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. 2021 Sep 2;7(2):veab076.
doi: 10.1093/ve/veab076. eCollection 2021.

Ancient viral integrations in marsupials: a potential antiviral defence

Emma F Harding  1 , Alice G Russo  1 , Grace J H Yan  1 , Paul D Waters  1 , Peter A White  1
Affiliations

Ancient viral integrations in marsupials: a potential antiviral defence

Emma F Harding et al. Virus Evol. .

Abstract

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

Keywords: RNAi; endogenous viral element; marsupial; paleovirology; piRNA; small RNA; virus evolution.

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Conflict of interest statement

None declared.

Figures

Figure 1.
Figure 1.
The bioinformatics workflow used in this study to identify viral transcripts from thirty-five marsupial RNA-Seq data sets. Publicly available data sets were downloaded from NCBI SRA and quality checked using FastQC before assembly into transcriptomes, using Trinity. DIAMOND was used to annotate each assembled transcript as host, viral, or other. Viral transcripts were filtered to merge overlapping hits and remove duplicate hits. To confirm the viral origin of each transcript, a reciprocal BLASTx search of the NCBI nr protein database was performed. Transcripts were classified as EVEs if they mapped to the representative marsupial genome (where available), or putative EVEs if they had interrupted reading frames, identity to confirmed EVEs, or were from frequently endogenised viral families. Small RNA analysis was undertaken to identify if any EVEs gave rise to small RNA molecules. Figure created using BioRender.com.
Figure 2.
Figure 2.
Classification of viral-like transcripts present in Australian marsupial data sets. Thirty five RNA-Seq data sets 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.
Figure 3.
Figure 3.
Alignment of endogenous viral element (EVE) transcripts with similarity to Bornaviridae, Filoviridae, and Parvoviridae sequences. Sequences were identified from marsupial RNA-Seq reads using a bioinformatics workflow and the viral identity was determined with a BLASTx search against the NCBI non-redundant database. Sequences were translated and mapped to the viral protein with which they share the highest identity. Grey lines represent transcripts from the thirty-five marsupial transcriptomes. Genes coloured blue are structural genes, and genes coloured red are non-structural genes. The proteins are gradated based on the conservation of amino acids between members of the viral families; light green represents conserved regions and grey represents more variable regions. Red boxes highlight areas containing recognised viral enzymatic motifs.
Figure 4.
Figure 4.
Viral gene of origin for EVEs identified in this study. EVE transcripts were identified using a bioinformatics pipeline and classified into family and gene of origin based on the top hit in a BLASTx search of the NCBI non-redundant database. Colours represent the different gene categories and are detailed in the figure legend. Viral icons depicting the basic viral structure for each family are shown in the centre of the pie chart. The number of EVE transcripts that mapped to each family are shown below the pie chart.
Figure 5.
Figure 5.
Analysis of small RNA reads that map to viral integrations in the koala genome. Small RNA data sets were downloaded from NCBI SRA and mapped to EVEs in the koala genome identified in this study using HISAT2. (A) Distribution of mapped small RNA between genomic EVEs in the koala, grouped by tissue type. Viral family and genes are coloured and detailed in the key. (B) Orientation of small RNA mapped to EVE-derived transcripts in the koala. (C) Sequence logo of secondary piRNA-length molecules, showing 1ʹU bias and 10ʹA bias. Created using WebLogo 3 (Crooks et al. 2004).
Figure 6.
Figure 6.
Estimated integration times of endogenous viral elements identified in this study. BLASTn searches of marsupial genomes using Ensembl and transcriptomes using Geneious Prime was conducted to identify EVEs present in Australian marsupials. EVEs present in three or more marsupial species were identified and their integration times estimated using an evolutionary tree of marsupials generated by TimeTree. The coloured lines on the tree represent estimated integration times with the colours corresponding to the different EVEs as depicted in the figure legend. Animals with available genomes are shaded grey. Double helices denote the presence of the EVE in the animal genome, and single lines represent active transcription of the EVE. The scale bar on the tree represents million years of evolution.
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