# Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan

Syun-Ichi Urayama, Akihito Fukudome, Miho Hirai, Tomoyo Okumura, Yosuke Nishimura, Yoshihiro Takaki, Norio Kurosawa, Eugene V Koonin, Mart Krupovic, Takuro Nunoura

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## To cite this version:

Syun-Ichi Urayama, Akihito Fukudome, Miho Hirai, Tomoyo Okumura, Yosuke Nishimura, et al. Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan. Nature Microbiology, 2024, 9 (2), pp.514 - 523. 10.1038/s41564-023-01579-5. pasteur-04447423

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https://pasteur.hal.science/pasteur-04447423

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# Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan

Syun-Ichi Urayama, Akihito Fukudome, Miho Hirai, Tomoyo Okumura, Yosuke Nishimura, Yoshihiro Takaki, Norio Kurosawa, Eugene V Koonin, Mart Krupovic, Takuro Nunoura

► To cite this version:

Syun-Ichi Urayama, Akihito Fukudome, Miho Hirai, Tomoyo Okumura, Yosuke Nishimura, et al. Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan. Nature Microbiology, 2024, 9 (2), pp.514 - 523. 10.1038/s41564-023-01579-5. pasteur-04447423

HAL Id: pasteur-04447423

https://pasteur.hal.science/pasteur-04447423v1

Submitted on 8 Feb 2024

**HAL** is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire **HAL**, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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Distributed under a Creative Commons Attribution 4.0 International License
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# Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan

Received: 3 July 2023

Accepted: 8 December 2023

Published online: 17 January 2024

<figure><img src="image_4.png" /></figure>

Check for updates

Syun-ichi Urayama Ⓓ1,2, Akihito Fukudome Ⓒ3, Miho Hirai Ⓒ4,
Tomoyo Okumura Ⓒ5, Yosuke Nishimura Ⓒ6, Yoshihiro Takaki Ⓒ4,
Norio Kurosawa Ⓒ7, Eugene V. Koonin Ⓒ8, Mart Krupovic Ⓒ9 & Takuro Nunoura Ⓒ6

Metatranscriptome sequencing expanded the known diversity of the bacterial RNA virome, suggesting that additional riboviruses infecting bacterial hosts remain to be discovered. Here we employed double-stranded RNA sequencing to recover complete genome sequences of two ribovirus groups from acidic hot springs in Japan. One group, denoted hot spring riboviruses (HsRV), consists of viruses with distinct RNA-directed RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. This group forms a distinct phylum, *Artimaviricota*, or even kingdom within the realm *Riboviria*. We identified viruses encoding HsRV-like RdRPs in marine water, river sediments and salt marshes, indicating that this group is widespread beyond extreme ecosystems. The second group, denoted hot spring partiti-like viruses (HsPV), forms a distinct branch within the family *Partitiviridae*. The genome architectures of HsRV and HsPV and their identification in bacteria-dominated habitats suggest that these viruses infect thermoacidophilic bacteria.

Recent metagenomics and metatranscriptomics analyses transformed the study of viromes. These approaches that do not require laborious virus cultivation have become the principal source of virus discovery<fnref n="1" />. Indeed, numerous virus groups across all taxonomic levels have been discovered. In particular, the diversity of RNA viruses that, in the current virus taxonomy, comprise the kingdom *Orthornavirae* within the realm *Riboviria* has expanded more than an order of magnitude through global metatranscriptome surveys²⁻⁹.

Only one hallmark gene encoding the RNA-directed RNA polymerase (RdRP) is conserved across the entire kingdom *Orthornavirae*. Therefore, detection of the RdRP, typically using search methods based on sequence profiles, is the principal approach employed in metatranscriptome mining for riboviruses, and phylogenetic analysis of the RdRP is the basis of ribovirus taxonomy. Before the advent of massive metatranscriptome analysis, the viruses in this kingdom have been classified into 5 large phyla corresponding to

<footnote n="1">¹Department of Life and Environmental Sciences, Laboratory of Fungal Interaction and Molecular Biology (donated by IFO), University of Tsukuba, Tsukuba, Japan. ²Microbiology Research Center for Sustainability (MiCS), University of Tsukuba, Tsukuba, Japan. ³Howard Hughes Medical Institute, Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA. ⁴Super-cutting-edge Grand and Advanced Research (SUGAR) Program, Japan Agency for Marine Science and Technology (JAMSTEC), Yokosuka, Japan. ⁵Marine Core Research Institute, Kochi University, Nankoku, Kochi, Japan. ⁶Research Center for Bioscience and Nanoscience (CeBN), JAMSTEC, Yokosuka, Japan. ⁷Faculty of Science and Engineering, Soka University, Hachioji, Japan. ⁸National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA. ⁹Institut Pasteur, Université Paris Cité, Archaeal Virology Unit, Paris, France. ✉e-mail: urayama.shunichi.gn@u.tsukuba.ac.jp</footnote>
<figure><img src="image_2.png" /></figure>

# Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan

Received: 3 July 2023

Accepted: 8 December 2023

Published online: 17 January 2024

<figure><img src="image_3.png" /></figure>

Check for updates

Syun-ichi Urayama Ⓓ1,2, Akihito Fukudome Ⓒ3, Miho Hirai Ⓒ4,
Tomoyo Okumura Ⓒ5, Yosuke Nishimura Ⓒ6, Yoshihiro Takaki Ⓒ4,
Norio Kurosawa Ⓒ7, Eugene V. Koonin Ⓒ8, Mart Krupovic Ⓒ9 & Takuro Nunoura Ⓒ6

Metatranscriptome sequencing expanded the known diversity of the bacterial RNA virome, suggesting that additional riboviruses infecting bacterial hosts remain to be discovered. Here we employed double-stranded RNA sequencing to recover complete genome sequences of two ribovirus groups from acidic hot springs in Japan. One group, denoted hot spring riboviruses (HsRV), consists of viruses with distinct RNA-directed RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. This group forms a distinct phylum, *Artimaviricota*, or even kingdom within the realm *Riboviria*. We identified viruses encoding HsRV-like RdRPs in marine water, river sediments and salt marshes, indicating that this group is widespread beyond extreme ecosystems. The second group, denoted hot spring partiti-like viruses (HsPV), forms a distinct branch within the family *Partitiviridae*. The genome architectures of HsRV and HsPV and their identification in bacteria-dominated habitats suggest that these viruses infect thermoacidophilic bacteria.

Recent metagenomics and metatranscriptomics analyses transformed the study of viromes. These approaches that do not require laborious virus cultivation have become the principal source of virus discovery<fnref n="1" />. Indeed, numerous virus groups across all taxonomic levels have been discovered. In particular, the diversity of RNA viruses that, in the current virus taxonomy, comprise the kingdom *Orthornavirae* within the realm *Riboviria* has expanded more than an order of magnitude through global metatranscriptome surveys²⁻⁹.

Only one hallmark gene encoding the RNA-directed RNA polymerase (RdRP) is conserved across the entire kingdom *Orthornavirae*. Therefore, detection of the RdRP, typically using search methods based on sequence profiles, is the principal approach employed in metatranscriptome mining for riboviruses, and phylogenetic analysis of the RdRP is the basis of ribovirus taxonomy. Before the advent of massive metatranscriptome analysis, the viruses in this kingdom have been classified into 5 large phyla corresponding to

<footnote n="1">¹Department of Life and Environmental Sciences, Laboratory of Fungal Interaction and Molecular Biology (donated by IFO), University of Tsukuba, Tsukuba, Japan. ²Microbiology Research Center for Sustainability (MiCS), University of Tsukuba, Tsukuba, Japan. ³Howard Hughes Medical Institute, Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA. ⁴Super-cutting-edge Grand and Advanced Research (SUGAR) Program, Japan Agency for Marine Science and Technology (JAMSTEC), Yokosuka, Japan. ⁵Marine Core Research Institute, Kochi University, Nankoku, Kochi, Japan. ⁶Research Center for Bioscience and Nanoscience (CeBN), JAMSTEC, Yokosuka, Japan. ⁷Faculty of Science and Engineering, Soka University, Hachioji, Japan. ⁸National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA. ⁹Institut Pasteur, Université Paris Cité, Archaeal Virology Unit, Paris, France. ✉e-mail: urayama.shunichi.gn@u.tsukuba.ac.jp</footnote>
major clades in the RdRP phylogeny¹⁰. Metatranscriptome studies largely validated the robustness of these phyla and additionally identified several candidate smaller phyla. The diversity of riboviruses across the lower taxonomy ranks demonstrated a nearly uniform increase, for example, roughly fivefold in one study that provided quantitative estimates⁸.

Metatranscriptome mining yielded qualitative insights into the global view of the RNA virome. Traditionally, riboviruses have been recognized as the major component of the eukaryote virome, whereas the viromes of bacteria and archaea were dominated by DNA viruses¹¹,¹². For many years, only two small families of RNA viruses, each infecting a narrow range of bacteria, have been known: *Leviviridae* (single-stranded RNA (ssRNA) bacteriophages) and *Cystoviridae* (double-stranded RNA (dsRNA) bacteriophages). Metatranscriptome analyses revealed a much greater diversity of leviviruses than previously suspected, elevating this family to the rank of the class *Leviviricetes* that includes multiple orders and families⁸,¹³⁻¹⁵. The family *Cystoviridae* was substantially expanded as well⁸. For uncharacterized groups of viruses without a close relationship to any known groups, host assignment becomes a challenge. Nevertheless, several lines of evidence including (nearly) exclusive co-occurrence with bacteria, prediction of multiple virus genes preceded by prokaryote-type (Shine–Dalgarno (SD)) ribosome-binding sequences (RBS), identification of virus-encoded cell wall degrading enzymes, and most notably, targeting by reverse transcriptase (RT)-containing type III CRISPR systems strongly suggest that several previously uncharacterized groups of riboviruses infect prokaryotes⁸. Thus, the diversity of riboviruses infecting bacteria has been substantially underestimated and additional groups of such viruses most probably remain to be discovered.

Long dsRNA is a molecular marker of RNA virus infection¹⁶. The recently developed method of Fragmented and primer-Ligated DsRNA Sequencing (FLDS) made it possible to capitalize on the presence of (nearly) identical terminal sequences in genome segments of the same virus. This information enables one to identify multisegmented RNA virus genomes even if they did not show sequence similarity to known viruses¹⁷⁻¹⁹. Here we used FLDS to identify riboviruses associated with microbial consortia dominated by bacteria and archaea in several acidic hot springs in Japan. This analysis resulted in the identification of two distinct groups of riboviruses with multisegmented RNA genomes with organization typical of bacterial riboviruses.

## Composition of small subunit ribosomal RNA and identification of RNA virus

To determine the composition of active microbial consortia in the hot spring water samples, total ssRNA sequencing reads were mapped on the small subunit (SSU) ribosomal RNA (rRNA) sequences from the Silva database (SILVA SSU v.138) using phyloFlash²⁰ (Fig. 1 and Supplementary Text). All samples were dominated by prokaryotes, with the H4, H5, Y66 and Oi samples, where RNA viruses were identified, containing <1% of eukaryotic SSU rRNA reads (Extended Data Table 1).

In FLDS, potential complete genomes of multipartite RNA viruses were obtained from samples H4, H5, Y66 and Oi (Extended Data Table 2). For the samples from the other stations, sequence libraries were successfully constructed except for the Ob sample, but no contigs representing potential complete genomes of RNA viruses in FLDS read mapping¹⁸ were obtained.

## Bipartite RNA virus from the hot spring and other ecosystems

FLDS of the Oi sample (79.3 °C, pH 2.2) yielded three populations of contigs (Fig. 2a) which collectively recruited ~50% of the clean FLDS reads from the Oi library. Among the contigs, we identified similar 5'- and 3'-terminal sequences (Fig. 2b), a characteristic feature of segmented RNA viruses²¹. On the basis of the similarity of the 5'- and

<figure><img src="image_5.png" /><figcaption>**Fig. 1 | Composition of microbiomes associated with the hot spring samples.**
The composition was analysed on the basis of the mapped sequence reads on the rRNA sequences using PhyloFlash. Details are given in Extended Data Table 1.</figcaption></figure>

3'-terminal sequences, lengths of the segments and gene content, we concluded that two sets of contigs constituted genomes of a distinct group of bipartite RNA viruses. The segments were denoted RNA1, RNA2 and RNA2* (Supplementary Text and Extended Data Table 3). In total, we obtained complete sequences for 4, 4 and 2 divergent variants of segments RNA1, RNA2 and RNA2*, respectively (Fig. 2a). The similarity between the termini of the segments precluded assignment of all sets of segments to particular virus strains. However, segments RNA1a and RNA2a were most abundant and had longer conserved terminal sequences and were thus assigned to the same virus strain with a bisegmented genome.

RNA1, RNA2 and RNA2* harboured 4–5, 5–6 and 5–7 open reading frames (ORFs), respectively (Fig. 2a). None of the predicted proteins encoded by these RNAs showed significant similarity (BLASTP E-value = 5 × 10⁻³⁰) to any protein sequences in public databases. Even the most sensitive profile–profile searches using HHpred yielded no significant (HHpred probability >90%) hits for any of the predicted proteins. However, HHpred searches queried with the amino acid sequence of ORF4 from the RNA1 segment produced a partial hit to several RdRPs. Although the hits were not significant (HHpred probability <90%) and encompassed only a small region of the RdRP (~15% of the target profile), the aligned region covered the diagnostic RdRP motifs B (SGxxxT, x – any amino acid) and C (GDD) (Extended Data Fig. 1a), so we pursued this clue further. However, despite several attempts, we were unable to convincingly identify RNA1_ORF4 of HsRV as an RdRP (Supplementary Text). Thus, we set out to enrich the sequence diversity of RNA1_ORF4 by reanalyzing the entire FLDS dataset. To this end, unmapped sequence reads were assembled and RNA1_ORF4 protein sequences were used as queries to search against the assembled contigs using BLASTX. This search yielded 10 additional RNA1_ORF4-like sequences encoded by H5_contig_1 from H5 and Oi_contigs_1–9 from Oi samples (E-value ≤ 1 × 10⁻⁰⁵) (Extended Data Table 4). The additional homologues detected in this search were combined with the 4 initially identified RNA1_ORF4 sequences and the produced multiple sequence alignment (MSA) was used as a query in an HHpred search against the PDB70 database. This search yielded significant hits (probability >90%) to various ribovirus RdRPs, although the aligned region remained limited (~15% of the target profiles). Collectively, these searches suggested that RNA1_ORF4 homologues are highly divergent RdRPs.

Using the MSA that included the identified RNA1_ORF4 homologues, a high-quality (average per-residue Local Distance Difference Test (pLDDT) = 90.7) AF2 model of the putative RdRP was obtained (Fig. 2c). Examination of this model revealed a topology typical of the
major clades in the RdRP phylogeny¹⁰. Metatranscriptome studies largely validated the robustness of these phyla and additionally identified several candidate smaller phyla. The diversity of riboviruses across the lower taxonomy ranks demonstrated a nearly uniform increase, for example, roughly fivefold in one study that provided quantitative estimates⁸.

Metatranscriptome mining yielded qualitative insights into the global view of the RNA virome. Traditionally, riboviruses have been recognized as the major component of the eukaryote virome, whereas the viromes of bacteria and archaea were dominated by DNA viruses¹¹,¹². For many years, only two small families of RNA viruses, each infecting a narrow range of bacteria, have been known: *Leviviridae* (single-stranded RNA (ssRNA) bacteriophages) and *Cystoviridae* (double-stranded RNA (dsRNA) bacteriophages). Metatranscriptome analyses revealed a much greater diversity of leviviruses than previously suspected, elevating this family to the rank of the class *Leviviricetes* that includes multiple orders and families⁸,¹³⁻¹⁵. The family *Cystoviridae* was substantially expanded as well⁸. For uncharacterized groups of viruses without a close relationship to any known groups, host assignment becomes a challenge. Nevertheless, several lines of evidence including (nearly) exclusive co-occurrence with bacteria, prediction of multiple virus genes preceded by prokaryote-type (Shine–Dalgarno (SD)) ribosome-binding sequences (RBS), identification of virus-encoded cell wall degrading enzymes, and most notably, targeting by reverse transcriptase (RT)-containing type III CRISPR systems strongly suggest that several previously uncharacterized groups of riboviruses infect prokaryotes⁸. Thus, the diversity of riboviruses infecting bacteria has been substantially underestimated and additional groups of such viruses most probably remain to be discovered.

Long dsRNA is a molecular marker of RNA virus infection¹⁶. The recently developed method of Fragmented and primer-Ligated DsRNA Sequencing (FLDS) made it possible to capitalize on the presence of (nearly) identical terminal sequences in genome segments of the same virus. This information enables one to identify multisegmented RNA virus genomes even if they did not show sequence similarity to known viruses¹⁷⁻¹⁹. Here we used FLDS to identify riboviruses associated with microbial consortia dominated by bacteria and archaea in several acidic hot springs in Japan. This analysis resulted in the identification of two distinct groups of riboviruses with multisegmented RNA genomes with organization typical of bacterial riboviruses.

## Composition of small subunit ribosomal RNA and identification of RNA virus

To determine the composition of active microbial consortia in the hot spring water samples, total ssRNA sequencing reads were mapped on the small subunit (SSU) ribosomal RNA (rRNA) sequences from the Silva database (SILVA SSU v.138) using phyloFlash²⁰ (Fig. 1 and Supplementary Text). All samples were dominated by prokaryotes, with the H4, H5, Y66 and Oi samples, where RNA viruses were identified, containing <1% of eukaryotic SSU rRNA reads (Extended Data Table 1).

In FLDS, potential complete genomes of multipartite RNA viruses were obtained from samples H4, H5, Y66 and Oi (Extended Data Table 2). For the samples from the other stations, sequence libraries were successfully constructed except for the Ob sample, but no contigs representing potential complete genomes of RNA viruses in FLDS read mapping¹⁸ were obtained.

## Bipartite RNA virus from the hot spring and other ecosystems

FLDS of the Oi sample (79.3 °C, pH 2.2) yielded three populations of contigs (Fig. 2a) which collectively recruited ~50% of the clean FLDS reads from the Oi library. Among the contigs, we identified similar 5'- and 3'-terminal sequences (Fig. 2b), a characteristic feature of segmented RNA viruses²¹. On the basis of the similarity of the 5'- and

<figure><img src="image_4.png" /><figcaption>**Fig. 1 | Composition of microbiomes associated with the hot spring samples.**
The composition was analysed on the basis of the mapped sequence reads on the rRNA sequences using PhyloFlash. Details are given in Extended Data Table 1.</figcaption></figure>

3'-terminal sequences, lengths of the segments and gene content, we concluded that two sets of contigs constituted genomes of a distinct group of bipartite RNA viruses. The segments were denoted RNA1, RNA2 and RNA2* (Supplementary Text and Extended Data Table 3). In total, we obtained complete sequences for 4, 4 and 2 divergent variants of segments RNA1, RNA2 and RNA2*, respectively (Fig. 2a). The similarity between the termini of the segments precluded assignment of all sets of segments to particular virus strains. However, segments RNA1a and RNA2a were most abundant and had longer conserved terminal sequences and were thus assigned to the same virus strain with a bisegmented genome.

