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. 2021 May 31;13(6):1042.
doi: 10.3390/v13061042.

Discovery and Characterization of Actively Replicating DNA and Retro-Transcribing Viruses in Lower Vertebrate Hosts Based on RNA Sequencing

Xin-Xin Chen  1 , Wei-Chen Wu  1 , Mang Shi  1
Affiliations

Discovery and Characterization of Actively Replicating DNA and Retro-Transcribing Viruses in Lower Vertebrate Hosts Based on RNA Sequencing

Xin-Xin Chen et al. Viruses. .

Abstract

In a previous study, a metatranscriptomics survey of RNA viruses in several important lower vertebrate host groups revealed huge viral diversity, transforming the understanding of the evolution of vertebrate-associated RNA virus groups. However, the diversity of the DNA and retro-transcribing viruses in these host groups was left uncharacterized. Given that RNA sequencing is capable of revealing viruses undergoing active transcription and replication, we collected previously generated datasets associated with lower vertebrate hosts, and searched them for DNA and retro-transcribing viruses. Our results revealed the complete genome, or "core gene sets", of 18 vertebrate-associated DNA and retro-transcribing viruses in cartilaginous fishes, ray-finned fishes, and amphibians, many of which had high abundance levels, and some of which showed systemic infections in multiple organs, suggesting active transcription or acute infection within the host. Furthermore, these new findings recharacterized the evolutionary history in the families Hepadnaviridae, Papillomaviridae, and Alloherpesviridae, confirming long-term virus-host codivergence relationships for these virus groups. Collectively, our results revealed reliable and sufficient information within metatranscriptomics sequencing to characterize not only RNA viruses, but also DNA and retro-transcribing viruses, and therefore established a key methodology that will help us to understand the composition and evolution of the total "infectome" within a diverse range of vertebrate hosts.

Keywords: codivergence; metatranscriptomics; vertebrate-associated DNA virus; virome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Discovery and characterization of vertebrate-associated DNA, RT, and RNA viruses in lower vertebrate hosts. (A) Heat map showing the presence and abundance of major DNA, RT, and RNA virus groups discovered from the 125 metatranscriptomics sequencing data belonging to 6 major lower vertebrate host groups. The RNA virus genomes used here were derived from the 2018 study [7]. (B) Bar plot showing the types and positive rates of DNA/RT viruses discovered in each host group. (C) Left: Box plot and scatter plots showing the abundance distribution of DNA/RT virus families discovered in this study; each box has the upper, median, and lower quartiles, and each circle point represents one library; Right: bar plot showing the positive rate for each of the virus families. (D) Comparisons of the pool positive rate between DNA/RT and RNA viruses. (E) Comparisons of the number of DNA/RT and RNA virus species detected. (F) Box plot and scatter plots showing the distributions of abundance levels of DNA/RT (purple) and RNA viruses (blue), respectively. (G) The distribution of the number of transcriptomics libraries positive for DNA/RT viruses.
Figure 2
Figure 2
Genomic, transcriptomics, and phylogenetic analyses of the newly identified ranid herpesvirus 4. (A) Comparison of the genome structures of ranid herpesvirus 2 (RHV2, NC_008210.1) and RHV4. Genes are shown as rectangles with arrow tips showing the transcription direction, and those conserved in both viruses are connected with dotted lines. (B) Bar graph showing the expression levels of different genes by RHV4 in lung (HWWF) and liver/spleen (HWWGP) tissues. The horizontal axis represents the abundance of each gene as measured by RPKM. (C) The maximum likelihood phylogenetic trees based on the capsid maturational protease (capsid), ATPase subunit of terminase, and protein kinase proteins, respectively, showing the positions of RHV4 (solid red circle) within the diversity of the family Alloherpesviridae. Four amphibian-associated herpesviruses discovered previously are marked in different colors. All trees are midpoint rooted, and the taxonomy information is labelled to the right of each tree.
Figure 3
Figure 3
Genomic structures and evolutionary histories of novel small DNA viruses detected from metatranscriptomics and transcriptomics libraries. These include five potential new viruses from three families—namely (A) Parvoviridae, (B) Circoviridae, and (C) Papillomaviridae. The phylogenetic trees were reconstructed based on non-structural protein 1 (NS1), replicase protein, and L1 protein, respectively. All trees are midpoint rooted and scaled to the number amino acid substitutions per site. The virus names are color coded to reflect their host group. The positions of newly discovered viruses are shown in solid red circles in each tree and the host. Branch support values (>70%) are shown at the key nodes. All of the genomic structures are shown above the corresponding phylogenetic tree and the potential proteins or protein domains they encode are labeled in the predicted ORFs of these genomes.
Figure 4
Figure 4
Genome organization and evolutionary analyses of hepadnaviruses discovered from metatranscriptomics and transcriptomics data. Upper panel: Genome structures of five complete hepadnavirus genomes, including Rhinobatos hynnicephalus HBV (RHHBV), Chimaera phantasma HBV (CPHBV), Anguilliformes HBV 3 (AHBV 3), Schizopygopsis younghusbandi HBV (SYHBV), and Hoplobatrachus rugulosus HBV (HRHBV); the predicted coding sequences are shown in blue (polymerase), brown (surface), and red (core). Lower panel: Maximum likelihood phylogenetic tree including all 12 hepadnaviruses within the context of enveloped and non-enveloped hepadna-like viruses. Tree is midpoint rooted and scaled to the number of amino acid substitutions per site. Viruses discovered in our study are shown in solid circles in each tree. Branch support values (>70%) are shown at the key nodes. The virus names and dots are color coded to reflect their host group.
Figure 5
Figure 5
Genome structure and evolutionary analysis of retrovirus-like elements discovered from metatranscriptomics and transcriptomics data. (A) Maximum likelihood phylogenetic tree based on newly discovered retro-like virus elements and members of the family Retroviridae. (B) The structures of ORFs predicted from the retro-like virus elements in lower vertebrate hosts. Tree is midpoint rooted and scaled to the number of amino acid substitutions per site. Newly discovered EVEs are shown in sold circles in each tree, which are color coded to reflect the host group of each virus or EVE. Branch support values (>70%) are shown at the key nodes.
Figure 6
Figure 6
Codivergence relationships between vertebrate hosts and their associated DNA/RT viruses. (A) Comparison of the virus and host phylogenetic structures for herpesviruses, papillomaviruses, hepadnaviruses, and parvoviruses. More detailed presentations containing taxon labels are shown in Supplementary Figures S1–S4. (B) The box plot showing the estimation of codivergence events across the phylogeny of vertebrate-associated DNA viruses. Each box plot has the estimated median (center line) and upper and lower quartiles (box limits).
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