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Classification of viral-like transcripts present in Australian marsupial datasets. Thirty five RNA-Seq datasets from thirteen Australian marsupial species were screened for the presence of viral-like transcripts using a genome-independent bioinformatics workflow. Viral-like transcripts were grouped into families based on the top hit of the reciprocal BLASTx nr search. The colours represent the viral family of each transcript top BLASTx hit indicated in the figure legend. The total number of viral-like transcripts identified in each species is shown on the right of the bar graph.
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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 d...
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... read datasets (n=35) were quality trimmed using Trimmomatic (version 0.38, (Bolger, et al. 2014)) and assembled de novo using Trinity (v.2.5.1, Grabherr, et al. 2011) ( Figure 1, step 2). ...
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... RNA-Seq datasets were screened for the presence of viral transcripts to determine the diversity of viral families represented. A total of 200 viral transcripts were identified in 13 Australian marsupial species, from 35 RNA-Seq datasets ( Figure 2). All sequences, when translated in silico, exhibited similarity to proteins from one of six viral families: Bornaviridae (n=85, 42%), Filoviridae (n=60, 30%), Parvoviridae (n=39, 20%), Herpesviridae (n=6, 3%), Hepadnaviridae (n=4, 2%) and Anelloviridae (n=2, 1%), with the remaining four transcripts unclassifiable beyond the Riboviria realm (Figure 2). ...
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... total of 200 viral transcripts were identified in 13 Australian marsupial species, from 35 RNA-Seq datasets ( Figure 2). All sequences, when translated in silico, exhibited similarity to proteins from one of six viral families: Bornaviridae (n=85, 42%), Filoviridae (n=60, 30%), Parvoviridae (n=39, 20%), Herpesviridae (n=6, 3%), Hepadnaviridae (n=4, 2%) and Anelloviridae (n=2, 1%), with the remaining four transcripts unclassifiable beyond the Riboviria realm (Figure 2). ...
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... most numerous transcripts mapped to negative ssRNA (-ssRNA) viruses (145/188 transcripts) (Figure 3 and Supplementary Table S2). Of these, Bornaviridae-like transcripts were the most ubiquitous (n=85/188, 45%), present in 9/13 marsupial species (Figure 2 and Supplementary Table S2). These transcripts mapped to the RNA-dependent RNA polymerase (RdRp) domain of the L (large) protein (n=54/85), nucleoprotein (n=16/85), glycoprotein (n=6/85), matrix protein (n=5/85) or phosphoprotein (n=4/85) ( Figure 3, 4 and Supplementary Table S2). ...
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... transcripts that mapped to the -ssRNA Filoviridae genes were also abundant; present in 9/13 species, with a total of 60/188 (32%) sequences identified (Figure 2). Filoviridae-like transcripts were identified in the wombat, koala, long-nosed bandicoot, sugar glider, tammar wallaby, striped possum, Western pygmy possum, brushtail possum and yellow-footed rock wallaby species ( Figure 2, Supplementary Table S2). ...
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... transcripts that mapped to the -ssRNA Filoviridae genes were also abundant; present in 9/13 species, with a total of 60/188 (32%) sequences identified (Figure 2). Filoviridae-like transcripts were identified in the wombat, koala, long-nosed bandicoot, sugar glider, tammar wallaby, striped possum, Western pygmy possum, brushtail possum and yellow-footed rock wallaby species ( Figure 2, Supplementary Table S2). These transcripts, when translated in silico, were all similar to human filovirus proteins and mapped to the nucleocapsid (n=56/60), VP35 (n=3/60) and L (n=1/60) genes (Figures 3, 4 and Supplementary Table S2). ...
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... addition to -ssRNA viruses, numerous transcripts mapped to ssDNA Parvoviridae family genes (n=39/188) (Supplementary Table S2). Parvoviridae-like transcripts were common throughout the datasets, appearing in five of the 13 marsupial species studied: the fat-tailed dunnart, the koala, the long-nosed bandicoot, the tammar wallaby and the Tasmanian devil ( Figure 2 Table S2). ...
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... transcripts from the koala and wombat had BLASTx hits to viral proteins from the Riboviria realm and could not be further classified with this methodology (Figure 2). These transcripts included three putative nucleoprotein encoding sequences with identity to sequences from Guangdong red-banded snake chuvirus-like virus (n=3; 44.2% identity over 74 aa, 42.9% identity over 62 aa, 36.4% over 76 aa, GenPept: AVM87274), as well as one hypothetical protein encoding sequence similar to Sanxia atyid shrimp virus 4 (n=1; 45.5% identity over 43 aa, GenPept: YP_009337430). ...
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... from these families have only previously been identified in the tammar wallaby and Tasmanian devil genomes (Kapoor, et al. 2010;Taylor, et al. 2010). This study provides novel evidence that EVEs are transcriptionally active in a range of marsupial hosts and tissues, including: the fat-tailed dunnart, long-nosed bandicoot, Southern brown bandicoot, striped possum, brushtail possum, Western pygmy possum, yellowfooted rock wallaby and false antechinus (Figure 2 and Supplementary Table S2). This workflow also identified four possible infecting viruses. ...