RNA1, RNA2 and RNA2* harboured 4–5, 5–6 and 5–7 open reading frames (ORFs), respectively (Fig. 2a). None of the predicted proteins encoded by these RNAs showed significant similarity (BLASTP E-value = 5 × 10⁻³⁰) to any protein sequences in public databases. Even the most sensitive profile–profile searches using HHpred yielded no significant (HHpred probability >90%) hits for any of the predicted proteins. However, HHpred searches queried with the amino acid sequence of ORF4 from the RNA1 segment produced a partial hit to several RdRPs. Although the hits were not significant (HHpred probability <90%) and encompassed only a small region of the RdRP (~15% of the target profile), the aligned region covered the diagnostic RdRP motifs B (SGxxxT, x – any amino acid) and C (GDD) (Extended Data Fig. 1a), so we pursued this clue further. However, despite several attempts, we were unable to convincingly identify RNA1_ORF4 of HsRV as an RdRP (Supplementary Text). Thus, we set out to enrich the sequence diversity of RNA1_ORF4 by reanalyzing the entire FLDS dataset. To this end, unmapped sequence reads were assembled and RNA1_ORF4 protein sequences were used as queries to search against the assembled contigs using BLASTX. This search yielded 10 additional RNA1_ORF4-like sequences encoded by H5_contig_1 from H5 and Oi_contigs_1–9 from Oi samples (E-value ≤ 1 × 10⁻⁰⁵) (Extended Data Table 4). The additional homologues detected in this search were combined with the 4 initially identified RNA1_ORF4 sequences and the produced multiple sequence alignment (MSA) was used as a query in an HHpred search against the PDB70 database. This search yielded significant hits (probability >90%) to various ribovirus RdRPs, although the aligned region remained limited (~15% of the target profiles). Collectively, these searches suggested that RNA1_ORF4 homologues are highly divergent RdRPs.

Using the MSA that included the identified RNA1_ORF4 homologues, a high-quality (average per-residue Local Distance Difference Test (pLDDT) = 90.7) AF2 model of the putative RdRP was obtained (Fig. 2c). Examination of this model revealed a topology typical of the
a

<figure><img src="image_6.png" /></figure>

b

<figure><img src="image_7.png" /></figure>

d

<figure><img src="image_8.png" /></figure>

c

<figure><img src="image_9.png" /></figure>

f

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

e

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>Qβ RdRP</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>phi6 RdRP</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>MMLV RT</figcaption></figure>

**Fig. 2 | Unusual bipartite RNA virus genomes from the Oi hot spring.**
*a, Genome organization and conservation of the three genomic segments (RNA1, RNA2 and RNA2*) of HsRV. ORFs encoding homologous proteins are shown as arrows with identical colours. Yellow circles represent predicted SD RBS. Asterisks denote putative genes encoding predicted transmembrane domain (TMD)-containing proteins. **b, MSA** of the 5'- and 3'-terminal regions of the coding strands of reconstructed genome segments. Black shading, 100% nucleotide identity; grey shading, >50% nucleotide identity. **c, Quality** assessment of the AlphaFold2 model of the HsRV RdRP. The structural model is coloured on the basis of the pLDDT scores (average pLDDT = 90.7), with the

colour key shown at the bottom left corner. **d,e,** Domain organization of the HsRV RdRP. **d,** Schematic representation of the domain organization, with exact coordinates of each subdomain, including the five 'Fingers', indicated. **M,** middle finger. The positions of the motifs A, B and C are indicated. **e,** The structural model of HsRV RdRP coloured using the same scheme as in **d,f,** Comparison of the HsRV RdRP with homologues from other RNA viruses, including hepatitis C virus (HCV; PDB: **6GP9**), Norwalk virus (NV; PDB: **1SHO**), Qβ (PDB: **3MMP**), phi6 (PDB: **1HHS**) as well as RT from Moloney murine leukaemia virus (MMLV; PDB: **4MH8**). The structures are coloured using the rainbow scheme, from blue N terminus to red C terminus.

palm-domain polymerases, with readily discernible 'Fingers', 'Palm' and 'Thumb' subdomains (Fig. 2d,e) and overall architecture similar to that of viral RdRPs (Fig. 2f), albeit with some unique structural features. In particular, the RNA1_ORF4 model displayed an extended and highly ordered 'Fingers' subdomain, with the 'fingertips' forming a 5-stranded β-sheet that is missing in other RdRPs and interacts with the 'Thumb' subdomain. The conserved motifs B and C identified by HHpred were located within the Palm subdomain, at positions equivalent to those in other RdRPs. Structural superposition of the Palm subdomains from different RdRPs allowed identification of the third core motif, A, in RNA1_ORF4 (see below). Thus, we concluded that RNA1_ORF4 encodes an RdRP and provisionally named the discovered bipartite virus 'hot spring RNA virus (HsRV)', with the strain harbouring segments RNA1a and RNA2a denoted HsRV1. The four RdRPs encoded by the complete RNA1 segments shared 37 to 75% pairwise amino acid sequence identity and thus appear to represent four distinct virus species (or even higher

taxa). To characterize the diversity of HsRV-related RdRP in our FLDS data, the minor contigs including the aforementioned 10 sequences were analysed (Extended Data Fig. 2a). This analysis yielded several contigs with a high (>90%) identity to HsRV RNA1b RdRP. In addition, several contigs with moderate (>60%) identity to HsRV RNA1a or RNA1b were detected. Y66 and Y86 also included a few contigs related to HsRV RdRP.

The sequence profile of the HsRV RdRP was used to search the previously described FLDS sequence data from coastal seawater samples¹⁹, leading to the identification of two additional contigs (GenBank accessions: **BDQA01000957** and **BDQA01004869**) encoding incomplete HsRV-like RdRPs. Searches against the IMG/VR database queried with these RdRPs yielded significant hits (E-value ≤ 1 × 10⁻⁵⁰) to three additional putative RdRPs encoded by apparently complete or near-complete 5.3–5.6-kb-long genome segments (Ga0456180_000042, Ga0393213_00017, Ga0169446_00510; Fig. 3a, Extended Data Fig. 1b, Table 5 and Supplementary Text).
a

<figure><img src="image_5.png" /></figure>

b

<figure><img src="image_6.png" /></figure>

d

<figure><img src="image_7.png" /></figure>

c

<figure><img src="image_8.png" /></figure>

f

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

e

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

**Fig. 2 | Unusual bipartite RNA virus genomes from the Oi hot spring.**
*a, Genome organization and conservation of the three genomic segments (RNA1, RNA2 and RNA2*) of HsRV. ORFs encoding homologous proteins are shown as arrows with identical colours. Yellow circles represent predicted SD RBS. Asterisks denote putative genes encoding predicted transmembrane domain (TMD)-containing proteins. **b, MSA** of the 5'- and 3'-terminal regions of the coding strands of reconstructed genome segments. Black shading, 100% nucleotide identity; grey shading, >50% nucleotide identity. **c, Quality** assessment of the AlphaFold2 model of the HsRV RdRP. The structural model is coloured on the basis of the pLDDT scores (average pLDDT = 90.7), with the

colour key shown at the bottom left corner. **d,e, Domain organization of the** HsRV RdRP. **d, Schematic representation of the domain organization, with exact** coordinates of each subdomain, including the five 'Fingers', indicated. **M, middle** finger. The positions of the motifs A, B and C are indicated. **e, The structural** model of HsRV RdRP coloured using the same scheme as in **d, f, Comparison of** the HsRV RdRP with homologues from other RNA viruses, including hepatitis C virus (HCV; PDB: 6GP9), Norwalk virus (NV; PDB: 1SHO), Qβ (PDB: 3MMP), phi6 (PDB: 1HHS) as well as RT from Moloney murine leukaemia virus (MMLV; PDB: 4MH8). The structures are coloured using the rainbow scheme, from blue N terminus to red C terminus.

palm-domain polymerases, with readily discernible 'Fingers', 'Palm' and 'Thumb' subdomains (Fig. 2d,e) and overall architecture similar to that of viral RdRPs (Fig. 2f), albeit with some unique structural features. In particular, the RNA1_ORF4 model displayed an extended and highly ordered 'Fingers' subdomain, with the 'fingertips' forming a 5-stranded β-sheet that is missing in other RdRPs and interacts with the 'Thumb' subdomain. The conserved motifs B and C identified by HHpred were located within the Palm subdomain, at positions equivalent to those in other RdRPs. Structural superposition of the Palm subdomains from different RdRPs allowed identification of the third core motif, A, in RNA1_ORF4 (see below). Thus, we concluded that RNA1_ORF4 encodes an RdRP and provisionally named the discovered bipartite virus 'hot spring RNA virus (HsRV)', with the strain harbouring segments RNA1a and RNA2a denoted HsRV1. The four RdRPs encoded by the complete RNA1 segments shared 37 to 75% pairwise amino acid sequence identity and thus appear to represent four distinct virus species (or even higher

taxa). To characterize the diversity of HsRV-related RdRP in our FLDS data, the minor contigs including the aforementioned 10 sequences were analysed (Extended Data Fig. 2a). This analysis yielded several contigs with a high (>90%) identity to HsRV RNA1b RdRP. In addition, several contigs with moderate (>60%) identity to HsRV RNA1a or RNA1b were detected. Y66 and Y86 also included a few contigs related to HsRV RdRP.

The sequence profile of the HsRV RdRP was used to search the previously described FLDS sequence data from coastal seawater samples¹⁹, leading to the identification of two additional contigs (GenBank accessions: BDQA01000957 and BDQA01004869) encoding incomplete HsRV-like RdRPs. Searches against the IMG/VR database queried with these RdRPs yielded significant hits (E-value ≤ 1 × 10⁻⁵) to three additional putative RdRPs encoded by apparently complete or near-complete 5.3–5.6-kb-long genome segments (Ga0456180_000042, Ga0393213_00017, Ga0169446_00510; Fig. 3a, Extended Data Fig. 1b, Table 5 and Supplementary Text).
a

<figure><img src="image_17.png" /></figure>

c

<figure><img src="image_18.png" /></figure>

d

<figure><img src="image_19.png" /></figure>

b

<figure><img src="image_20.png" /></figure>

e

<figure><img src="image_21.png" /></figure>

**Fig. 3 | HsRV-like viruses from moderate environments.** a, RdRP-encoding segments of HsRV-like viruses from non-extreme aquatic ecosystems. ORFs encoding homologous proteins are shown as arrows with identical colours. b, Maximum-likelihood phylogeny of the HsRV-like RdRPs encoded by viruses from extreme (pink) and moderate (blue) ecosystems. Node support was assessed using the SH-aLRT, with the corresponding values (%) shown on the branches. The scale bar represents the number of substitutions per site. c, Comparison of the HsRV RdRP with the homologues encoded by viruses from moderate aquatic ecosystems. The model was produced using AlphaFold2. The models are coloured using the rainbow scheme, from blue N terminus to red C terminus. d, Comparison of the catalytic cores encompassing the conserved RdRP motifs A (yellow), B (green) and C (cyan). Top: the structure of bacteriophage phi6 RdRP with the substrate nucleoside triphosphates (NTP) and template RNA strand (blue ribbon). Bottom: the HsRV RdRP. Middle: structurally superposed HsRV-like RdRPs from moderate ecosystems. The NTP and active

site residues of motifs A and C are shown using the stick representation. The conserved aspartate residues of motif A are circled, with structurally equivalent residues indicated with red asterisks, whereas the non-conserved residue located in the loop facing away from the motif C in HsRV and related RdRP is indicated with the black asterisk. e, MSA of the conserved motifs of HsRV-like RdRPs from extreme (red shading) and moderate (blue shading) ecosystems with the corresponding regions from RdRPs and RT from other viruses (grey shading), including Moloney murine leukaemia virus (MMLV), hepatitis C virus (HCV), Norwalk virus (NV), PCV1 and hot spring partiti-like virus H5 (HsPV-H5). The sequences are indicated with the PDB or GenBank accession numbers. The conserved residues are shaded yellow, green and cyan, respectively, matching those in d. The conserved aspartate residues of motif A are highlighted in yellow, with structurally equivalent residues indicated with red asterisks, whereas the non-conserved residue in HsRV-like RdRPs located at the equivalent position as the second aspartate in other RdRPs is indicated with the black asterisk.

Ga0456180, Ga0393213 and Ga0169446 originate from floodplain (river sediments), salt marsh and aquarium samples, respectively. Phylogenetic analysis of HsRV-like RdRPs showed clear separation between viruses from the hot spring and those from moderate aquatic environments (Fig. 3b). Collectively, these results indicate that HsRV-like viruses are broadly distributed in both hot springs and non-extreme aquatic ecosystems.

Structural similarities between HsRV-like RdRPs and RTs

AF2 models of the three HsRV-like RdRPs from moderate ecosystems showed clear structural similarity with the HsRV RdRP, including the extended 'Fingers' subdomain (Fig. 3c). Another signature feature of these proteins is an unusual, extended RdRP motif A. In the canonical motif A, the two conserved Asp residues involved in catalysis and substrate discrimination<sup>22,23</sup>, respectively, are separated by 4–5 residues and bracket the catalytic GDD residues of motif C (Fig. 3d,e). By contrast, in HsRV-like RdRPs, the second Asp residue of motif A is not

conserved, and the corresponding residue is located in a loop facing perpendicularly away from motif C, suggesting that it cannot perform the same function. However, all analysed HsRV-like RdRPs contain an Asp (Asp*) which is located 12–14 residues away from the first Asp of motif A (Fig. 3e). Despite the extended spacing in the protein sequence, Asp* occupies a position equivalent to that of the second Asp of the canonical motif A (Fig. 3d,e) and is likely to be its counterpart involved in substrate discrimination.

We next performed structural clustering on the basis of the pairwise DALI Z-scores of the HsRV-like RdRPs together with selected RdRPs of other riboviruses, including putative phyla of RNA phages identified in recent metatranscriptome analyses<sup>7,24</sup> and RT encoded by eukaryotic viruses of the order *Ortervirales*<sup>25</sup> as well as non-viral RTs from bacteria and eukaryotes (Fig. 4a). The HsRV-like RdRPs from both hot springs and moderate aquatic ecosystems formed a tight cluster, underscoring their relatedness despite high sequence divergence. All previously known viral RdRPs formed a clade in the structure-based
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

**Fig. 3** | HsRV-like viruses from moderate environments. **a**, RdRP-encoding segments of HsRV-like viruses from non-extreme aquatic ecosystems. ORFs encoding homologous proteins are shown as arrows with identical colours. **b**, Maximum-likelihood phylogeny of the HsRV-like RdRPs encoded by viruses from extreme (pink) and moderate (blue) ecosystems. Node support was assessed using the SH-aLRT, with the corresponding values (%) shown on the branches. The scale bar represents the number of substitutions per site. **c**, Comparison of the HsRV RdRP with the homologues encoded by viruses from moderate aquatic ecosystems. The model was produced using AlphaFold2. The models are coloured using the rainbow scheme, from blue N terminus to red C terminus. **d**, Comparison of the catalytic cores encompassing the conserved RdRP motifs A (yellow), B (green) and C (cyan). Top: the structure of bacteriophage phi6 RdRP with the substrate nucleoside triphosphates (NTP) and template RNA strand (blue ribbon). Bottom: the HsRV RdRP. Middle: structurally superposed HsRV-like RdRPs from moderate ecosystems. The NTP and active

site residues of motifs A and C are shown using the stick representation. The conserved aspartate residues of motif A are circled, with structurally equivalent residues indicated with red asterisks, whereas the non-conserved residue located in the loop facing away from the motif C in HsRV and related RdRP is indicated with the black asterisk. **e**, MSA of the conserved motifs of HsRV-like RdRPs from extreme (red shading) and moderate (blue shading) ecosystems with the corresponding regions from RdRPs and RT from other viruses (grey shading), including Moloney murine leukaemia virus (MMLV), hepatitis C virus (HCV), Norwalk virus (NV), PCV1 and hot spring partiti-like virus H5 (HsPV-H5). The sequences are indicated with the PDB or GenBank accession numbers. The conserved residues are shaded yellow, green and cyan, respectively, matching those in **d**. The conserved aspartate residues of motif A are highlighted in yellow, with structurally equivalent residues indicated with red asterisks, whereas the non-conserved residue in HsRV-like RdRPs located at the equivalent position as the second aspartate in other RdRPs is indicated with the black asterisk.

Ga0456180, Ga0393213 and Ga0169446 originate from floodplain (river sediments), salt marsh and aquarium samples, respectively. Phylogenetic analysis of HsRV-like RdRPs showed clear separation between viruses from the hot spring and those from moderate aquatic environments (Fig. 3b). Collectively, these results indicate that HsRV-like viruses are broadly distributed in both hot springs and non-extreme aquatic ecosystems.

Structural similarities between HsRV-like RdRPs and RTs

AF2 models of the three HsRV-like RdRPs from moderate ecosystems showed clear structural similarity with the HsRV RdRP, including the extended 'Fingers' subdomain (Fig. 3c). Another signature feature of these proteins is an unusual, extended RdRP motif A. In the canonical motif A, the two conserved Asp residues involved in catalysis and substrate discrimination<sup>22,23</sup>, respectively, are separated by 4–5 residues and bracket the catalytic GDD residues of motif C (Fig. 3d,e). By contrast, in HsRV-like RdRPs, the second Asp residue of motif A is not

conserved, and the corresponding residue is located in a loop facing perpendicularly away from motif C, suggesting that it cannot perform the same function. However, all analysed HsRV-like RdRPs contain an Asp (Asp*) which is located 12–14 residues away from the first Asp of motif A (Fig. 3e). Despite the extended spacing in the protein sequence, Asp* occupies a position equivalent to that of the second Asp of the canonical motif A (Fig. 3d,e) and is likely to be its counterpart involved in substrate discrimination.

