Virus: Rapid and Disjunct Evolution of a Recent Viral Lineage in Australia.

Myxoma virus (MYXV) has been evolving in a novel host species-European rabbits-in Australia since 1950. Previous studies of viruses sampled from 1950 to 1999 revealed a remarkably clock-like evolutionary process across all Australian lineages of MYXV. Through an analysis of 49 newly generated MYXV genome sequences isolated in Australia between 2008 and 2017, we show that MYXV evolution in Australia can be characterized by three lineages, one of which exhibited a greatly elevated rate of evolutionary change and a dramatic breakdown of temporal structure. Phylogenetic analysis revealed that this apparently punctuated evolutionary event occurred between 1996 and 2012. The branch leading to the rapidly evolving lineage contained a relatively high number of nonsynonymous substitutions, and viruses in this lineage reversed a mutation found in the progenitor standard laboratory strain (SLS) and all previous sequences that disrupts the reading frame of the M005L/R gene. Analysis of genes encoding proteins involved in DNA synthesis or RNA transcription did not reveal any mutations likely to cause rapid evolution. Although there was some evidence for recombination across the MYXV phylogeny, this was not associated with the increase in the evolutionary rate. The period from 1996 to 2012 saw significant declines in wild rabbit numbers, due to the introduction of rabbit hemorrhagic disease and prolonged drought in southeastern Australia, followed by the partial recovery of populations. It is therefore possible that a rapidly changing environment for virus transmission changed the selection pressures faced by MYXV, altering the course and pace of virus evolution.IMPORTANCE The coevolution of myxoma virus (MYXV) and European rabbits in Australia is one of the most important natural experiments in evolutionary biology, providing insights into virus adaptation to new hosts and the evolution of virulence. Previous studies of MYXV evolution have also shown that the virus evolves both relatively rapidly and in a strongly clock-like manner. Using newly acquired MYXV genome sequences from Australia, we show that the virus has experienced a dramatic change in evolutionary behavior over the last 20 years, with a breakdown in clock-like structure, the appearance of a rapidly evolving virus lineage, and the accumulation of multiple nonsynonymous and indel mutations. We suggest that this punctuated evolutionary event may reflect a change in selection pressures as rabbit numbers declined following the introduction of rabbit hemorrhagic disease virus and drought in the geographic regions inhabited by rabbits.

Extensive Diversity of RNA Viruses in Australian Ticks.

Understanding the microbiome of ticks in Australia is of considerable interest given the ongoing debate over whether Lyme disease and its causative agent, the bacterium Borrelia burgdorferisensu lato, are present in Australia. The diversity of bacteria infecting Australian ticks has been studied using both culture- and metagenomics-based techniques. However, little is known about the virome of Australian ticks, including whether this includes viruses with the potential to infect mammals. We used a meta-transcriptomics approach to reveal the diversity and evolution of viruses from Australian ticks collected from two locations on the central east coast of Australia, including metropolitan Sydney. From this we identified 19 novel RNA viruses belonging to 12 families, as well as 1 previously described RNA virus. The majority of these viruses were related to arthropod-associated viruses, suggesting that they do not utilize mammalian hosts. However, two novel viruses discovered in ticks feeding on bandicoot marsupials clustered closely within the mammal-associated hepacivirus and pestivirus groups (family Flaviviridae). Another bandicoot tick yielded a novel coltivirus (family Reoviridae), a group of largely tick-associated viruses containing the known human pathogen Colorado tick fever virus and its relative, Eyach virus. Importantly, our transcriptomic data provided no evidence for the presence of B. burgdorferisensu lato in any tick sample, providing further evidence against the presence of Lyme disease in Australia. In sum, this study reveals that Australian ticks harbor a diverse virome, including some viruses that merit additional screening in the context of emerging infectious disease.IMPORTANCE Each year a growing number of individuals along the east coast of Australia experience debilitating disease following tick bites. As there is no evidence for the presence of the causative agent of Lyme disease, Borrelia burgdorferisensu lato, in Australian ticks, the etiological basis of this disease syndrome remains controversial. To characterize the viruses associated with Australian ticks, particularly those that might be associated with mammalian infection, we performed unbiased RNA sequencing on 146 ticks collected across two locations along the coast of New South Wales, Australia. This revealed 19 novel RNA viruses from a diverse set of families. Notably, three of these viruses clustered with known mammalian viruses, including a novel coltivirus that was related to the human pathogen Colorado tick fever virus.