Citations
... Among these, Rhabdoviridae-derived nrEVEs were the most abundant, as previously observed in mosquitos [22]. Interestingly, this pattern extends beyond the phylum Arthropoda, since negative ssRNA viruses from the Bornaviridae and Filoviridae families represented the 72% of the nrEVEs discovered in the genomes of thirteen Australian marsupial species [43]. Our study also confirms the prevalence of structural gene-coding regions in nrEVEs, as previously reported in other insect species [22,23]. ...
A wide variety of insect-specific non-retroviral RNA viruses specifically infect insects. During viral infection, fragments of viral sequences can integrate into the host genomes creating non-retroviral endogenous viral elements (nrEVEs). Although the exact function of nrEVEs is so far unknown, some studies suggest that nrEVEs may interfere with virus replication by producing PIWI-interacting RNAs (piRNAs) that recognize and degrade viral RNAs through sequence complementarity. In this article, we identified the nrEVEs repertoire of ten species within the dipteran family Tephritidae (true fruit flies), which are considered a major threat to agriculture worldwide. Our results suggest that each of these species contains nrEVEs, although in limited numbers, and that nrEVE integration may have occurred both before and after speciation. Furthermore, the majority of nrEVEs originated from viruses with negative single-stranded RNA genomes and represent structural viral functions. Notably, these nrEVEs exhibit low similarity to currently known circulating viruses. To explore the potential role of nrEVEs, we investigated their transcription pattern and the production of piRNAs in different tissues of Ceratitis capitata. We successfully identified piRNAs that are complementary to the sequence of one nrEVE in C. capitata, thereby highlighting a potential link between nrEVEs and the piRNA pathway. Overall, our results provide valuable insights into the comparative landscape of nrEVEs in true fruit flies, contributing to the understanding of the intimate relation between fruit flies and their past and present viral pathogens.
... There have also been a small number of serological and bioinformatic screening studies targeting or including marsupial carnivores. For example, the molecular and serological study of marsupial tissues for the identification of herpesviruses led to the identification of a novel herpesvirus in a Tasmanian devil (Stalder et al. 2015), while an analysis of available transcriptomic data and reference host genomes characterised the endogenous viral elements (EVEs) of marsupial carnivores (Harding et al. 2021). ...
... The de novo assembler and FindORFs tool, available in Geneious (Kearse et al. 2012), were used to further assemble genomes where necessary and to identify ORFs in potential virus sequences, respectively. EVEs were identified by referencing Harding et al. (2021) and removed manually. NCBI Web BLAST (https:// www.ncbi.nlm.nih.gov/BLAST) was then used to check for false positives, disrupted open reading frames (ORFs), and to manually assess alignment to virus motifs. ...
... Although this is a risk when analysing SRA data, the use of taxonomic classification software such as CCMetagen (Marcelino et al. 2020) can be used to identify contaminating host sequences. We can also be confident that the viruses identified here are exogenous, rather than endogenous, as high-quality host genomes are available for three of the four Dasyurid species studied here, and a comprehensive analysis of EVEs in marsupials has previously been undertaken (Harding et al. 2021). ...
Although Australian marsupials are characterised by unique biology and geographic isolation, little is known about the viruses present in these iconic wildlife species. The Dasyuromorphia are an order of marsupial carnivores found only in Australia that include both the extinct Tasmanian tiger (thylacine) and the highly threatened Tasmanian devil. Several other members of the order are similarly under threat of extinction due to habitat loss, hunting, disease, and competition and predation by introduced species such as feral cats. We utilised publicly available RNA-seq data from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database to document the viral diversity within four Dasyuromorph species. Accordingly, we identified fifteen novel virus sequences from five DNA virus families (Adenoviridae, Anelloviridae, Gammaherpesvirinae, Papillomaviridae, and Polyomaviridae) and three RNA virus taxa: the order Jingchuvirales, the genus Hepacivirus, and the delta-like virus group. Of particular note was the identification of a marsupial-specific clade of delta-like viruses that may indicate an association of deltaviruses with marsupial species. In addition, we identified a highly divergent hepacivirus in a numbat liver transcriptome that falls outside of the larger mammalian clade. We also detect what may be the first Jingchuvirales virus in a mammalian host—a chu-like virus in Tasmanian devils—thereby expanding the host range beyond invertebrates and ectothermic vertebrates. As many of these Dasyuromorphia species are currently being used in translocation efforts to reseed populations across Australia, understanding their virome is of key importance to prevent the spread of viruses to naive populations.
... Although this is a 392 risk when analysing SRA data, the use of taxonomic classification software such as 393 CCMetagen (Marcelino et al. 2020) can be used to identify contaminating host sequences.394We can also be confident that the viruses identified here are exogenous, rather than 395 endogenous, as high-quality host genomes are available for three of the four Dasyurid 396 species studied here, and a comprehensive analysis of endogenous viral elements in 397 marsupials has previously been undertaken(Harding et al. 2021). ...