We next performed structural clustering on the basis of the pairwise DALI Z-scores of the HsRV-like RdRPs together with selected RdRPs of other riboviruses, including putative phyla of RNA phages identified in recent metatranscriptome analyses<sup>7,24</sup> and RT encoded by eukaryotic viruses of the order *Ortervirales*<sup>25</sup> as well as non-viral RTs from bacteria and eukaryotes (Fig. 4a). The HsRV-like RdRPs from both hot springs and moderate aquatic ecosystems formed a tight cluster, underscoring their relatedness despite high sequence divergence. All previously known viral RdRPs formed a clade in the structure-based
a

<figure><img src="image_22.png" /></figure>

**Fig. 4 |** *Structural relationships between RdRPs and RTs.* *a.* Matrix and cluster dendrogram were constructed on the basis of the pairwise Z-score comparisons calculated using DALI. Different protein groups are highlighted with different background colours on the dendrogram: green, RdRPs from previously characterized viruses; blue, viral and non-viral RTs; red, HsRV-like RdRPs. The colour scale indicates the corresponding Z-scores. hPBV, human picobirnavirus; FMDV, foot-and-mouth disease virus; ReoV, reovirus; LACV, La Crosse virus;

b

<figure><img src="image_23.png" /></figure>

HIV-1, human immunodeficiency virus 1; TERT, telomerase RT; non-LTR R2 retroel., non-long terminal repeat R2 retroelement; AF2, AlphaFold2 model. For experimentally determined structures, the corresponding PDB accession numbers are indicated at the bottom of the matrix. **b**, Structural comparison of the core domain of RdRPs and RT encompassing the conserved motifs A–C. The structures are coloured using the rainbow scheme, from blue N terminus to red C terminus.

dendrogram, but the HsRV-like RdRPs remained separated from those (Fig. 4a). The two viral RdRP clusters were interspersed with the RTs, such that the viral RTs were the closest structural neighbours of the HsRV-like RdRPs. This result confirms the extreme divergence of the HsRV-like RdRPs and might reflect a closer relationship to viral RTs. This unexpected link was strengthened by the comparison of the ‘Palm’ subdomain of HsRV-like RdRPs with homologues from other riboviruses as well as viral and non-viral RTs. In RdRPs of riboviruses from 5 established phyla¹⁰, the first β-strand (blue in Fig. 4b) containing motif A and the motif B-containing α-helix are separated by a characteristic helix-turn-helix (HTH) region followed by a β-hairpin corresponding to the ‘Middle’ finger subdomain (Fig. 2d,e). However, the HTH motif is absent in both the HsRV-like RdRPs and viral RTs. Notably, non-viral RTs, such as those from group II introns or retrons, contain the HTH motif but lack the β-hairpin region, which is compatible with the intermediate position of RTs between the two clades of viral RdRPs. Thus, the HsRV-like RdRPs might comprise an evolutionary intermediate between viral RdRPs and RTs. A BLASTN search against the metagenomic DNA sequences obtained from the hot springs did not detect HsRV-like sequences, suggesting that HsRV-like viruses are bona fide riboviruses that lack a DNA intermediate stage (Supplementary Text).

A thermoacidophilic partiti-like virus

Analysis of the FLDS RNA sequencing data from the stations H4 (68.8 °C, pH 3.2), H5 (69.7 °C, pH 3.1) and Y66 (68.7 °C, pH 2.7) revealed a bipartite virus genome unrelated to HsRV (Fig. 5a, Extended Data Table 2 and Fig. 2b). The genomic segments, RNA1 and RNA2, shared conserved 5' terminal sequences and encoded one and two proteins, respectively (Fig. 5b). ORF1 of RNA1 was unambiguously identified as an RdRP, yielding significant BLASTP hits to RdRPs of members of the *Partitiviridae* family, with the best hit being to the unclassified Driatsky virus (QIS87951; E-value = $1 \times 10^{-95}$). We denoted this virus as hot spring partiti-like virus (HsPV). The similarity between the termini of

the segments precluded assignment of all sets of segments to particu-
lar virus strains. However, on the basis of co-occurrence in the same
sample and similar abundances, segment pairs RNA1_a and RNA2_b
from sample H5 could be assigned to the same virus strain, HsPV1.
Phylogenetic analysis of the RdRP sequence from diverse classified
and unclassified partiti-like viruses showed that HsPVs and Driatsky
virus (see below) were nested within genPartiti.0029 (Fig. 5c), a highly
diverse, unclassified group defined in a recent metatranscriptome
study⁸. The genPartiti.0029, including HsPV and Driatsky virus and
several other subclades, formed a deep clade separate from all other
partitiviruses. Thus, genPartiti.0029 can be considered a separate sister
family to the bona fide *Partitiviridae*. AF2 modelling yielded an HsPV
RdRP model closely similar to that of the RdRP of the deltapartitivirus
pepper cryptic virus 1 (PCV1; Fig. 5d and Extended Data Fig. 3a), which
was confirmed by DALI Z-score-based clustering (Fig. 4a), where the
two viruses formed a clade next to picobirnaviruses.

Structural modelling of RNA2 ORF1 of different HsPV strains and Driatsky virus yielded a high-quality model (pLDDT = 78.8), with only the terminal regions being of lower quality (Extended Data Fig. 3b and Supplementary Text). Structure similarity searches against the PDB database using DALI produced significant hits to capsid proteins (CPs) of partitiviruses and picobirnaviruses²⁶⁻²⁸, with the best match (Z-score = 8.2) to the CP of PCV1 (Fig. 5e; PDB ID: 7ncr; Deltapartitivirus). Thus, the RdRP phylogeny and structural similarity of the CPs indicate that HsPV is related to members of the family *Partitiviridae*. The phylogenetic relationship between amino acid sequences of HsPVs is shown in Extended Data Fig. 4.

HsRV and HsPV probably infect prokaryotic hosts

All samples in which HsRV and HsPV were detected nearly exclusively
contained rRNA sequences from prokaryotes, with eukaryotic presence
being below 1%. This is consistent with eukaryotes being unable to
thrive in polyextremophilic conditions combining high temperatures
and acidic pH. The microbial communities in all 4 samples (H4, H5,
<figure><img src="image_20.png" /><figcaption>**Fig. 4 |** *Structural relationships between RdRPs and RTs.* *a.* Matrix and cluster dendrogram were constructed on the basis of the pairwise Z-score comparisons calculated using DALI. Different protein groups are highlighted with different background colours on the dendrogram: green, RdRPs from previously characterized viruses; blue, viral and non-viral RTs; red, HsRV-like RdRPs. The colour scale indicates the corresponding Z-scores. hPBV, human picobirnavirus; FMDV, foot-and-mouth disease virus; ReoV, reovirus; LACV, La Crosse virus;</figcaption></figure>

<figure><img src="image_21.png" /></figure>

HIV-1, human immunodeficiency virus 1; TERT, telomerase RT; non-LTR R2 retroel., non-long terminal repeat R2 retroelement; AF2, AlphaFold2 model. For experimentally determined structures, the corresponding PDB accession numbers are indicated at the bottom of the matrix. **b.** Structural comparison of the core domain of RdRPs and RT encompassing the conserved motifs A–C. The structures are coloured using the rainbow scheme, from blue N terminus to red C terminus.

dendrogram, but the HsRV-like RdRPs remained separated from those (Fig. 4a). The two viral RdRP clusters were interspersed with the RTs, such that the viral RTs were the closest structural neighbours of the HsRV-like RdRPs. This result confirms the extreme divergence of the HsRV-like RdRPs and might reflect a closer relationship to viral RTs. This unexpected link was strengthened by the comparison of the ‘Palm’ subdomain of HsRV-like RdRPs with homologues from other riboviruses as well as viral and non-viral RTs. In RdRPs of riboviruses from 5 established phyla¹⁰, the first β-strand (blue in Fig. 4b) containing motif A and the motif B-containing α-helix are separated by a characteristic helix-turn-helix (HTH) region followed by a β-hairpin corresponding to the ‘Middle’ finger subdomain (Fig. 2d,e). However, the HTH motif is absent in both the HsRV-like RdRPs and viral RTs. Notably, non-viral RTs, such as those from group II introns or retrons, contain the HTH motif but lack the β-hairpin region, which is compatible with the intermediate position of RTs between the two clades of viral RdRPs. Thus, the HsRV-like RdRPs might comprise an evolutionary intermediate between viral RdRPs and RTs. A BLASTN search against the metagenomic DNA sequences obtained from the hot springs did not detect HsRV-like sequences, suggesting that HsRV-like viruses are bona fide riboviruses that lack a DNA intermediate stage (Supplementary Text).

## A thermoacidophilic partiti-like virus

Analysis of the FLDS RNA sequencing data from the stations H4 (68.8 °C, pH 3.2), H5 (69.7 °C, pH 3.1) and Y66 (68.7 °C, pH 2.7) revealed a bipartite virus genome unrelated to HsRV (Fig. 5a, Extended Data Table 2 and Fig. 2b). The genomic segments, RNA1 and RNA2, shared conserved 5' terminal sequences and encoded one and two proteins, respectively (Fig. 5b). ORF1 of RNA1 was unambiguously identified as an RdRP, yielding significant BLASTP hits to RdRPs of members of the *Partitiviridae* family, with the best hit being to the unclassified Driatsky virus (QIS87951; E-value = $1 \times 10^{-95}$). We denoted this virus as hot spring partiti-like virus (HsPV). The similarity between the termini of

the segments precluded assignment of all sets of segments to particular virus strains. However, on the basis of co-occurrence in the same sample and similar abundances, segment pairs RNA1_a and RNA2_b from sample H5 could be assigned to the same virus strain, HsPV1. Phylogenetic analysis of the RdRP sequence from diverse classified and unclassified partiti-like viruses showed that HsPVs and Driatsky virus (see below) were nested within genPartiti.0029 (Fig. 5c), a highly diverse, unclassified group defined in a recent metatranscriptome study⁸. The genPartiti.0029, including HsPV and Driatsky virus and several other subclades, formed a deep clade separate from all other partitiviruses. Thus, genPartiti.0029 can be considered a separate sister family to the bona fide *Partitiviridae*. AF2 modelling yielded an HsPV RdRP model closely similar to that of the RdRP of the deltapartitivirus pepper cryptic virus 1 (PCV1; Fig. 5d and Extended Data Fig. 3a), which was confirmed by DALI Z-score-based clustering (Fig. 4a), where the two viruses formed a clade next to picobirnaviruses.

Structural modelling of RNA2 ORF1 of different HsPV strains and Driatsky virus yielded a high-quality model (pLDDT = 78.8), with only the terminal regions being of lower quality (Extended Data Fig. 3b and Supplementary Text). Structure similarity searches against the PDB database using DALI produced significant hits to capsid proteins (CPs) of partitiviruses and picobirnaviruses²⁶⁻²⁸, with the best match (Z-score = 8.2) to the CP of PCV1 (Fig. 5e; PDB ID: 7ncr; Deltapartitivirus). Thus, the RdRP phylogeny and structural similarity of the CPs indicate that HsPV is related to members of the family *Partitiviridae*. The phylogenetic relationship between amino acid sequences of HsPVs is shown in Extended Data Fig. 4.

## HsRV and HsPV probably infect prokaryotic hosts

All samples in which HsRV and HsPV were detected nearly exclusively contained rRNA sequences from prokaryotes, with eukaryotic presence being below 1%. This is consistent with eukaryotes being unable to thrive in polyextremophilic conditions combining high temperatures and acidic pH. The microbial communities in all 4 samples (H4, H5,
a

<figure><img src="image_24.png" /></figure>

b

<figure><img src="image_25.png" /></figure>

c

<figure><img src="image_26.png" /></figure>

d

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

e

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

PCV1 CP
(PDB: 7NCR)

**Fig. 5** | A thermoacidophilic partiti-like virus. **a**, Genome organization and conservation of the two genome segments, RNA1 and RNA2, of HsPV. ORFs encoding homologous proteins are shown as arrows with identical colours. Yellow circles represent predicted SD RBS. Asterisks denote putative genes encoding predicted TMD-containing proteins. **b**, MSA of the 5' and 3'-terminal regions of the coding strands of reconstructed genome segments. Black shading, 100% nucleotide identity; grey shading, >50% nucleotide identity. **c**, Maximum- likelihood phylogeny of the RdRP proteins from representative members of the family *Partitiviridae* and related sequences (including all HsPV strains, shown in orange). Clades corresponding to the official *Partitiviridae* genera are shown

in blue, whereas those corresponding to unclassified groups are in grey. Node supports were assessed using the SH-aLRT; circles indicate nodes with ≥90% supports. The scale bar represents the number of substitutions per site. GenBank accession numbers of used sequences are shown in Supplementary Text.
**d**, Comparison of the RdRP from HsPV-H5 with a homologue from deltapartitivirus PCV1. **e**, Comparison of the CP from HsPV-H4 with a homologue from deltapartitivirus PCV1. The structures are coloured using the rainbow scheme, from blue N terminus to red C terminus. The HsPV RdRP and CP structures coloured on the basis of the pLDDT quality scores can be found in Extended Data Fig. 3.

Y66 and Oi) were dominated by bacteria (Supplementary Text). Thus, HsRV and HsPV most probably infect bacteria. To test this inference, we predicted ribosome-binding SD motifs in all HsRV and HsPV strains. SD motifs are essential for translation initiation in many prokaryotes, and their conservation is a diagnostic feature of prokaryotic genes that has been used to assign bacterial hosts to several groups of RNA viruses, namely, picobirnaviruses and partitiviruses, including gen-Partiti.0019 and genPartiti.0029 (refs. 8,29). Analysis of the HsRV and HsPV genomes showed that nearly every gene in these viruses is pre-ceded by an SD motif (Figs. 2a, 3a and 5a and Extended Data Table 6), further suggesting that both HsRV and HsPV infect prokaryotic hosts. Bacteria of the genus *Hydrogenobaculum* (family *Aquificaceae*) were

predominant (>95%) in samples H4 and H5 and highly abundant in Y66 (>85%), suggesting that HsPV detected in all three samples infects *Hydrogenobaculum* sp.

No CRISPR spacers matching the HsRV and HsPV genomes were identified in the public databases or the 919 CRISPR spacer sequences obtained by metagenomic DNA sequencing of the hot spring samples (Supplementary Text). Nevertheless, the lack of eukaryotes in the hot spring samples, contrasted by the dominance of bacteria, together with the presence of typical prokaryotic SD motifs upstream of the predicted virus genes and the polycistronic organization of the viral genomes, strongly suggest that HsRV and HsPV are viruses of thermo-philic bacteria.
a

<figure><img src="image_22.png" /></figure>

b

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

c

<figure><img src="image_26.png" /></figure>

**Fig. 5 | A thermoacidophilic partiti-like virus.** a, Genome organization and conservation of the two genome segments, RNA1 and RNA2, of HsPV. ORFs encoding homologous proteins are shown as arrows with identical colours. Yellow circles represent predicted SD RBS. Asterisks denote putative genes encoding predicted TMD-containing proteins. b, MSA of the 5' and 3'-terminal regions of the coding strands of reconstructed genome segments. Black shading, 100% nucleotide identity; grey shading, >50% nucleotide identity. c, Maximum-likelihood phylogeny of the RdRP proteins from representative members of the family *Partitiviridae* and related sequences (including all HsPV strains, shown in orange). Clades corresponding to the official *Partitiviridae* genera are shown

d

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

e

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

PCV1 CP
(PDB: 7NCR)

in blue, whereas those corresponding to unclassified groups are in grey. Node supports were assessed using the SH-aLRT; circles indicate nodes with ≥90% supports. The scale bar represents the number of substitutions per site. GenBank accession numbers of used sequences are shown in Supplementary Text.
**d.** Comparison of the RdRP from HsPV-H5 with a homologue from deltapartitivirus PCV1. **e.** Comparison of the CP from HsPV-H4 with a homologue from deltapartitivirus PCV1. The structures are coloured using the rainbow scheme, from blue N terminus to red C terminus. The HsPV RdRP and CP structures coloured on the basis of the pLDDT quality scores can be found in Extended Data Fig. 3.

Y66 and Oi) were dominated by bacteria (Supplementary Text). Thus, HsRV and HsPV most probably infect bacteria. To test this inference, we predicted ribosome-binding SD motifs in all HsRV and HsPV strains. SD motifs are essential for translation initiation in many prokaryotes, and their conservation is a diagnostic feature of prokaryotic genes that has been used to assign bacterial hosts to several groups of RNA viruses, namely, picobirnaviruses and partitiviruses, including gen-Partiti.0019 and genPartiti.0029 (refs. 8,29). Analysis of the HsRV and HsPV genomes showed that nearly every gene in these viruses is pre-ceded by an SD motif (Figs. 2a, 3a and 5a and Extended Data Table 6), further suggesting that both HsRV and HsPV infect prokaryotic hosts. Bacteria of the genus *Hydrogenobaculum* (family *Aquificaceae*) were

predominant (>95%) in samples H4 and H5 and highly abundant in Y66 (>85%), suggesting that HsPV detected in all three samples infects *Hydrogenobaculum* sp.