Correction: The paradox of HBV evolution as revealed from a 16th century mummy.

[This corrects the article DOI: 10.1371/journal.ppat.1006750.].

The paradox of HBV evolution as revealed from a 16th century mummy.

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen.

Meta-transcriptomics reveals a diverse antibiotic resistance gene pool in avian microbiomes.

BACKGROUND: Antibiotic resistance is rendering common bacterial infections untreatable. Wildlife can incorporate and disperse antibiotic-resistant bacteria in the environment, such as water systems, which in turn serve as reservoirs of resistance genes for human pathogens. Anthropogenic activity may contribute to the spread of bacterial resistance cycling through natural environments, including through the release of human waste, as sewage treatment only partially removes antibiotic-resistant bacteria. However, empirical data supporting these effects are currently limited. Here we used bulk RNA-sequencing (meta-transcriptomics) to assess the diversity and expression levels of functionally viable resistance genes in the gut microbiome of birds with aquatic habits in diverse locations. RESULTS: We found antibiotic resistance genes in birds from all localities, from penguins in Antarctica to ducks in a wastewater treatment plant in Australia. Comparative analysis revealed that birds feeding at the wastewater treatment plant carried the greatest resistance gene burden, including genes typically associated with multidrug resistance plasmids as the aac(6)-Ib-cr gene. Differences in resistance gene burden also reflected aspects of bird ecology, taxonomy, and microbial function. Notably, ducks, which feed by dabbling, carried a higher abundance and diversity of resistance genes than turnstones, avocets, and penguins, which usually prey on more pristine waters. CONCLUSIONS: These transcriptome data suggest that human waste, even if it undergoes treatment, might contribute to the spread of antibiotic resistance genes to the wild. Differences in microbiome functioning across different bird lineages may also play a role in the antibiotic resistance burden carried by wild birds. In summary, we reveal the complex factors explaining the distribution of resistance genes and their exchange routes between humans and wildlife, and show that meta-transcriptomics is a valuable tool to access functional resistance genes in whole microbial communities.

Viral Discovery in the Invasive Australian Cane Toad (Rhinella marina) Using Metatranscriptomic and Genomic Approaches.

Cane toads are a notorious invasive species, inhabiting over 1.2 million km2 of Australia and threatening native biodiversity. The release of pathogenic cane toad viruses is one possible biocontrol strategy yet is currently hindered by the poorly described cane toad virome. Metatranscriptomic analysis of 16 cane toad livers revealed the presence of a novel and full-length picornavirus, Rhimavirus A (RhiV-A), a member of a reptile- and amphibian-specific cluster of the Picornaviridae basal to the Kobuvirus-like group. In the combined liver transcriptome, we also identified a complete genome sequence of a distinct epsilonretrovirus, Rhinella marina endogenous retrovirus (RMERV). The recently sequenced cane toad genome contains 8 complete RMERV proviruses as well as 21 additional truncated insertions. The oldest full-length RMERV provirus was estimated to have inserted 1.9 million years ago (MYA). To screen for these viral sequences in additional toads, we analyzed publicly available transcriptomes from six diverse Australian locations. RhiV-A transcripts were identified in toads sampled from three locations across 1,000 km of Australia, stretching to the current Western Australia (WA) invasion front, while RMERV transcripts were observed at all six sites. Finally, we scanned the cane toad genome for nonretroviral endogenous viral elements, finding three sequences related to small DNA viruses in the family Circoviridae This shows ancestral circoviral infection with subsequent genomic integration. The identification of these current and past viral infections enriches our knowledge of the cane toad virome, an understanding of which will facilitate future work on infection and disease in this important invasive species.IMPORTANCE Cane toads are poisonous amphibians that were introduced to Australia in 1935 for insect control. Since then, their population has increased dramatically, and they now threaten many native Australian species. One potential method to control the population is to release a cane toad virus with high mortality rates, yet few cane toad viruses have been characterized. This study samples cane toads from different Australian locations and uses an RNA sequencing and computational approach to find new viruses. We report novel complete picornavirus and retrovirus sequences that were genetically similar to viruses infecting frogs, reptiles, and fish. Using data generated in other studies, we show that these viral sequences are present in cane toads from distinct Australian locations. Three sequences related to circoviruses were also found in the toad genome. The identification of new viral sequences will aid future studies that investigate their prevalence and potential as agents for biocontrol.