Although Australian marsupials are characterised by unique biology and geographic isolation, little is known about the viruses present in these iconic wildlife species. The Dasyuromorphia are an order of marsupial carnivores found only in Australia that include both the extinct Tasmanian tiger (Thylacine) and the highly threatened Tasmanian devil. Several other members of the order are similarly under threat of extinction due to habitat loss, hunting, disease, and competition and predation by introduced species such as feral cats. We utilised publicly available RNA-seq data from the NCBI Sequence Read Archive (SRA) database to document the viral diversity within four Dasyuromorphia species. Accordingly, we identified 15 novel virus species from five DNA virus families ( Adenoviridae , Anelloviridae , Herpesviridae , Papillomaviridae and Polyomaviridae ) and three RNA virus taxa: the order Jingchuvirales, the genus Hepacivirus , and the delta-like virus group. Of particular note was the identification of a marsupial specific clade of delta-like viruses that may indicate an association of deltaviruses and with marsupial species dating back to their origin some 160 million years ago. In addition, we identified a highly divergent hepacivirus in a numbat liver transcriptome that falls outside of the larger mammalian clade, as well as the first detection of the Jingchuvirales in a mammalian host – a chu-like virus in Tasmanian devils – thereby expanding the host range beyond invertebrates and ectothermic vertebrates. As many of these Dasyuromorphia species are currently being used in translocation efforts to reseed populations across Australia, understanding their virome is of key importance to prevent the spread of viruses to naive populations.
... Out of these results, the top viral hit for each virus-like transcript was compiled. Two custom Python scripts were used throughout the workflow to consolidate hits and filter output (Harding et al., 2021). The nucleotide sequences of the This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. ...
... EVEs that produce transcripts can be differentiated from infecting viruses due to the presence of stop codons in the RNA (Harding et al., 2021). ...
We report identification and analysis of a full sequence of a novel polyomavirus from a brushtail possum (Trichosurus vulpecula) termed possum polyomavirus (PPyV). The sequence was obtained from the next-generation sequencing assembly during an investigation into the aetiological agent for a neurological disease of possums termed wobbly possum disease (WPD), but the virus was not aetiologically involved in WPD. The PPyV genome was 5,224 nt long with the organisation typical for polyomaviruses including early (large and small T antigens) and late (VP1, VP2 and VP3) coding regions separated by the non-coding control region of 465 nt. Possum polyomavirus clustered with betapolyomaviruses in the WUKI clade but showed less than 60% identity to any of the members of this clade. We propose that PPyV is classified within a new species in the genus Betapolyomavirus. These data add to our limited knowledge of the marsupial viruses and their evolution.
Amphibians and non-avian reptiles represent a significant proportion of terrestrial vertebrates, however knowledge of their viruses is not proportional to their abundance. Many amphibians and reptiles have strict habitual environments and localised populations and are vulnerable to viral outbreaks and potential elimination as a result. We sought to identify viruses that were hidden in amphibian and reptile metatranscriptomic data by screening 235 RNA-sequencing datasets from a 122 species covering 25 countries. We identified 26 novel viruses and eight previously characterised viruses from fifteen different viral families. Twenty-five viruses had RNA genomes with identity to Arteriviridae, Tobaniviridae, Hantaviridae, Rhabdoviridae, Astroviridae, Arenaviridae, Hepeviridae, Picornaviridae, Orthomyxoviridae, Reoviridae, Flaviviridae and Caliciviridae. In addition to RNA viruses, we also screened datasets for DNA viral transcripts, which are commonly excluded from transcriptomic analysis. We identified ten DNA viruses with identity to Papillomaviridae, Parvoviridae, Circoviridae and Adomaviridae. With the addition of these viruses, we expand the global amphibian and reptile virome and identify new potentially pathogenic viruses that could challenge populations. We speculate that amphibian viruses often have simpler genomes than those in amniotes, as in the case of the Secondpapillomavirinae and Orthomyxoviridae viruses identified in this study. In addition, we find evidence of inter-family recombination in RNA viruses, and we also identify new members of the recombinant Adomaviridae family. Overall, we provide insights into the uncharacterised diversity of amphibian and reptile viruses with the aim of improving population management, treatment and conservation into the future.
Genomic viral integrations, termed endogenous viral elements (EVEs), are fragments of viruses in host chromosomes that provide information about viral evolution and could even help protect the host from infection. In the present study we examined EVEs in thirteen different Australian marsupial species to identify trends in their integration, commonality and to investigate their possible cellular function. We found that marsupial EVEs are commonly derived from viruses of the Bornaviridae, Filoviridae and Parvoviridae families, and circulated up to 160 million years ago. We also show the EVEs are actively transcribed into both long and short RNA molecules in marsupials, and propose they are involved in a cellular defence mechanism to protect the germline from viral genomic invasion.