No CRISPR spacers matching the HsRV and HsPV genomes were identified in the public databases or the 919 CRISPR spacer sequences obtained by metagenomic DNA sequencing of the hot spring samples (Supplementary Text). Nevertheless, the lack of eukaryotes in the hot spring samples, contrasted by the dominance of bacteria, together with the presence of typical prokaryotic SD motifs upstream of the predicted virus genes and the polycistronic organization of the viral genomes, strongly suggest that HsRV and HsPV are viruses of thermo-philic bacteria.
<table><caption>Table 1 | Characteristics of hot spring water samples</caption><thead><tr><th>Code</th><th>Geographical coordinates</th><th>Area</th><th>Temp (°C)</th><th>pH</th><th>DO (mg l<sup>-1</sup>)</th><th>H<sub>2</sub>S (mM)</th><th>Sampling date</th><th>Site characteristics</th></tr></thead><tbody><tr><td>H4</td><td>31°54′07.5"N, 130°50′06.2"E</td><td>Hayashida</td><td>68.8</td><td>3.2</td><td>2.1</td><td>1.3</td><td>10 Mar 2017</td><td>Transparent water pool with sulfur precipitates</td></tr><tr><td>H5</td><td>31°54′07.5"N, 130°50′06.2"E</td><td>Hayashida</td><td>69.7</td><td>3.1</td><td>2.0</td><td>1.8</td><td>10 Mar 2017</td><td>Transparent water pool with sulfur precipitates</td></tr><tr><td>T1</td><td>31°54′37.7"N, 130°49′00.6"E</td><td>Tearai</td><td>92.1</td><td>2.9</td><td>-</td><td>0.0</td><td>09 Mar 2017</td><td>Yellowish grey water pool with active venting</td></tr><tr><td>T2</td><td></td><td>Tearai</td><td>95.9</td><td>2.1</td><td>-</td><td>0.0</td><td>09 Mar 2017</td><td>Yellowish grey vent pool</td></tr><tr><td>T3</td><td></td><td>Tearai</td><td>94.4</td><td>2.4</td><td>0.0</td><td>0.0</td><td>09 Mar 2017</td><td>Slightly grey water vent pool</td></tr><tr><td>T4</td><td></td><td>Tearai</td><td>92.8</td><td>2.7</td><td>0.0</td><td>0.0</td><td>09 Mar 2017</td><td>Yellowish grey water vent pool</td></tr><tr><td>Y66</td><td>31°55′03.8"N, 130°48′40.4"E</td><td>Yunoike</td><td>68.7</td><td>2.7</td><td>2.1</td><td>0.0</td><td>10 Mar 2017</td><td>Yellowish grey vent pool</td></tr><tr><td>Y80</td><td></td><td>Yunoike</td><td>75-86<sup>a</sup></td><td>2.5</td><td>1.5</td><td>0.0</td><td>10 Mar 2017</td><td>Muddy small vent pool</td></tr><tr><td>Y86</td><td></td><td>Yunoike</td><td>86.5</td><td>2.5</td><td>0.0</td><td>0.0</td><td>10 Mar 2017</td><td>Muddy boiling vent pool</td></tr><tr><td>Oi</td><td>32°44′25.3"N, 130°15′48.4"E</td><td>Unzen</td><td>79.3</td><td>2.2</td><td>0.0</td><td>0.4</td><td>18 Nov 2015</td><td>Yellowish grey vent pool</td></tr><tr><td>Ob</td><td>32°43′33.0"N, 130°12′24.7"E</td><td>Obama</td><td>72.8</td><td>7.9</td><td>0.0</td><td>0.0</td><td>17 Nov 2015</td><td>Transparent water pool under hot spring water tank</td></tr></tbody></table>

## Discussion

The discovery of the HsRV-like group of riboviruses recapitulates previous findings of several small groups of riboviruses that are predicted to infect bacteria and might become distinct phyla<sup>7,8</sup>. However, the RdRPs of HsRV and its relatives seem to deviate from the RdRP consensus farther than any of the other recently discovered putative phyla, with none of which they appear to be affiliated, and possess unusual (predicted) structural features that appear to link them to viral RTs. Whether this connection reflects an intermediate position of the HsRV-like viruses between the kingdoms *Orthornavirae* and *Pararnavirae*, or results from convergent evolution, remains uncertain and should be clarified by sequencing and structural analysis of additional members of this group, or possibly, other groups of riboviruses with similar features. Furthermore, although we did not detect any evidence of the formation of DNA copies of the genomes of HsRV-like viruses, it will be of interest to determine whether their RdRPs possess RT activity, as shown for some viral RdRPs<sup>30</sup>. Regardless, HsRV-like viruses are strong candidates for a separate phylum in the kingdom *Orthornavirae*, which we propose to name *Artimaviricota* after the potential link to viral RTs (arti) and ‘artima’ which means ‘close’ in Lithuanian, or even a third kingdom within the realm *Riboviria*.

This report is a proof of concept for the discovery of multiple, perhaps many groups of riboviruses with unexpected properties by obtaining complete genomes of segmented riboviruses from meta-dsRNA-seq data and mining metatranscriptomes from habitats with distinct conditions. Information on non-RdRP segments is unavailable for most of the RNA virus lineages identified only from metatranscriptomes, whereas riboviruses that are distantly related to known RNA viruses can be missed altogether. Our approach helps to overcome these limitations and contributes to a more complete characterization of RNA viromes.

## Methods

### Sample collection

A total of 11 samples were collected from five hot springs regions in southern Japan, in proximity to active volcanoes (Table 1 and Supplementary Text), according to the instructions of Unzen City, Unzen Nature Conservation Bureau and private companies that maintain each hot spring region. Temperature, pH and dissolved oxygen (DO) were measured in situ by using a multiple electrode sensor (D-55, Horiba). H₂S concentration was calculated from the spectrophotometric absorbance at 680 nm of methylene blue formed from a reaction with N,N-dimethyl-p-phenylenediamine in FeCl₃-HCl solution. Typical measurement errors are 0.1 for pH, 0.1 mg l⁻¹ for DO

and 5% for H₂S. Dissolved chemicals and water isotope ratios of the geothermal waters were also measured and are summarized in Supplementary Text.

Most of the sampling sites were characterized by high temperatures above 65 °C, acidic pH (2–3, except for Site Ob with a slightly alkaline pH of 7.9) and lower level of DO with accompanying grey mud or light-yellow sulfur deposits. At each sampling station, ~10 l of hot spring water was collected in a sterilized plastic bag and then filtered with 0.2-µm-pore-size cellulose acetate membrane filters in 47 mm diameter (Advantecn) within 0.5–3 h after sampling. The filters were stored at –80 °C until nucleic acid extraction.

### RNA extraction

Cells collected on a portion of the 0.2-µm-pore-size filters corresponding to ~2 l of hot spring water were pulverized in a mortar in liquid nitrogen and suspended in dsRNA extraction buffer (20 mM Tris-HCl, pH 6.8, 200 mM NaCl, 2 mM EDTA, 1% SDS and 0.1% (v/v) β-mercaptoethanol) or TRIzol buffer for ds- and ssRNA purification, respectively. For dsRNA purification, total nucleic acids were manually extracted with SDS-phenol. dsRNA was purified using the cellulose resin chromatography method<sup>16,31</sup>. The remaining DNA and ssRNA were removed by DNase I (Invitrogen) and S1 nuclease (Invitrogen) treatment<sup>19</sup>. For ssRNA purification, the ssRNA fraction was collected using the TRIzol Plus RNA purification kit (Invitrogen) according to manufacturer protocol. The ssRNA fraction was treated with DNase I (Invitrogen) and concentrated using the RNA Clean and Concentrator-5 kit (Zymoresearch).

### Complementary DNA synthesis

Complementary DNA (cDNA) was synthesized from purified dsRNA and ssRNA as described previously<sup>19</sup>. In brief, purified dsRNA was physically fragmented into ~1.5 kbp and adapter oligonucleotide (U2: 5'-GAC GTA AGA ACG TCG CAC CA-3') was ligated to the 3'-end of fragmented dsRNAs. After heat denaturation with an oligonucleotide primer (U2-comp: 5'-TGG TGC GAC GTT CTT ACG TC-3'), that has complementary sequence to the adapter oligonucleotide, cDNA was synthesized using SMARTer RACE 5'/3' kit (Takara Bio). ssRNA was converted into cDNA using SMARTer Universal Low Input RNA kit according to manufacturer protocol (Takara Bio). After PCR amplification, cDNA was fragmented by a Covaris S220 ultrasonicator.

### Illumina sequencing library construction and sequencing

Illumina sequencing libraries were then constructed using KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems) from the physically shared environmental cDNAs. The libraries were sequenced using the

<footnote>*There were temperature gradients in the pool site: surface layer 75.0°C; bottom layer 81.6°C, 80.3°C, 85.9°C; middle layer 81.0°C.</footnote>
<table><caption>Table 1 | Characteristics of hot spring water samples</caption><thead><tr><th>Code</th><th>Geographical coordinates</th><th>Area</th><th>Temp (°C)</th><th>pH</th><th>DO (mg l<sup>-1</sup>)</th><th>H<sub>2</sub>S (mM)</th><th>Sampling date</th><th>Site characteristics</th></tr></thead><tbody><tr><td>H4</td><td>31°54′07.5"N, 130°50′06.2"E</td><td>Hayashida</td><td>68.8</td><td>3.2</td><td>2.1</td><td>1.3</td><td>10 Mar 2017</td><td>Transparent water pool with sulfur precipitates</td></tr><tr><td>H5</td><td>31°54′07.5"N, 130°50′06.2"E</td><td>Hayashida</td><td>69.7</td><td>3.1</td><td>2.0</td><td>1.8</td><td>10 Mar 2017</td><td>Transparent water pool with sulfur precipitates</td></tr><tr><td>T1</td><td>31°54′37.7"N, 130°49′00.6"E</td><td>Tearai</td><td>92.1</td><td>2.9</td><td>-</td><td>0.0</td><td>09 Mar 2017</td><td>Yellowish grey water pool with active venting</td></tr><tr><td>T2</td><td></td><td>Tearai</td><td>95.9</td><td>2.1</td><td>-</td><td>0.0</td><td>09 Mar 2017</td><td>Yellowish grey vent pool</td></tr><tr><td>T3</td><td></td><td>Tearai</td><td>94.4</td><td>2.4</td><td>0.0</td><td>0.0</td><td>09 Mar 2017</td><td>Slightly grey water vent pool</td></tr><tr><td>T4</td><td></td><td>Tearai</td><td>92.8</td><td>2.7</td><td>0.0</td><td>0.0</td><td>09 Mar 2017</td><td>Yellowish grey water vent pool</td></tr><tr><td>Y66</td><td>31°55′03.8"N, 130°48′40.4"E</td><td>Yunoike</td><td>68.7</td><td>2.7</td><td>2.1</td><td>0.0</td><td>10 Mar 2017</td><td>Yellowish grey vent pool</td></tr><tr><td>Y80</td><td></td><td>Yunoike</td><td>75–86<sup>a</sup></td><td>2.5</td><td>1.5</td><td>0.0</td><td>10 Mar 2017</td><td>Muddy small vent pool</td></tr><tr><td>Y86</td><td></td><td>Yunoike</td><td>86.5</td><td>2.5</td><td>0.0</td><td>0.0</td><td>10 Mar 2017</td><td>Muddy boiling vent pool</td></tr><tr><td>Oi</td><td>32°44′25.3"N, 130°15′48.4"E</td><td>Unzen</td><td>79.3</td><td>2.2</td><td>0.0</td><td>0.4</td><td>18 Nov 2015</td><td>Yellowish grey vent pool</td></tr><tr><td>Ob</td><td>32°43′33.0"N, 130°12′24.7"E</td><td>Obama</td><td>72.8</td><td>7.9</td><td>0.0</td><td>0.0</td><td>17 Nov 2015</td><td>Transparent water pool under hot spring water tank</td></tr></tbody></table>

## Discussion

The discovery of the HsRV-like group of riboviruses recapitulates previous findings of several small groups of riboviruses that are predicted to infect bacteria and might become distinct phyla<sup>7,8</sup>. However, the RdRPs of HsRV and its relatives seem to deviate from the RdRP consensus farther than any of the other recently discovered putative phyla, with none of which they appear to be affiliated, and possess unusual (predicted) structural features that appear to link them to viral RTs. Whether this connection reflects an intermediate position of the HsRV-like viruses between the kingdoms *Orthornavirae* and *Pararnavirae*, or results from convergent evolution, remains uncertain and should be clarified by sequencing and structural analysis of additional members of this group, or possibly, other groups of riboviruses with similar features. Furthermore, although we did not detect any evidence of the formation of DNA copies of the genomes of HsRV-like viruses, it will be of interest to determine whether their RdRPs possess RT activity, as shown for some viral RdRPs<sup>30</sup>. Regardless, HsRV-like viruses are strong candidates for a separate phylum in the kingdom *Orthornavirae*, which we propose to name *Artimaviricota* after the potential link to viral RTs (arti) and ‘artima’ which means ‘close’ in Lithuanian, or even a third kingdom within the realm *Riboviria*.

This report is a proof of concept for the discovery of multiple, perhaps many groups of riboviruses with unexpected properties by obtaining complete genomes of segmented riboviruses from meta-dsRNA-seq data and mining metatranscriptomes from habitats with distinct conditions. Information on non-RdRP segments is unavailable for most of the RNA virus lineages identified only from metatranscriptomes, whereas riboviruses that are distantly related to known RNA viruses can be missed altogether. Our approach helps to overcome these limitations and contributes to a more complete characterization of RNA viromes.

## Methods

### Sample collection

A total of 11 samples were collected from five hot springs regions in southern Japan, in proximity to active volcanoes (Table 1 and Supplementary Text), according to the instructions of Unzen City, Unzen Nature Conservation Bureau and private companies that maintain each hot spring region. Temperature, pH and dissolved oxygen (DO) were measured in situ by using a multiple electrode sensor (D-55, Horiba). H₂S concentration was calculated from the spectrophotometric absorbance at 680 nm of methylene blue formed from a reaction with N,N-dimethyl-p-phenylenediamine in FeCl₃·HCl solution. Typical measurement errors are 0.1 for pH, 0.1 mg l⁻¹ for DO

and 5% for H₂S. Dissolved chemicals and water isotope ratios of the geothermal waters were also measured and are summarized in Supplementary Text.

Most of the sampling sites were characterized by high temperatures above 65 °C, acidic pH (2–3, except for Site Ob with a slightly alkaline pH of 7.9) and lower level of DO with accompanying grey mud or light-yellow sulfur deposits. At each sampling station, ~10 l of hot spring water was collected in a sterilized plastic bag and then filtered with 0.2-µm-pore-size cellulose acetate membrane filters in 47 mm diameter (Advantecn) within 0.5–3 h after sampling. The filters were stored at –80 °C until nucleic acid extraction.

### RNA extraction

Cells collected on a portion of the 0.2-µm-pore-size filters corresponding to ~2 l of hot spring water were pulverized in a mortar in liquid nitrogen and suspended in dsRNA extraction buffer (20 mM Tris–HCl, pH 6.8, 200 mM NaCl, 2 mM EDTA, 1% SDS and 0.1% (v/v) β-mercaptoethanol) or TRIzol buffer for ds- and ssRNA purification, respectively. For dsRNA purification, total nucleic acids were manually extracted with SDS-phenol. dsRNA was purified using the cellulose resin chromatography method<sup>16,31</sup>. The remaining DNA and ssRNA were removed by DNase I (Invitrogen) and S1 nuclease (Invitrogen) treatment<sup>19</sup>. For ssRNA purification, the ssRNA fraction was collected using the TRIzol Plus RNA purification kit (Invitrogen) according to manufacturer protocol. The ssRNA fraction was treated with DNase I (Invitrogen) and concentrated using the RNA Clean and Concentrator-5 kit (Zymoresearch).

### Complementary DNA synthesis

Complementary DNA (cDNA) was synthesized from purified dsRNA and ssRNA as described previously<sup>19</sup>. In brief, purified dsRNA was physically fragmented into ~1.5 kbp and adapter oligonucleotide (U2: 5'-GAC GTA AGA ACG TCG CAC CA-3') was ligated to the 3'-end of fragmented dsRNAs. After heat denaturation with an oligonucleotide primer (U2-comp: 5'-TGG TGC GAC GTT CTT ACG TC-3'), that has complementary sequence to the adapter oligonucleotide, cDNA was synthesized using SMARTer RACE 5'/3' kit (Takara Bio). ssRNA was converted into cDNA using SMARTer Universal Low Input RNA kit according to manufacturer protocol (Takara Bio). After PCR amplification, cDNA was fragmented by a Covaris S220 ultrasonicator.

### Illumina sequencing library construction and sequencing

Illumina sequencing libraries were then constructed using KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems) from the physically shared environmental cDNAs. The libraries were sequenced using the

<footnote>*There were temperature gradients in the pool site: surface layer 75.0°C; bottom layer 81.6°C, 80.3°C, 85.9°C; middle layer 81.0°C.</footnote>
Illumina MiSeq v3 Reagent kit (600 cycles) with 300-bp paired-end reads on the Illumina MiSeq platform.

## Data processing

Trimmed reads were obtained using a custom Perl pipeline script (https://github.com/takakiy/FLDS) from dsRNA raw sequence reads¹⁷. The clean reads were subjected to de novo assembly using CLC GENOMICS WORKBENCH v.11.0 (Qiagen) with the following parameters: a minimum contig length of 500, word value set to auto and bubble size set to auto. The full-length sequences were manually extracted using CLC GENOMICS WORKBENCH v.11.0 (Qiagen), Genetyx v.14 (Genetyx) and Tablet viewer v.1.19.09.03 (ref. 32) as described previously³³. In brief, contigs for which both termini were determined to be the ends were identified as full-length sequences. In cases of dominant reads (more than 10 reads) that stopped in the same position around the ends of contigs in the mapping analysis, that position was recognized as the segment (genome) end. In this study, major full-length sequences with >1,000 average coverage were analysed, except for the Oi sample where all full-length sequences were recovered. From ssRNA raw sequence reads, trimmed reads were also obtained using a custom Perl pipeline script (https://github.com/takakiy/FLDS). The resultant clean reads were applied to phyloFlash²⁰ to identify active microbes in our samples.

## Sequence analyses

RNA viral genes were identified using the BLASTX programme against the NCBI non-redundant (nr) database with an *E*-value ≤ 1 × 10⁻⁵⁰. The ribosome-binding SD motifs were identified using Prodigal³⁴. Remote homology searches were performed using HHpred against the PDB70, Pfam, UniProt-SwissProt-viral70 and NCBI-CD (conserved domains) databases³⁵. MSA of HsRV RNA1_ORF4s was built using MEGA6 (ref. 36). The alignment was then used as input in HHblits 3.3.0, which compared the alignments to the PDB70 (pdb70_from_mmcif_220313) database. Transmembrane domains were predicted using TMHMM³⁷.

## Search for HsRV homologues in public databases

To identify viruses related to HsRV in the IMG/VR database³⁸, BLASTP searches (*E*-value ≤ 1 × 10⁻⁵⁰) queried with the RdRP sequences encoded by HsRV-like contigs previously deposited to GenBank (accessions: BDQA01000957 and BDQA01004869) were performed on the IMG/VR website (https://img.jgi.doe.gov/cgi-bin/vr/main.cgi?section=Viral&page findViralGenesBlast). The nucleotide sequences of the contigs encoding the related RdRPs were downloaded and annotated as described above for the HsRVs.

## Modelling protein structures with AlphaFold2 and structural comparisons

Structural predictions for HsRV and HsRV-like RdRP amino acid sequences were performed using ColabFold 1.5.1 installed locally through LocalColabFold (https://github.com/YoshitakaMo/local-colabfold). A custom MSA with ten HsRV (HsRV_La~d, H5_contig_1, Oi_contig_1, Oi_contig_3, Oi_contig_5, Oi_contig_8, Oi_contig_9) and five HsRV-like (BDQA01000957, BDQA01004869, Ga0456180, Ga0393213, Ga0169446) RdRP amino acid sequences was used as input. The number of recycles used for HsRV_La ORF4 and HsRV-like RdRP predictions were 6 and 10, respectively. For the core (motifs A–C) region of marine HsRV-like RdRP BDQA01004869 (Fig. 3d), 20 recycles were used. For Fig. 4a, Ambiviricota RdRP model (pLDDT 95, predicted template modeling (pTM) score 0.938) was generated with 3 recycles using a custom MSA of 422 Ambivirus RdRP sequences available at https://github.com/ababaian/serratus/wiki/ambivirusExtended_data (ref. 24). Paraxenoviricota (TARA_132_DCM_0.22-3_k119_33585_1_799) RdRP model (pLDDT 88.6, pTM 0.882) was generated with 20 recycles using a custom MSA of 12 amino acid sequences obtained by running BLASTP against ORFs from 44779_RdRP_contigs available at

https://datacommon.s.cyverse.org/browse/iplant/home/shared/iVirus/ZayedWainainaDominguez-Huerta RNAevolution_Dec2021/Contigs (ref. 7). Similarly, Wamoviricota (84SUR2MMQQ14_2_ERRI712161_contig_61452_3_468) RdRP model (pLDDT 84.5, pTM 0.822) was modelled with 20 recycles using a custom MSA of 6 sequences from the 44779_RdRP_contigs⁷ and 56 additional sequences obtained from a BLASTP search against the IMG/VR database. p.0002 (ND_055403_2847-982) RdRP model (pLDDT 84.3, pTM 0.864) was generated with 12 recycles using a custom MSA with 107 p.0002 RdRP sequences kindly provided by Dr Yuri I. Wolf⁸. The RdRPs of HsPV-H5 and PCV1 (GenBank ID: YP_009466859) were modelled using AlphaFold 2 through Colb-Fold (v.1.5.2)³⁹,⁴⁰ with 6 recycles each. For the HsPV-H4 CP modelling, an alignment of RNA2 ORF1 homologues from HsPV-like viruses and Driatsky virus was used as a template with 12 recycles. The obtained model had a medium quality (average pLDDT = 57.3), although the central region was modelled with higher quality (average pLDDT > 70). This model was used as a query in DALI search, which identified the CP of PCV1 (PDB ID: 7ncr) as the best hit with a Z-score of 6.5. Thus, to improve the quality of the HsPV-H4 CP model, we repeated the modelling using the same sequence alignment and providing the PDB structure of the PCV1 CP as a template, with 24 recycles. The obtained model had an average pLDDT score of 78.1. Model display, structural alignment, colouring and figure preparation were performed using UCSF ChimeraX software⁴¹.

## Phylogenetic analysis

Amino acid sequences of RdRP encoded by identified viruses and viruses related to the family Partitiviridae were aligned using MAFFT (G-INS-1)⁴². The ambiguous positions in the alignment were removed using TrimAl (gap threshold 0.2)⁴³. The maximum-likelihood tree was constructed using IQ-TREE (v.2.0.6)⁴⁴. The best-fitting substitution model was selected using ModelFinder⁴⁵ and was LG + F + R8. Node supports were estimated using the SH-like approximate likelihood-ratio test (SH-aLRT) with 1,000 replicates. For phylogenetic analysis of the HsRV-like RdRPs, the proteins were aligned using PROMALS3D⁴⁶ and uninformative positions we removed using TrimAl with the gap-pyout functions⁴³. The final alignment contained 520 positions. The maximum-likelihood tree was constructed using IQ-TREE (v.2.0.6)⁴⁴. The best-fitting substitution model was selected using ModelFinder⁴⁵ and was LG + I + G4. Node supports were estimated using SH-aLRT (1,000 replicates).

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

Datasets obtained in this study have been made available in the Gen-Bank database repository (accession nos. HsRV: BTCN01000001-BTCN01000010; HsPV-H4: BTC001000001-BTC001000006; HsPV-H5: BTCP01000001-BTCP01000005; HsPV-Y66: BTCQ01000001-BTCQ01000004; H5_contig_1: BTCR01000001; Oi_contig_1-9: BTCS01000001-BTCS01000009) and Short Read Archive database (accession no. DRA016131). Datasets (PDB70 mm cif_2023-10-24, Pfam v.35, UniProt-SwissProt-viral70_Nov_2021 and NCBI-CD v.3.19) are available at http://ftp.tuebingen.mpg.de/pub/protevo/toolkit/databases/hhsuite dbs/. Searches using the IMG/VR dataset were available only at https://img.jgi.doe.gov/cgi-bin/vr/main.cgi?section=WorkspaceBlast&page=viralform. Datasets (SILVA SSU v.138, Neo-HMM v.1.1 and RVDB-HMM v.23.0) are publicly available.

## Code availability

A custom code used in this study has been made available in a git repository publicly available on GitHub at https://github.com/takakiy/FLDS ( Cleanup FLDS).
Illumina MiSeq v3 Reagent kit (600 cycles) with 300-bp paired-end reads on the Illumina MiSeq platform.

## Data processing

Trimmed reads were obtained using a custom Perl pipeline script (https://github.com/takakiy/FLDS) from dsRNA raw sequence reads¹⁷. The clean reads were subjected to de novo assembly using CLC GENOMICS WORKBENCH v.11.0 (Qiagen) with the following parameters: a minimum contig length of 500, word value set to auto and bubble size set to auto. The full-length sequences were manually extracted using CLC GENOMICS WORKBENCH v.11.0 (Qiagen), Genetyx v.14 (Genetyx) and Tablet viewer v.1.19.09.03 (ref. 32) as described previously³³. In brief, contigs for which both termini were determined to be the ends were identified as full-length sequences. In cases of dominant reads (more than 10 reads) that stopped in the same position around the ends of contigs in the mapping analysis, that position was recognized as the segment (genome) end. In this study, major full-length sequences with >1,000 average coverage were analysed, except for the Oi sample where all full-length sequences were recovered. From ssRNA raw sequence reads, trimmed reads were also obtained using a custom Perl pipeline script (https://github.com/takakiy/FLDS). The resultant clean reads were applied to phyloFlash²⁰ to identify active microbes in our samples.

## Sequence analyses

RNA viral genes were identified using the BLASTX programme against the NCBI non-redundant (nr) database with an *E*-value ≤ 1 × 10⁻⁵⁰. The ribosome-binding SD motifs were identified using Prodigal³⁴. Remote homology searches were performed using HHpred against the PDB70, Pfam, UniProt-SwissProt-viral70 and NCBI-CD (conserved domains) databases³⁵. MSA of HsRV RNA1_ORF4s was built using MEGA6 (ref. 36). The alignment was then used as input in HHblits 3.3.0, which compared the alignments to the PDB70 (pdb70_from_mmcif_220313) database. Transmembrane domains were predicted using TMHMM³⁷.

## Search for HsRV homologues in public databases

To identify viruses related to HsRV in the IMG/VR database³⁸, BLASTP searches (*E*-value ≤ 1 × 10⁻⁵⁰) queried with the RdRP sequences encoded by HsRV-like contigs previously deposited to GenBank (accessions: BDQA01000957 and BDQA01004869) were performed on the IMG/VR website (https://img.jgi.doe.gov/cgi-bin/vr/main.cgi?section=Viral&page findViralGenesBlast). The nucleotide sequences of the contigs encoding the related RdRPs were downloaded and annotated as described above for the HsRVs.

## Modelling protein structures with AlphaFold2 and structural comparisons

Structural predictions for HsRV and HsRV-like RdRP amino acid sequences were performed using ColabFold 1.5.1 installed locally through LocalColabFold (https://github.com/YoshitakaMo/local-colabfold). A custom MSA with ten HsRV (HsRV_La~d, H5_contig_1, Oi_contig_1, Oi_contig_3, Oi_contig_5, Oi_contig_8, Oi_contig_9) and five HsRV-like (BDQA01000957, BDQA01004869, Ga0456180, Ga0393213, Ga0169446) RdRP amino acid sequences was used as input. The number of recycles used for HsRV_La ORF4 and HsRV-like RdRP predictions were 6 and 10, respectively. For the core (motifs A–C) region of marine HsRV-like RdRP BDQA01004869 (Fig. 3d), 20 recycles were used. For Fig. 4a, Ambiviricota RdRP model (pLDDT 95, predicted template modeling (pTM) score 0.938) was generated with 3 recycles using a custom MSA of 422 Ambivirus RdRP sequences available at https://github.com/ababaian/serratus/wiki/ambivirusExtended_data (ref. 24). Paraxenoviricota (TARA_132_DCM_0.22-3_k119_33585_1_799) RdRP model (pLDDT 88.6, pTM 0.882) was generated with 20 recycles using a custom MSA of 12 amino acid sequences obtained by running BLASTP against ORFs from 44779_RdRP_contigs available at

https://datacommon.s.cyverse.org/browse/iplant/home/shared/iVirus/ZayedWainainaDominguez-Huerta RNAevolution_Dec2021/Contigs (ref. 7). Similarly, Wamoviricota (84SUR2MMQQ14_2_ERRI712161_contig_61452_3_468) RdRP model (pLDDT 84.5, pTM 0.822) was modelled with 20 recycles using a custom MSA of 6 sequences from the 44779_RdRP_contigs⁷ and 56 additional sequences obtained from a BLASTP search against the IMG/VR database. p.0002 (ND_055403_2847-982) RdRP model (pLDDT 84.3, pTM 0.864) was generated with 12 recycles using a custom MSA with 107 p.0002 RdRP sequences kindly provided by Dr Yuri I. Wolf⁸. The RdRPs of HsPV-H5 and PCV1 (GenBank ID: YP_009466859) were modelled using AlphaFold 2 through Colb-Fold (v.1.5.2)³⁹,⁴⁰ with 6 recycles each. For the HsPV-H4 CP modelling, an alignment of RNA2 ORF1 homologues from HsPV-like viruses and Driatsky virus was used as a template with 12 recycles. The obtained model had a medium quality (average pLDDT = 57.3), although the central region was modelled with higher quality (average pLDDT > 70). This model was used as a query in DALI search, which identified the CP of PCV1 (PDB ID: 7ncr) as the best hit with a Z-score of 6.5. Thus, to improve the quality of the HsPV-H4 CP model, we repeated the modelling using the same sequence alignment and providing the PDB structure of the PCV1 CP as a template, with 24 recycles. The obtained model had an average pLDDT score of 78.1. Model display, structural alignment, colouring and figure preparation were performed using UCSF ChimeraX software⁴¹.

## Phylogenetic analysis

Amino acid sequences of RdRP encoded by identified viruses and viruses related to the family Partitiviridae were aligned using MAFFT (G-INS-1)⁴². The ambiguous positions in the alignment were removed using TrimAl (gap threshold 0.2)⁴³. The maximum-likelihood tree was constructed using IQ-TREE (v.2.0.6)⁴⁴. The best-fitting substitution model was selected using ModelFinder⁴⁵ and was LG + F + R8. Node supports were estimated using the SH-like approximate likelihood-ratio test (SH-aLRT) with 1,000 replicates. For phylogenetic analysis of the HsRV-like RdRPs, the proteins were aligned using PROMALS3D⁴⁶ and uninformative positions we removed using TrimAl with the gap-pyout functions⁴³. The final alignment contained 520 positions. The maximum-likelihood tree was constructed using IQ-TREE (v.2.0.6)⁴⁴. The best-fitting substitution model was selected using ModelFinder⁴⁵ and was LG + I + G4. Node supports were estimated using SH-aLRT (1,000 replicates).

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

Datasets obtained in this study have been made available in the Gen-Bank database repository (accession nos. HsRV: BTCN01000001-BTCN01000010; HsPV-H4: BTC001000001-BTC001000006; HsPV-H5: BTCP01000001-BTCP01000005; HsPV-Y66: BTCQ01000001-BTCQ01000004; H5_contig_1: BTCR01000001; Oi_contig_1-9: BTCS01000001-BTCS01000009) and Short Read Archive database (accession no. DRA016131). Datasets (PDB70 mm cif_2023-10-24, Pfam v.35, UniProt-SwissProt-viral70_Nov_2021 and NCBI-CD v.3.19) are available at http://ftp.tuebingen.mpg.de/pub/protevo/toolkit/databases/hhsuite dbs/. Searches using the IMG/VR dataset were available only at https://img.jgi.doe.gov/cgi-bin/vr/main.cgi?section=WorkspaceBlast&page=viralform. Datasets (SILVA SSU v.138, Neo-HMM v.1.1 and RVDB-HMM v.23.0) are publicly available.

## Code availability

A custom code used in this study has been made available in a git repository publicly available on GitHub at https://github.com/takakiy/FLDS ( Cleanup FLDS).
## References

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4. Wolf, Y. I. et al. Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome. *Nat. Microbiol.* **5**, 1262–1270 (2020).

5. Shi, M., Zhang, Y. Z. & Holmes, E. C. Meta-transcriptomics and the evolutionary biology of RNA viruses. *Virus Res.* **243**, 83–90 (2018).

6. Shi, M. et al. The evolutionary history of vertebrate RNA viruses. *Nature* **556**, 197–202 (2018).

7. Zayed, A. A. et al. Cryptic and abundant marine viruses at the evolutionary origins of Earth's RNA virome. *Science* **376**, 156–162 (2022).

8. Neri, U. et al. Expansion of the global RNA virome reveals diverse clades of bacteriophages. *Cell* **185**, 4023–4037 (2022).

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10. Koonin, E. V. et al. Global organization and proposed megataxisomy of the virus world. *Microbiol. Mol. Biol. Rev.* **84**, e00061-19 (2020).

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19. Urayama, S. et al. Unveiling the RNA virosphere associated with marine microorganisms. *Mol. Ecol. Resour.* **18**, 1444–1455 (2018).

20. Gruber-Vodicka, H. R., Seah, B. K. & Pruesse, E. phyloFlash: rapid small-subunit rRNA profiling and targeted assembly from metagenomes. *mSystems* **5**, e00920 (2020).

21. Yang, Y. et al. Characterization of the first double-stranded RNA bacteriophage infecting *Pseudomonas aeruginosa*. *Sci. Rep.* **6**, 38795 (2016).

22. Venkataraman, S., Prasad, B. & Selvarajan, R. RNA dependent RNA polymerases: insights from structure, function and evolution. *Viruses* **10**, 76 (2018).

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25. Krupovic, M. et al. Ortervirales: new virus order unifying five families of reverse-transcribing viruses. *J. Virol.* **92**, e00515-18 (2018).

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27. Byrne, M., Kashyap, A., Esquirol, L., Ranson, N. & Sainsbury, F. The structure of a plant-specific partitivirus capsid reveals a unique coat protein domain architecture with an intrinsically disordered protrusion. *Commun. Biol.* **4**, 1155 (2021).

28. Duquerroy, S. et al. The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry. *EMBO J.* **28**, 1655–1665 (2009).

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30. Peyambari, M., Guan, S. & Roossinck, M. J. RdRp or RT, that is the question. *Mol. Biol. Evol.* **38**, 5082–5091 (2021).

31. Okada, R., Kiyota, E., Moriyama, H., Fukuhara, T. & Natsuaki, T. A simple and rapid method to purify viral dsRNA from plant and fungal tissue. *J. Gen. Plant Pathol.* **81**, 103–107 (2015).

32. Milne, I. et al. Tablet—next generation sequence assembly visualization. *Bioinformatics* **26**, 401–402 (2010).

33. Urayama, S., Takaki, Y., Hagiwara, D. & Nunoura, T. dsRNA-seq reveals novel RNA virus and virus-like putative complete genome sequences from *Hymeniacidon* sp. sponge. *Microbes Environ.* **35**, ME19132 (2020).

34. Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. *BMC Bioinformatics* **11**, 119 (2010).

35. Gabler, F. et al. Protein sequence analysis using the MPI bioinformatics toolkit. *Curr. Protoc. Bioinformatics* **72**, e108 (2020).

36. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. *Mol. Biol. Evol.* **30**, 2725–2729 (2013).

37. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. *J. Mol. Biol.* **305**, 567–580 (2001).

38. Camargo, A. P. et al. IMG/VR v4: an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata. *Nucleic Acids Res.* **51**, D733–D743 (2023).

39. Mirdita, M. et al. ColabFold: making protein folding accessible to all. *Nat. Methods* **19**, 679–682 (2022).

40. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. *Nature* **596**, 583–589 (2021).

41. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. *Protein Sci.* **30**, 70–82 (2021).

42. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Mol. Biol. Evol.* **30**, 772–780 (2013).

43. Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. *Bioinformatics* **25**, 1972–1973 (2009).

44. Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. *Mol. Biol. Evol.* **37**, 1530–1534 (2020).

45. Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. *Nat. Methods* **14**, 587–589 (2017).

46. Pei, J. & Grishin, N. V. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. *Methods Mol. Biol.* **1079**, 263–271 (2014).

## Acknowledgements

We thank NITTETSU MINING CO., LTD KAGOSHIMA GEOTHERMAL FACILITY, NIPPON PAPER LUMBER CO., LTD and Kirishima Iwasaki
## References

1. Simmonds, P. et al. Consensus statement: virus taxonomy in the age of metagenomics. *Nat. Rev. Microbiol.* **15**, 161–168 (2017).

2. Shi, M. et al. Redefining the invertebrate RNA virosphere. *Nature* **540**, 539–543 (2016).

3. Wolf, Y. I. et al. Origins and evolution of the global RNA virome. *mBio* **9**, e02329-18 (2018).

4. Wolf, Y. I. et al. Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome. *Nat. Microbiol.* **5**, 1262–1270 (2020).

5. Shi, M., Zhang, Y. Z. & Holmes, E. C. Meta-transcriptomics and the evolutionary biology of RNA viruses. *Virus Res.* **243**, 83–90 (2018).

6. Shi, M. et al. The evolutionary history of vertebrate RNA viruses. *Nature* **556**, 197–202 (2018).

7. Zayed, A. A. et al. Cryptic and abundant marine viruses at the evolutionary origins of Earth's RNA virome. *Science* **376**, 156–162 (2022).

8. Neri, U. et al. Expansion of the global RNA virome reveals diverse clades of bacteriophages. *Cell* **185**, 4023–4037 (2022).

9. Edgar, R. C. et al. Petabase-scale sequence alignment catalyses viral discovery. *Nature* **602**, 142–147 (2022).

10. Koonin, E. V. et al. Global organization and proposed megataxisomy of the virus world. *Microbiol. Mol. Biol. Rev.* **84**, e00061-19 (2020).

11. Koonin, E. V., Dolja, V. V. & Krupovic, M. Origins and evolution of viruses of eukaryotes: the ultimate modularity. *Virology* **479-480**, 2–25 (2015).

12. Nasir, A., Forterre, P., Kim, K. M. & Caetano-Anolles, G. The distribution and impact of viral lineages in domains of life. *Front. Microbiol.* **5**, 194 (2014).

13. Callanan, J. et al. Leviviricetes: expanding and restructuring the taxonomy of bacteria-infecting single-stranded RNA viruses. *Microb. Genomics* **7**, 000686 (2021).

14. Callanan, J. et al. Expansion of known ssRNA phage genomes: from tens to over a thousand. *Sci. Adv.* **6**, eaay5981 (2020).

15. Krishnamurthy, S. R., Janowski, A. B., Zhao, G., Barouch, D. & Wang, D. Hyperexpansion of RNA bacteriophage diversity. *PLoS Biol.* **14**, e1002409 (2016).

16. Morris, T. J. & Dodds, J. A. Isolation and analysis of double-stranded-RNA from virus-infected plant and fungal tissue. *Phytopathology* **69**, 854–858 (1979).

17. Hirai, M. et al. RNA viral metagenome analysis of subnanogram dsRNA using fragmented and primer ligated dsRNA sequencing (FLDS). *Microbes Environ.* **36**, ME20152 (2021).

18. Urayama, S., Takaki, Y. & Nunoura, T. FLDS: a comprehensive dsRNA sequencing method for intracellular RNA virus surveillance. *Microbes Environ.* **31**, 33–40 (2016).

19. Urayama, S. et al. Unveiling the RNA virosphere associated with marine microorganisms. *Mol. Ecol. Resour.* **18**, 1444–1455 (2018).

20. Gruber-Vodicka, H. R., Seah, B. K. & Pruesse, E. phyloFlash: rapid small-subunit rRNA profiling and targeted assembly from metagenomes. *mSystems* **5**, e00920 (2020).

21. Yang, Y. et al. Characterization of the first double-stranded RNA bacteriophage infecting *Pseudomonas aeruginosa*. *Sci. Rep.* **6**, 38795 (2016).

22. Venkataraman, S., Prasad, B. & Selvarajan, R. RNA dependent RNA polymerases: insights from structure, function and evolution. *Viruses* **10**, 76 (2018).

23. Te Velthuis, A. J. Common and unique features of viral RNA-dependent polymerases. *Cell. Mol. Life Sci.* **71**, 4403–4420 (2014).

24. Forgia, M. et al. Hybrids of RNA viruses and viroid-like elements replicate in fungi. *Nat. Commun.* **14**, 2591 (2023).

25. Krupovic, M. et al. Ortervirales: new virus order unifying five families of reverse-transcribing viruses. *J. Virol.* **92**, e00515-18 (2018).

26. Luque, D., Mata, C. P., Suzuki, N., Ghabrial, S. A. & Castón, J. R. Capsid structure of dsRNA fungal viruses. *Viruses* **10**, 481 (2018).

27. Byrne, M., Kashyap, A., Esquirol, L., Ranson, N. & Sainsbury, F. The structure of a plant-specific partitivirus capsid reveals a unique coat protein domain architecture with an intrinsically disordered protrusion. *Commun. Biol.* **4**, 1155 (2021).

28. Duquerroy, S. et al. The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry. *EMBO J.* **28**, 1655–1665 (2009).

29. Krishnamurthy, S. R. & Wang, D. Extensive conservation of prokaryotic ribosomal binding sites in known and novel picobirnaviruses. *Virology* **516**, 108–114 (2018).

30. Peyambari, M., Guan, S. & Roossinck, M. J. RdRp or RT, that is the question. *Mol. Biol. Evol.* **38**, 5082–5091 (2021).

31. Okada, R., Kiyota, E., Moriyama, H., Fukuhara, T. & Natsuaki, T. A simple and rapid method to purify viral dsRNA from plant and fungal tissue. *J. Gen. Plant Pathol.* **81**, 103–107 (2015).

32. Milne, I. et al. Tablet—next generation sequence assembly visualization. *Bioinformatics* **26**, 401–402 (2010).

33. Urayama, S., Takaki, Y., Hagiwara, D. & Nunoura, T. dsRNA-seq reveals novel RNA virus and virus-like putative complete genome sequences from *Hymeniacidon* sp. sponge. *Microbes Environ.* **35**, ME19132 (2020).

34. Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. *BMC Bioinformatics* **11**, 119 (2010).

35. Gabler, F. et al. Protein sequence analysis using the MPI bioinformatics toolkit. *Curr. Protoc. Bioinformatics* **72**, e108 (2020).

36. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. *Mol. Biol. Evol.* **30**, 2725–2729 (2013).

37. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. *J. Mol. Biol.* **305**, 567–580 (2001).

38. Camargo, A. P. et al. IMG/VR v4: an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata. *Nucleic Acids Res.* **51**, D733–D743 (2023).

39. Mirdita, M. et al. ColabFold: making protein folding accessible to all. *Nat. Methods* **19**, 679–682 (2022).

40. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. *Nature* **596**, 583–589 (2021).

41. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. *Protein Sci.* **30**, 70–82 (2021).

42. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Mol. Biol. Evol.* **30**, 772–780 (2013).

43. Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. *Bioinformatics* **25**, 1972–1973 (2009).

44. Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. *Mol. Biol. Evol.* **37**, 1530–1534 (2020).

45. Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. *Nat. Methods* **14**, 587–589 (2017).

46. Pei, J. & Grishin, N. V. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. *Methods Mol. Biol.* **1079**, 263–271 (2014).

## Acknowledgements

We thank NITTETSU MINING CO., LTD KAGOSHIMA GEOTHERMAL FACILITY, NIPPON PAPER LUMBER CO., LTD and Kirishima Iwasaki
Hotel for support for field sampling; S. Kawagucci, M. Yoshida, Y. Yoshida-Takashima, M. Tsuda and F. Kondo for discussions, suggestions, sample collections and preliminary experiments related to this study; and Y. I. Wolf for technical help. This study was supported by JSPS KAKENHI (Grant Nos. 15H05468 to T.O. and 20K20377 to T.N.) and by Grants-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (Grant Nos. 22H04879 and 20H05579 to S.U.; 19H05684, 16H06429, 16K21723 and 16H06437 to T.N). This research was also supported in part by Lilly Endowment, Inc., through its support for the Indiana University Pervasive Technology Institute which provided supercomputing resources for protein structure modelling, and by a grant from the Institute for Fermentation, Osaka, Japan. E.V.K. was supported by the Intramural Research Program of the US National Institutes of Health (National Library of Medicine).

## Author contributions

All authors had a substantial contribution to this work. S.U. and T.N. were responsible for the design of the work and the acquisition, analysis and interpretation of data, and drafted the initial work. S.U., A.F., E.V.K., M.K. and T.N. substantively revised the work. A.F., Y.N., Y.T. and M.K. performed bioinformatic analysis. M.H. and T.O. performed experiments, and analysed and interpreted the data. S.U., A.F., T.O., Y.N., N.K., E.V.K., M.K. and T.N. wrote the paper.

## Competing interests

JAMSTEC holds a patent for the 'Double-stranded RNA fragmentation method and use thereof', with S.U. and T.N. listed as inventors. These patents include European Patent (EP) Registration No. 3363898, registered on 30 November 2022; China Registration No. ZL201680060127.X, registered on 8 February 2022; US Registration No. 10894981, registered on 19 January 2021; and Japanese patent No. 6386678, registered on 17 August 2018. The other authors declare no competing interests.

## Additional information

**Extended data** is available for this paper at
https://doi.org/10.1038/s41564-023-01579-5.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41564-023-01579-5.

**Correspondence and requests for materials** should be addressed to Syun-ichi Urayama.

**Peer review information** *Nature Microbiology* thanks Vanessa Marcelino and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

**Reprints and permissions information** is available at
www.nature.com/reprints.

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024
Hotel for support for field sampling; S. Kawagucci, M. Yoshida, Y. Yoshida-Takashima, M. Tsuda and F. Kondo for discussions, suggestions, sample collections and preliminary experiments related to this study; and Y. I. Wolf for technical help. This study was supported by JSPS KAKENHI (Grant Nos. 15H05468 to T.O. and 20K20377 to T.N.) and by Grants-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (Grant Nos. 22H04879 and 20H05579 to S.U.; 19H05684, 16H06429, 16K21723 and 16H06437 to T.N). This research was also supported in part by Lilly Endowment, Inc., through its support for the Indiana University Pervasive Technology Institute which provided supercomputing resources for protein structure modelling, and by a grant from the Institute for Fermentation, Osaka, Japan. E.V.K. was supported by the Intramural Research Program of the US National Institutes of Health (National Library of Medicine).

## Author contributions

All authors had a substantial contribution to this work. S.U. and T.N. were responsible for the design of the work and the acquisition, analysis and interpretation of data, and drafted the initial work. S.U., A.F., E.V.K., M.K. and T.N. substantively revised the work. A.F., Y.N., Y.T. and M.K. performed bioinformatic analysis. M.H. and T.O. performed experiments, and analysed and interpreted the data. S.U., A.F., T.O., Y.N., N.K., E.V.K., M.K. and T.N. wrote the paper.

## Competing interests

JAMSTEC holds a patent for the 'Double-stranded RNA fragmentation method and use thereof', with S.U. and T.N. listed as inventors. These patents include European Patent (EP) Registration No. 3363898, registered on 30 November 2022; China Registration No. ZL201680060127.X, registered on 8 February 2022; US Registration No. 10894981, registered on 19 January 2021; and Japanese patent No. 6386678, registered on 17 August 2018. The other authors declare no competing interests.

## Additional information

**Extended data** is available for this paper at
https://doi.org/10.1038/s41564-023-01579-5.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41564-023-01579-5.

**Correspondence and requests for materials** should be addressed to Syun-ichi Urayama.

**Peer review information** *Nature Microbiology* thanks Vanessa Marcelino and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

**Reprints and permissions information** is available at
www.nature.com/reprints.

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024
a

<figure><img src="image_31.png" /></figure>

b

<figure><img src="image_32.png" /></figure>

Extended Data Fig. 2 | See next page for caption.
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /><figcaption>Extended Data Fig. 2 | See next page for caption.</figcaption></figure>
**Extended Data Fig. 2 | Distribution of minor contigs related to the RdRPs of HsRV and HsPV.** Distribution of minor contigs related to the RdRPs of a, HsRV and b, HsPV. Each bar represents the position of predicted amino acid sequences of contigs aligned to the HsRV or HsPV RdRP shown at the top of the panel, and their identities to the reference RdRP sequences are indicated by the colors in the heatmap. The name of source libraries are shown in the right-side panel.

Trimmed reads from each sample were assembled using CLC assembler, followed by the removal of sequences identical to HsRV or HsPV. Using the amino acid sequences of RdRPs from HsRV and HsPV as queries, tBLASTN searches were performed on the remaining contigs. Sequences with > 60% amino acid identity and > 100 aa hit were shown.
**Extended Data Fig. 2** | Distribution of minor contigs related to the RdRPs of HsRV and HsPV. Distribution of minor contigs related to the RdRPs of a, HsRV and b, HsPV. Each bar represents the position of predicted amino acid sequences of contigs aligned to the HsRV or HsPV RdRP shown at the top of the panel, and their identities to the reference RdRP sequences are indicated by the colors in the heatmap. The name of source libraries are shown in the right-side panel.

Trimmed reads from each sample were assembled using CLC assembler, followed by the removal of sequences identical to HsRV or HsPV. Using the amino acid sequences of RdRPs from HsRV and HsPV as queries, tBLASTN searches were performed on the remaining contigs. Sequences with > 60% amino acid identity and > 100 aa hit were shown.
a

<figure><img src="image_33.png" /></figure>

HsPV-H5 RdRP (AF2)

b

<figure><img src="image_34.png" /></figure>

HsPV-H4 CP (AF2)

<figure><img src="image_35.png" /></figure>

**Extended Data Fig. 3** | pLDDT scores of HsPV RdRP and CP. Quality assessment of the AF2 model of the HsPV a, RdRP and b, CP. The structural model is colored based on the pLDDT scores, with the color key shown at the bottom right corner.
a

<figure><img src="image_33.png" /></figure>

HsPV-H5 RdRP (AF2)

b

<figure><img src="image_34.png" /></figure>

HsPV-H4 CP (AF2)

<figure><img src="image_35.png" /></figure>

**Extended Data Fig. 3** | pLDDT scores of HsPV RdRP and CP. Quality assessment of the AF2 model of the HsPV a, RdRP and b, CP. The structural model is colored based on the pLDDT scores, with the color key shown at the bottom right corner.
<figure><img src="image_36.png" /><figcaption>b RNA2 ORF2-1</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>c RNA2 ORF2-2</figcaption></figure>

<figure><img src="image_38.png" /></figure>

**Extended Data Fig. 4** | HsPV phylogeny. Maximum-likelihood trees of each ORF encoded by HsPVs and related sequences. Sequences were aligned using MEGA6. The ambiguous positions in the alignment were removed using TrimAl. The maximum likelihood tree was constructed using RAxML. The best-fitting

substitution model was selected by ProtTest. Numbers indicate the percentage bootstrap support from 1,000 RAxML bootstrap replicates. We used RAxML with the *a*, LG+G+I+F model for ORF1, *b*, LG+G model for ORF2-1 and *c*, LG+G+I+F model for ORF2-2.
<figure><img src="image_36.png" /><figcaption>b RNA2 ORF2-1</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>c RNA2 ORF2-2</figcaption></figure>

<figure><img src="image_38.png" /></figure>

**Extended Data Fig. 4** | HsPV phylogeny. Maximum-likelihood trees of each ORF encoded by HsPVs and related sequences. Sequences were aligned using MEGA6. The ambiguous positions in the alignment were removed using TrimAl. The maximum likelihood tree was constructed using RAxML. The best-fitting

substitution model was selected by ProtTest. Numbers indicate the percentage bootstrap support from 1,000 RAxML bootstrap replicates. We used RAxML with the *a*, LG+G+I+F model for ORF1, *b*, LG+G model for ORF2-1 and *c*, LG+G+I+F model for ORF2-2.
<table><caption>Extended Data Table 1 | Relative abundances of rRNA reads in ssRNA seq of representative microbial lineages</caption><thead><tr><th></th><th>H4</th><th>H5</th><th>T1</th><th>T2</th><th>T3</th><th>T4</th><th>Y66</th><th>Y80</th><th>Y86</th><th>Oi</th><th>Ob</th></tr></thead><tbody><tr><td>Eukaryota</td><td>0.59</td><td>0.19</td><td>1.64</td><td>0.99</td><td>7.31</td><td>1.51</td><td>0.11</td><td>1.53</td><td>5.16</td><td>0.75</td><td>4.02</td></tr><tr><td>Sulfolobaceae</td><td>1.00</td><td>0.23</td><td>54.50</td><td>72.32</td><td>31.19</td><td>66.31</td><td>7.89</td><td>5.28</td><td>59.04</td><td>10.94</td><td>0.00</td></tr><tr><td>other Archaea</td><td>1.01</td><td>0.19</td><td>43.23</td><td>26.29</td><td>17.83</td><td>30.36</td><td>7.06</td><td>15.11</td><td>20.99</td><td>4.83</td><td>0.14</td></tr><tr><td>Hydrogenobaculaceae</td><td>96.57</td><td>98.90</td><td>0.13</td><td>0.12</td><td>0.27</td><td>1.61</td><td>84.24</td><td>77.54</td><td>14.47</td><td>49.84</td><td>0.01</td></tr><tr><td>Aquificaceae</td><td>0.00</td><td>0.01</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.01</td><td>0.00</td><td>0.00</td><td>0.09</td><td>82.54</td></tr><tr><td>Comamonadaceae</td><td>0.05</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.38</td><td>0.00</td><td>0.01</td><td>0.00</td><td>0.00</td><td>25.21</td><td>0.00</td></tr><tr><td>other Bacteria</td><td>0.78</td><td>0.47</td><td>0.50</td><td>0.29</td><td>43.03</td><td>0.32</td><td>0.69</td><td>0.56</td><td>0.34</td><td>8.32</td><td>13.29</td></tr></tbody></table>
<table><caption>Extended Data Table 1 | Relative abundances of rRNA reads in ssRNA seq of representative microbial lineages</caption><thead><tr><th></th><th>H4</th><th>H5</th><th>T1</th><th>T2</th><th>T3</th><th>T4</th><th>Y66</th><th>Y80</th><th>Y86</th><th>Oi</th><th>Ob</th></tr></thead><tbody><tr><td>Eukaryota</td><td>0.59</td><td>0.19</td><td>1.64</td><td>0.99</td><td>7.31</td><td>1.51</td><td>0.11</td><td>1.53</td><td>5.16</td><td>0.75</td><td>4.02</td></tr><tr><td>Sulfolobaceae</td><td>1.00</td><td>0.23</td><td>54.50</td><td>72.32</td><td>31.19</td><td>66.31</td><td>7.89</td><td>5.28</td><td>59.04</td><td>10.94</td><td>0.00</td></tr><tr><td>other Archaea</td><td>1.01</td><td>0.19</td><td>43.23</td><td>26.29</td><td>17.83</td><td>30.36</td><td>7.06</td><td>15.11</td><td>20.99</td><td>4.83</td><td>0.14</td></tr><tr><td>Hydrogenobaculaceae</td><td>96.57</td><td>98.90</td><td>0.13</td><td>0.12</td><td>0.27</td><td>1.61</td><td>84.24</td><td>77.54</td><td>14.47</td><td>49.84</td><td>0.01</td></tr><tr><td>Aquificaceae</td><td>0.00</td><td>0.01</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.01</td><td>0.00</td><td>0.00</td><td>0.09</td><td>82.54</td></tr><tr><td>Comamonadaceae</td><td>0.05</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.38</td><td>0.00</td><td>0.01</td><td>0.00</td><td>0.00</td><td>25.21</td><td>0.00</td></tr><tr><td>other Bacteria</td><td>0.78</td><td>0.47</td><td>0.50</td><td>0.29</td><td>43.03</td><td>0.32</td><td>0.69</td><td>0.56</td><td>0.34</td><td>8.32</td><td>13.29</td></tr></tbody></table>
<table><caption>Extended Data Table 2 | Classification of NGS reads</caption><thead><tr><th>Sample</th><th>Library type</th><th>Raw reads (pair)</th><th>% of removed</th><th>% of rRNA</th><th>% of RNA viral (candidate)</th><th>% of other reads</th></tr></thead><tbody><tr><td>H4</td><td>dsRNA</td><td>930,952</td><td>31.8</td><td>9.8</td><td>47.7</td><td>10.6</td></tr><tr><td>H4</td><td>ssRNA</td><td>68,766</td><td>9.9</td><td>81.7</td><td>0.1</td><td>8.3</td></tr><tr><td>H5</td><td>dsRNA</td><td>1,009,144</td><td>10.3</td><td>3.6</td><td>81.8</td><td>4.3</td></tr><tr><td>H5</td><td>ssRNA</td><td>57,932</td><td>8.4</td><td>87.3</td><td>0.1</td><td>4.2</td></tr><tr><td>T1</td><td>dsRNA</td><td>1,276,072</td><td>42.1</td><td>34.2</td><td>0.0</td><td>23.7</td></tr><tr><td>T1</td><td>ssRNA</td><td>77,818</td><td>12.1</td><td>12.1</td><td>0.0</td><td>75.8</td></tr><tr><td>T2</td><td>dsRNA</td><td>1,069,831</td><td>19.2</td><td>80.0</td><td>0.0</td><td>0.8</td></tr><tr><td>T2</td><td>ssRNA</td><td>26,373</td><td>12.0</td><td>30.4</td><td>0.0</td><td>57.6</td></tr><tr><td>T3</td><td>dsRNA</td><td>1,203,878</td><td>17.0</td><td>81.2</td><td>0.0</td><td>1.8</td></tr><tr><td>T3</td><td>ssRNA</td><td>56,252</td><td>14.5</td><td>35.3</td><td>0.0</td><td>50.1</td></tr><tr><td>T4</td><td>dsRNA</td><td>1,433,410</td><td>62.5</td><td>19.1</td><td>0.0</td><td>18.4</td></tr><tr><td>T4</td><td>ssRNA</td><td>71,737</td><td>18.6</td><td>7.7</td><td>0.0</td><td>73.7</td></tr><tr><td>Y66</td><td>dsRNA</td><td>1,168,048</td><td>28.9</td><td>38.0</td><td>4.0</td><td>29.1</td></tr><tr><td>Y66</td><td>ssRNA</td><td>100,556</td><td>10.5</td><td>81.2</td><td>0.0</td><td>8.2</td></tr><tr><td>Y80</td><td>dsRNA</td><td>1,059,410</td><td>20.3</td><td>61.5</td><td>0.0</td><td>18.2</td></tr><tr><td>Y80</td><td>ssRNA</td><td>100,547</td><td>8.5</td><td>60.7</td><td>0.0</td><td>30.8</td></tr><tr><td>Y86</td><td>dsRNA</td><td>1,043,072</td><td>20.6</td><td>56.0</td><td>0.0</td><td>23.4</td></tr><tr><td>Y86</td><td>ssRNA</td><td>44,189</td><td>15.2</td><td>19.4</td><td>0.0</td><td>65.4</td></tr><tr><td>Ob</td><td>dsRNA</td><td>286,638</td><td>99.2</td><td>0.1</td><td>0.0</td><td>0.8</td></tr><tr><td>Ob</td><td>ssRNA</td><td>142,279</td><td>45.4</td><td>23.2</td><td>0.0</td><td>31.4</td></tr><tr><td>Oi</td><td>dsRNA</td><td>236,876</td><td>28.2</td><td>4.9</td><td>32.9</td><td>34.1</td></tr><tr><td>Oi</td><td>ssRNA</td><td>89,726</td><td>39.9</td><td>38.4</td><td>0.2</td><td>21.5</td></tr></tbody></table>
<table><caption>Extended Data Table 2 | Classification of NGS reads</caption><thead><tr><th>Sample</th><th>Library type</th><th>Raw reads (pair)</th><th>% of removed</th><th>% of rRNA</th><th>% of RNA viral (candidate)</th><th>% of other reads</th></tr></thead><tbody><tr><td>H4</td><td>dsRNA</td><td>930,952</td><td>31.8</td><td>9.8</td><td>47.7</td><td>10.6</td></tr><tr><td>H4</td><td>ssRNA</td><td>68,766</td><td>9.9</td><td>81.7</td><td>0.1</td><td>8.3</td></tr><tr><td>H5</td><td>dsRNA</td><td>1,009,144</td><td>10.3</td><td>3.6</td><td>81.8</td><td>4.3</td></tr><tr><td>H5</td><td>ssRNA</td><td>57,932</td><td>8.4</td><td>87.3</td><td>0.1</td><td>4.2</td></tr><tr><td>T1</td><td>dsRNA</td><td>1,276,072</td><td>42.1</td><td>34.2</td><td>0.0</td><td>23.7</td></tr><tr><td>T1</td><td>ssRNA</td><td>77,818</td><td>12.1</td><td>12.1</td><td>0.0</td><td>75.8</td></tr><tr><td>T2</td><td>dsRNA</td><td>1,069,831</td><td>19.2</td><td>80.0</td><td>0.0</td><td>0.8</td></tr><tr><td>T2</td><td>ssRNA</td><td>26,373</td><td>12.0</td><td>30.4</td><td>0.0</td><td>57.6</td></tr><tr><td>T3</td><td>dsRNA</td><td>1,203,878</td><td>17.0</td><td>81.2</td><td>0.0</td><td>1.8</td></tr><tr><td>T3</td><td>ssRNA</td><td>56,252</td><td>14.5</td><td>35.3</td><td>0.0</td><td>50.1</td></tr><tr><td>T4</td><td>dsRNA</td><td>1,433,410</td><td>62.5</td><td>19.1</td><td>0.0</td><td>18.4</td></tr><tr><td>T4</td><td>ssRNA</td><td>71,737</td><td>18.6</td><td>7.7</td><td>0.0</td><td>73.7</td></tr><tr><td>Y66</td><td>dsRNA</td><td>1,168,048</td><td>28.9</td><td>38.0</td><td>4.0</td><td>29.1</td></tr><tr><td>Y66</td><td>ssRNA</td><td>100,556</td><td>10.5</td><td>81.2</td><td>0.0</td><td>8.2</td></tr><tr><td>Y80</td><td>dsRNA</td><td>1,059,410</td><td>20.3</td><td>61.5</td><td>0.0</td><td>18.2</td></tr><tr><td>Y80</td><td>ssRNA</td><td>100,547</td><td>8.5</td><td>60.7</td><td>0.0</td><td>30.8</td></tr><tr><td>Y86</td><td>dsRNA</td><td>1,043,072</td><td>20.6</td><td>56.0</td><td>0.0</td><td>23.4</td></tr><tr><td>Y86</td><td>ssRNA</td><td>44,189</td><td>15.2</td><td>19.4</td><td>0.0</td><td>65.4</td></tr><tr><td>Ob</td><td>dsRNA</td><td>286,638</td><td>99.2</td><td>0.1</td><td>0.0</td><td>0.8</td></tr><tr><td>Ob</td><td>ssRNA</td><td>142,279</td><td>45.4</td><td>23.2</td><td>0.0</td><td>31.4</td></tr><tr><td>Oi</td><td>dsRNA</td><td>236,876</td><td>28.2</td><td>4.9</td><td>32.9</td><td>34.1</td></tr><tr><td>Oi</td><td>ssRNA</td><td>89,726</td><td>39.9</td><td>38.4</td><td>0.2</td><td>21.5</td></tr></tbody></table>
Extended Data Table 3 | Result of CDS clustering using a standard BLAST-mcl pipeline

<table><thead><tr><th>Cluster No. *1</th><th>Sequence No.</th><th>Virus</th><th>ORFs</th><th>Length</th><th>E-value</th><th>Identity</th></tr></thead><tbody><tr><td>Cluster 1</td><td>1</td><td>HsRV</td><td>RNA1a_ORF1</td><td>134</td><td></td><td></td></tr><tr><td rowspan="2">Cluster 2</td><td>2</td><td>HsRV</td><td>RNA1b_ORF1</td><td>110</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF1</td><td>104</td><td>1.00E-30</td><td>37</td></tr><tr><td>Cluster 3</td><td>1</td><td>HsRV</td><td>RNA1c_ORF1</td><td>246</td><td></td><td></td></tr><tr><td rowspan="4">Cluster 4</td><td>4</td><td>HsRV</td><td>RNA1a_ORF2</td><td>128</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1b_ORF2</td><td>130</td><td>3.00E-26</td><td>34</td></tr><tr><td></td><td>HsRV</td><td>RNA1c_ORF2</td><td>123</td><td>1.00E-14</td><td>26</td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF2</td><td>127</td><td>2.00E-31</td><td>42</td></tr><tr><td rowspan="3">Cluster 5</td><td>3</td><td>HsRV</td><td>RNA1a_ORF3</td><td>965</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1b_ORF3</td><td>933</td><td>0</td><td>33</td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF3</td><td>931</td><td>0</td><td>33</td></tr><tr><td>Cluster 6</td><td>1</td><td>HsRV</td><td>RNA1c_ORF3</td><td>1115</td><td></td><td></td></tr><tr><td>Cluster 7</td><td>1</td><td>HsRV</td><td>RNA1b_ORF5</td><td>77</td><td></td><td></td></tr><tr><td rowspan="4">Cluster 8</td><td>4</td><td>HsRV</td><td>RNA1a_ORF4</td><td>590</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1b_ORF4</td><td>581</td><td>3.00E-125</td><td>37</td></tr><tr><td></td><td>HsRV</td><td>RNA1c_ORF4</td><td>591</td><td>3.00E-128</td><td>37</td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF4</td><td>581</td><td>1.00E-134</td><td>39</td></tr><tr><td>Cluster 9</td><td>1</td><td>HsRV</td><td>RNA1c_ORF5</td><td>89</td><td></td><td></td></tr><tr><td rowspan="6">Cluster 10</td><td>6</td><td>HsRV</td><td>RNA2a_ORF1</td><td>162</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF1</td><td>195</td><td>2.00E-20</td><td>33</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF1</td><td>168</td><td>5.00E-76</td><td>70</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF1</td><td>167</td><td>2.00E-77</td><td>70</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF1</td><td>136</td><td>8.00E-10</td><td>36</td></tr><tr><td></td><td>HsRV</td><td>RNA2*b_ORF1</td><td>176</td><td>5.00E-06</td><td>29</td></tr><tr><td rowspan="6">Cluster 11</td><td>6</td><td>HsRV</td><td>RNA2a_ORF2</td><td>203</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF2</td><td>203</td><td>2.00E-83</td><td>62</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF2</td><td>205</td><td>6.00E-113</td><td>79</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF2</td><td>205</td><td>2.00E-123</td><td>80</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF2</td><td>257</td><td>5.00E-07</td><td>40</td></tr><tr><td></td><td>HsRV</td><td>RNA2*b_ORF2</td><td>252</td><td>4.00E-08</td><td>38</td></tr><tr><td rowspan="6">Cluster 12</td><td>6</td><td>HsRV</td><td>RNA2a_ORF3</td><td>397</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF3</td><td>391</td><td>4.00E-120</td><td>48</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF3</td><td>397</td><td>0</td><td>69</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF3</td><td>397</td><td>0</td><td>68</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF3</td><td>442</td><td>3.00E-07</td><td>23</td></tr><tr><td></td><td>HsRV</td><td>RNA2*b_ORF3</td><td>435</td><td>4.00E-09</td><td>25</td></tr><tr><td rowspan="4">Cluster 13</td><td>4</td><td>HsRV</td><td>RNA2a_ORF4</td><td>269</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF4</td><td>238</td><td>2.00E-09</td><td>32</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF4</td><td>269</td><td>2.00E-112</td><td>63</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF4</td><td>268</td><td>7.00E-112</td><td>62</td></tr><tr><td></td><td></td><td>HsRV</td><td>RNA2*b_ORF5</td><td>203</td><td></td><td></td></tr><tr><td rowspan="2">Cluster 14</td><td>3</td><td>HsRV</td><td>RNA2*a_ORF4</td><td>179</td><td>7.00E-24</td><td>34</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF5</td><td>380</td><td>1.00E-06</td><td>26</td></tr><tr><td>Cluster 15</td><td>1</td><td>HsRV</td><td>RNA2*b_ORF4</td><td>41</td><td></td><td></td></tr><tr><td rowspan="3">Cluster 16</td><td>3</td><td>HsRV</td><td>RNA2c_ORF5</td><td>231</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF5</td><td>225</td><td>1.00E-74</td><td>59</td></tr><tr><td></td><td>HsRV</td><td>RNA2a_ORF5</td><td>197</td><td>8.00E-12</td><td>31</td></tr><tr><td>Cluster 17</td><td>1</td><td>HsRV</td><td>RNA2b_ORF5</td><td>250</td><td></td><td></td></tr><tr><td>Cluster 18</td><td>1</td><td>HsRV</td><td>RNA2a_ORF6</td><td>43</td><td></td><td></td></tr><tr><td>Cluster 19</td><td>1</td><td>HsRV</td><td>RNA2*b_ORF6</td><td>51</td><td></td><td></td></tr><tr><td>Cluster 20</td><td>1</td><td>HsRV</td><td>RNA2*b_ORF6</td><td>167</td><td></td><td></td></tr><tr><td rowspan="6">Cluster 21</td><td>6</td><td>HsPV</td><td>H4_RNA1_a_ORF1</td><td>497</td><td></td><td></td></tr><tr><td></td><td>HsPV</td><td>H4_RNA1_b_ORF1</td><td>496</td><td>0</td><td>84</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA1_a_ORF1</td><td>497</td><td>0</td><td>99</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA1_b_ORF1</td><td>496</td><td>0</td><td>84</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA1_a_ORF1</td><td>386</td><td>0</td><td>76</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA1_b_ORF1</td><td>496</td><td>0</td><td>86</td></tr><tr><td rowspan="10">Cluster 22</td><td>9</td><td>HsPV</td><td>H4_RNA2_a_ORF1</td><td>252</td><td></td><td></td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_b_ORF1</td><td>251</td><td>2.00E-123</td><td>73</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_c_ORF1</td><td>254</td><td>9.00E-86</td><td>52</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_d_ORF1</td><td>251</td><td>7.00E-135</td><td>74</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_a_ORF1</td><td>252</td><td>6.00E-133</td><td>75</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_b_ORF1</td><td>252</td><td>0</td><td>99</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_c_ORF1</td><td>251</td><td>1.00E-125</td><td>75</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_a_ORF1</td><td>249</td><td>1.00E-82</td><td>52</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_b_ORF1</td><td>251</td><td>2.00E-136</td><td>73</td></tr><tr><td rowspan="10">Cluster 23</td><td>9</td><td>HsPV</td><td>H4_RNA2_a_ORF2</td><td>202</td><td></td><td></td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_b_ORF2</td><td>203</td><td>1.00E-32</td><td>38</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_c_ORF2</td><td>204</td><td>3.00E-18</td><td>24</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_d_ORF2</td><td>203</td><td>1.00E-50</td><td>45</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_a_ORF2</td><td>203</td><td>1.00E-32</td><td>38</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_b_ORF2</td><td>202</td><td>5.00E-146</td><td>98</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_c_ORF2</td><td>203</td><td>1.00E-53</td><td>46</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_a_ORF2</td><td>207</td><td>3.00E-15</td><td>24</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_b_ORF2</td><td>203</td><td>6.00E-33</td><td>36</td></tr></tbody></table>

<footnote>*The CDSs were clustered using a standard BLAST-mcl pipeline [BLASTP (v2.9.0) with default options, hits selected based on E-value < 1e-10, MCL clustering (v.14-137) with an inflation value of 2.8].</footnote>
Extended Data Table 3 | Result of CDS clustering using a standard BLAST-mcl pipeline

<table><thead><tr><th>Cluster No. *1</th><th>Sequence No.</th><th>Virus</th><th>ORFs</th><th>Length</th><th>E-value</th><th>Identity</th></tr></thead><tbody><tr><td>Cluster 1</td><td>1</td><td>HsRV</td><td>RNA1a_ORF1</td><td>134</td><td></td><td></td></tr><tr><td rowspan="2">Cluster 2</td><td>2</td><td>HsRV</td><td>RNA1b_ORF1</td><td>110</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF1</td><td>104</td><td>1.00E-30</td><td>37</td></tr><tr><td>Cluster 3</td><td>1</td><td>HsRV</td><td>RNA1c_ORF1</td><td>246</td><td></td><td></td></tr><tr><td rowspan="4">Cluster 4</td><td>4</td><td>HsRV</td><td>RNA1a_ORF2</td><td>128</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1b_ORF2</td><td>130</td><td>3.00E-26</td><td>34</td></tr><tr><td></td><td>HsRV</td><td>RNA1c_ORF2</td><td>123</td><td>1.00E-14</td><td>26</td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF2</td><td>127</td><td>2.00E-31</td><td>42</td></tr><tr><td rowspan="3">Cluster 5</td><td>3</td><td>HsRV</td><td>RNA1a_ORF3</td><td>965</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1b_ORF3</td><td>933</td><td>0</td><td>33</td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF3</td><td>931</td><td>0</td><td>33</td></tr><tr><td>Cluster 6</td><td>1</td><td>HsRV</td><td>RNA1c_ORF3</td><td>1115</td><td></td><td></td></tr><tr><td>Cluster 7</td><td>1</td><td>HsRV</td><td>RNA1b_ORF5</td><td>77</td><td></td><td></td></tr><tr><td rowspan="4">Cluster 8</td><td>4</td><td>HsRV</td><td>RNA1a_ORF4</td><td>590</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA1b_ORF4</td><td>581</td><td>3.00E-125</td><td>37</td></tr><tr><td></td><td>HsRV</td><td>RNA1c_ORF4</td><td>591</td><td>3.00E-128</td><td>37</td></tr><tr><td></td><td>HsRV</td><td>RNA1d_ORF4</td><td>581</td><td>1.00E-134</td><td>39</td></tr><tr><td>Cluster 9</td><td>1</td><td>HsRV</td><td>RNA1c_ORF5</td><td>89</td><td></td><td></td></tr><tr><td rowspan="6">Cluster 10</td><td>6</td><td>HsRV</td><td>RNA2a_ORF1</td><td>162</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF1</td><td>195</td><td>2.00E-20</td><td>33</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF1</td><td>168</td><td>5.00E-76</td><td>70</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF1</td><td>167</td><td>2.00E-77</td><td>70</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF1</td><td>136</td><td>8.00E-10</td><td>36</td></tr><tr><td></td><td>HsRV</td><td>RNA2*b_ORF1</td><td>176</td><td>5.00E-06</td><td>29</td></tr><tr><td rowspan="6">Cluster 11</td><td>6</td><td>HsRV</td><td>RNA2a_ORF2</td><td>203</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF2</td><td>203</td><td>2.00E-83</td><td>62</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF2</td><td>205</td><td>6.00E-113</td><td>79</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF2</td><td>205</td><td>2.00E-123</td><td>80</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF2</td><td>257</td><td>5.00E-07</td><td>40</td></tr><tr><td></td><td>HsRV</td><td>RNA2*b_ORF2</td><td>252</td><td>4.00E-08</td><td>38</td></tr><tr><td rowspan="6">Cluster 12</td><td>6</td><td>HsRV</td><td>RNA2a_ORF3</td><td>397</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF3</td><td>391</td><td>4.00E-120</td><td>48</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF3</td><td>397</td><td>0</td><td>69</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF3</td><td>397</td><td>0</td><td>68</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF3</td><td>442</td><td>3.00E-07</td><td>23</td></tr><tr><td></td><td>HsRV</td><td>RNA2*b_ORF3</td><td>435</td><td>4.00E-09</td><td>25</td></tr><tr><td rowspan="4">Cluster 13</td><td>4</td><td>HsRV</td><td>RNA2a_ORF4</td><td>269</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2b_ORF4</td><td>238</td><td>2.00E-09</td><td>32</td></tr><tr><td></td><td>HsRV</td><td>RNA2c_ORF4</td><td>269</td><td>2.00E-112</td><td>63</td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF4</td><td>268</td><td>7.00E-112</td><td>62</td></tr><tr><td></td><td></td><td>HsRV</td><td>RNA2*b_ORF5</td><td>203</td><td></td><td></td></tr><tr><td rowspan="2">Cluster 14</td><td>3</td><td>HsRV</td><td>RNA2*a_ORF4</td><td>179</td><td>7.00E-24</td><td>34</td></tr><tr><td></td><td>HsRV</td><td>RNA2*a_ORF5</td><td>380</td><td>1.00E-06</td><td>26</td></tr><tr><td>Cluster 15</td><td>1</td><td>HsRV</td><td>RNA2*b_ORF4</td><td>41</td><td></td><td></td></tr><tr><td rowspan="3">Cluster 16</td><td>3</td><td>HsRV</td><td>RNA2c_ORF5</td><td>231</td><td></td><td></td></tr><tr><td></td><td>HsRV</td><td>RNA2d_ORF5</td><td>225</td><td>1.00E-74</td><td>59</td></tr><tr><td></td><td>HsRV</td><td>RNA2a_ORF5</td><td>197</td><td>8.00E-12</td><td>31</td></tr><tr><td>Cluster 17</td><td>1</td><td>HsRV</td><td>RNA2b_ORF5</td><td>250</td><td></td><td></td></tr><tr><td>Cluster 18</td><td>1</td><td>HsRV</td><td>RNA2a_ORF6</td><td>43</td><td></td><td></td></tr><tr><td>Cluster 19</td><td>1</td><td>HsRV</td><td>RNA2*b_ORF6</td><td>51</td><td></td><td></td></tr><tr><td>Cluster 20</td><td>1</td><td>HsRV</td><td>RNA2*b_ORF6</td><td>167</td><td></td><td></td></tr><tr><td rowspan="6">Cluster 21</td><td>6</td><td>HsPV</td><td>H4_RNA1_a_ORF1</td><td>497</td><td></td><td></td></tr><tr><td></td><td>HsPV</td><td>H4_RNA1_b_ORF1</td><td>496</td><td>0</td><td>84</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA1_a_ORF1</td><td>497</td><td>0</td><td>99</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA1_b_ORF1</td><td>496</td><td>0</td><td>84</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA1_a_ORF1</td><td>386</td><td>0</td><td>76</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA1_b_ORF1</td><td>496</td><td>0</td><td>86</td></tr><tr><td rowspan="10">Cluster 22</td><td>9</td><td>HsPV</td><td>H4_RNA2_a_ORF1</td><td>252</td><td></td><td></td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_b_ORF1</td><td>251</td><td>2.00E-123</td><td>73</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_c_ORF1</td><td>254</td><td>9.00E-86</td><td>52</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_d_ORF1</td><td>251</td><td>7.00E-135</td><td>74</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_a_ORF1</td><td>252</td><td>6.00E-133</td><td>75</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_b_ORF1</td><td>252</td><td>0</td><td>99</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_c_ORF1</td><td>251</td><td>1.00E-125</td><td>75</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_a_ORF1</td><td>249</td><td>1.00E-82</td><td>52</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_b_ORF1</td><td>251</td><td>2.00E-136</td><td>73</td></tr><tr><td rowspan="10">Cluster 23</td><td>9</td><td>HsPV</td><td>H4_RNA2_a_ORF2</td><td>202</td><td></td><td></td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_b_ORF2</td><td>203</td><td>1.00E-32</td><td>38</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_c_ORF2</td><td>204</td><td>3.00E-18</td><td>24</td></tr><tr><td></td><td>HsPV</td><td>H4_RNA2_d_ORF2</td><td>203</td><td>1.00E-50</td><td>45</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_a_ORF2</td><td>203</td><td>1.00E-32</td><td>38</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_b_ORF2</td><td>202</td><td>5.00E-146</td><td>98</td></tr><tr><td></td><td>HsPV</td><td>H5_RNA2_c_ORF2</td><td>203</td><td>1.00E-53</td><td>46</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_a_ORF2</td><td>207</td><td>3.00E-15</td><td>24</td></tr><tr><td></td><td>HsPV</td><td>Y66_RNA2_b_ORF2</td><td>203</td><td>6.00E-33</td><td>36</td></tr></tbody></table>

<footnote>*The CDSs were clustered using a standard BLAST-mcl pipeline [BLASTP (v2.9.0) with default options, hits selected based on E-value < 1e-10, MCL clustering (v.14-137) with an inflation value of 2.8].</footnote>
Extended Data Table 4 | RNA virus and virus-like genomes identified in this study

<table><thead><tr><th>Group</th><th>Segment / Contig</th><th>Accession</th><th>Length (nt)</th><th>Ave. Cove.</th><th>Status</th><th>Top Hit (public DB)</th></tr></thead><tbody><tr><td rowspan="10">HsRV</td><td>RNA1a</td><td>BTCN01000001</td><td>6,117</td><td>1,621</td><td>full</td><td>No hit</td></tr><tr><td>RNA1b</td><td>BTCN01000002</td><td>6,013</td><td>401</td><td>full</td><td>No hit</td></tr><tr><td>RNA1c</td><td>BTCN01000003</td><td>6,560</td><td>66</td><td>full</td><td>No hit</td></tr><tr><td>RNA1d</td><td>BTCN01000004</td><td>6,008</td><td>47</td><td>full</td><td>No hit</td></tr><tr><td>RNA2a</td><td>BTCN01000005</td><td>4,417</td><td>2,518</td><td>full</td><td>No hit</td></tr><tr><td>RNA2b</td><td>BTCN01000006</td><td>4,302</td><td>1,605</td><td>full</td><td>No hit</td></tr><tr><td>RNA2c</td><td>BTCN01000007</td><td>4,398</td><td>395</td><td>full</td><td>No hit</td></tr><tr><td>RNA2d</td><td>BTCN01000008</td><td>4,427</td><td>199</td><td>full</td><td>No hit</td></tr><tr><td>RNA2*a</td><td>BTCN01000009</td><td>4,620</td><td>49</td><td>full</td><td>No hit</td></tr><tr><td>RNA2*b</td><td>BTCN01000010</td><td>4,272</td><td>44</td><td>full</td><td>No hit</td></tr><tr><td rowspan="10">HsRV-relates</td><td>Oi_contig_1</td><td>BTCS01000001</td><td>984</td><td>11</td><td>partial</td><td>RdRP [Ribovi<br>ria::Orthornavirae<br>(FAM010882)]</td></tr><tr><td>Oi_contig_2</td><td>BTCS01000002</td><td>617</td><td>113</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_3</td><td>BTCS01000003</td><td>3,057</td><td>13</td><td>partial</td><td>RdRP [Riboviria::Orthornavirae<br>(FAM010882)]</td></tr><tr><td>Oi_contig_4</td><td>BTCS01000004</td><td>622</td><td>40</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_5</td><td>BTCS01000005</td><td>3,243</td><td>9</td><td>partial</td><td>RdRP [Riboviria (FAM004495)]</td></tr><tr><td>Oi_contig_6</td><td>BTCS01000006</td><td>531</td><td>4</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_7</td><td>BTCS01000007</td><td>580</td><td>14</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_8</td><td>BTCS01000008</td><td>1,766</td><td>10</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_9</td><td>BTCS01000009</td><td>5,001</td><td>86</td><td>partial</td><td>No hit</td></tr><tr><td>H5_contig_1</td><td>BTCR01000001</td><td>2,030</td><td>17</td><td>partial</td><td>RdRP [Riboviria (FAM004495)]</td></tr><tr><td rowspan="4">HsPV (Y66)</td><td>RNA1_a</td><td>BTCQ01000001</td><td>1,638</td><td>2,997</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA1_b</td><td>BTCQ01000002</td><td>1,737</td><td>2,321</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA2_a</td><td>BTCQ01000003</td><td>1,925</td><td>2,674</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_b</td><td>BTCQ01000004</td><td>1,620</td><td>1,532</td><td>full</td><td>No hit</td></tr><tr><td rowspan="6">HsPV (H4)</td><td>RNA1_a</td><td>BTCO01000001</td><td>1,737</td><td>25,387</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA1_b</td><td>BTCO01000002</td><td>1,734</td><td>11,450</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA2_a</td><td>BTCO01000003</td><td>1,618</td><td>36,444</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_b</td><td>BTCO01000004</td><td>1,622</td><td>32,460</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_c</td><td>BTCO01000005</td><td>1,921</td><td>3,690</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_d</td><td>BTCO01000006</td><td>1,606</td><td>1,338</td><td>full</td><td>No hit</td></tr><tr><td rowspan="5">HsPV (H5)</td><td>RNA1_a</td><td>BTCP01000001</td><td>1,737</td><td>45,541</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA1_b</td><td>BTCP01000002</td><td>1,735</td><td>34,386</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA2_a</td><td>BTCP01000003</td><td>1,621</td><td>55,398</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_b</td><td>BTCP01000004</td><td>1,618</td><td>51,336</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_c</td><td>BTCP01000005</td><td>1,622</td><td>22,136</td><td>full</td><td>No hit</td></tr></tbody></table>
Extended Data Table 4 | RNA virus and virus-like genomes identified in this study

<table><thead><tr><th>Group</th><th>Segment / Contig</th><th>Accession</th><th>Length (nt)</th><th>Ave. Cove.</th><th>Status</th><th>Top Hit (public DB)</th></tr></thead><tbody><tr><td rowspan="10">HsRV</td><td>RNA1a</td><td>BTCN01000001</td><td>6,117</td><td>1,621</td><td>full</td><td>No hit</td></tr><tr><td>RNA1b</td><td>BTCN01000002</td><td>6,013</td><td>401</td><td>full</td><td>No hit</td></tr><tr><td>RNA1c</td><td>BTCN01000003</td><td>6,560</td><td>66</td><td>full</td><td>No hit</td></tr><tr><td>RNA1d</td><td>BTCN01000004</td><td>6,008</td><td>47</td><td>full</td><td>No hit</td></tr><tr><td>RNA2a</td><td>BTCN01000005</td><td>4,417</td><td>2,518</td><td>full</td><td>No hit</td></tr><tr><td>RNA2b</td><td>BTCN01000006</td><td>4,302</td><td>1,605</td><td>full</td><td>No hit</td></tr><tr><td>RNA2c</td><td>BTCN01000007</td><td>4,398</td><td>395</td><td>full</td><td>No hit</td></tr><tr><td>RNA2d</td><td>BTCN01000008</td><td>4,427</td><td>199</td><td>full</td><td>No hit</td></tr><tr><td>RNA2*a</td><td>BTCN01000009</td><td>4,620</td><td>49</td><td>full</td><td>No hit</td></tr><tr><td>RNA2*b</td><td>BTCN01000010</td><td>4,272</td><td>44</td><td>full</td><td>No hit</td></tr><tr><td rowspan="10">HsRV-relates</td><td>Oi_contig_1</td><td>BTCS01000001</td><td>984</td><td>11</td><td>partial</td><td>RdRP [Ribovi<br>ria::Orthornavirae<br>(FAM010882)]</td></tr><tr><td>Oi_contig_2</td><td>BTCS01000002</td><td>617</td><td>113</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_3</td><td>BTCS01000003</td><td>3,057</td><td>13</td><td>partial</td><td>RdRP [Riboviria::Orthornavirae<br>(FAM010882)]</td></tr><tr><td>Oi_contig_4</td><td>BTCS01000004</td><td>622</td><td>40</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_5</td><td>BTCS01000005</td><td>3,243</td><td>9</td><td>partial</td><td>RdRP [Riboviria (FAM004495)]</td></tr><tr><td>Oi_contig_6</td><td>BTCS01000006</td><td>531</td><td>4</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_7</td><td>BTCS01000007</td><td>580</td><td>14</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_8</td><td>BTCS01000008</td><td>1,766</td><td>10</td><td>partial</td><td>No hit</td></tr><tr><td>Oi_contig_9</td><td>BTCS01000009</td><td>5,001</td><td>86</td><td>partial</td><td>No hit</td></tr><tr><td>H5_contig_1</td><td>BTCR01000001</td><td>2,030</td><td>17</td><td>partial</td><td>RdRP [Riboviria (FAM004495)]</td></tr><tr><td rowspan="4">HsPV (Y66)</td><td>RNA1_a</td><td>BTCQ01000001</td><td>1,638</td><td>2,997</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA1_b</td><td>BTCQ01000002</td><td>1,737</td><td>2,321</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA2_a</td><td>BTCQ01000003</td><td>1,925</td><td>2,674</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_b</td><td>BTCQ01000004</td><td>1,620</td><td>1,532</td><td>full</td><td>No hit</td></tr><tr><td rowspan="6">HsPV (H4)</td><td>RNA1_a</td><td>BTCO01000001</td><td>1,737</td><td>25,387</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA1_b</td><td>BTCO01000002</td><td>1,734</td><td>11,450</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA2_a</td><td>BTCO01000003</td><td>1,618</td><td>36,444</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_b</td><td>BTCO01000004</td><td>1,622</td><td>32,460</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_c</td><td>BTCO01000005</td><td>1,921</td><td>3,690</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_d</td><td>BTCO01000006</td><td>1,606</td><td>1,338</td><td>full</td><td>No hit</td></tr><tr><td rowspan="5">HsPV (H5)</td><td>RNA1_a</td><td>BTCP01000001</td><td>1,737</td><td>45,541</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA1_b</td><td>BTCP01000002</td><td>1,735</td><td>34,386</td><td>full</td><td>RdRP [Driatsky virus]</td></tr><tr><td>RNA2_a</td><td>BTCP01000003</td><td>1,621</td><td>55,398</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_b</td><td>BTCP01000004</td><td>1,618</td><td>51,336</td><td>full</td><td>No hit</td></tr><tr><td>RNA2_c</td><td>BTCP01000005</td><td>1,622</td><td>22,136</td><td>full</td><td>No hit</td></tr></tbody></table>
Extended Data Table 5 | HHsearch hits for the IMG/VR virus proteins

<table><thead><tr><th>Protein</th><th>Annotation</th><th>HHsearch profile matched</th><th>HHsearch probability</th></tr></thead><tbody><tr><td>Ga0169446_00510_vOTU_07046706_5662_1</td><td>Predicted kinase</td><td>7E9V_A UMP-CMP kinase; catalytic activity, cytidylate kinase activity, kinase activity, transferase activity, TRANSFERASE; 2.1A {Homo sapiens} SCOP: c.37.1.0</td><td>97.55</td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_4</td><td>RdRP</td><td></td><td>58.29</td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_5</td><td>Potential zinc finger protein</td><td>5K2M_N Probable lysine biosynthesis protein; ATP -dependent amine/thiol ligase family Amino-group carrier protein Lysine biosynthesis Arginine biosynthesis, BIOSYNTHETIC PROTEIN; HET: ADP, UN1, SO4, PO4; 2.18A {Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1}}</td><td>93.62</td></tr><tr><td>Ga0393213_00017_vOTU_00596427_RC_5476_3</td><td>Phospholipase A2</td><td>PF08398.13; Phospholip A2_4; Phospholipase A2-like domain</td><td>95.08</td></tr><tr><td>Ga0393213_00017_vOTU_00596427_RC_5476_6</td><td>RdRP</td><td>5I62_A Potential RNA -dependent RNA polymerase; dsRNA, replication, transcription, insertion loop, viral protein; 2.001A {Human picobirnavirus (strain Human/Thailand/Hy005102/-)}</td><td>39.43</td></tr><tr><td>Ga0456180_000042_vOTU_00649204_RC_5304_1</td><td>Potential zinc finger protein</td><td>PF08792.13; A2L_zn_ribbon; A2L zinc ribbon domain</td><td>97.51</td></tr><tr><td>Ga0456180_000042_vOTU_00649204_RC_5304_5</td><td>RdRP</td><td></td><td>18.95</td></tr></tbody></table>
Extended Data Table 5 | HHsearch hits for the IMG/VR virus proteins

<table><thead><tr><th>Protein</th><th>Annotation</th><th>HHsearch profile matched</th><th>HHsearch probability</th></tr></thead><tbody><tr><td>Ga0169446_00510_vOTU_07046706_5662_1</td><td>Predicted kinase</td><td>7E9V_A UMP-CMP kinase; catalytic activity, cytidylate kinase activity, kinase activity, transferase activity, TRANSFERASE; 2.1A {Homo sapiens} SCOP: c.37.1.0</td><td>97.55</td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_4</td><td>RdRP</td><td></td><td>58.29</td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_5</td><td>Potential zinc finger protein</td><td>5K2M_N Probable lysine biosynthesis protein; ATP -dependent amine/thiol ligase family Amino-group carrier protein Lysine biosynthesis Arginine biosynthesis, BIOSYNTHETIC PROTEIN; HET: ADP, UN1, SO4, PO4; 2.18A {Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1}}</td><td>93.62</td></tr><tr><td>Ga0393213_00017_vOTU_00596427_RC_5476_3</td><td>Phospholipase A2</td><td>PF08398.13; Phospholip A2_4; Phospholipase A2-like domain</td><td>95.08</td></tr><tr><td>Ga0393213_00017_vOTU_00596427_RC_5476_6</td><td>RdRP</td><td>5I62_A Potential RNA -dependent RNA polymerase; dsRNA, replication, transcription, insertion loop, viral protein; 2.001A {Human picobirnavirus (strain Human/Thailand/Hy005102/-)}</td><td>39.43</td></tr><tr><td>Ga0456180_000042_vOTU_00649204_RC_5304_1</td><td>Potential zinc finger protein</td><td>PF08792.13; A2L_zn_ribbon; A2L zinc ribbon domain</td><td>97.51</td></tr><tr><td>Ga0456180_000042_vOTU_00649204_RC_5304_5</td><td>RdRP</td><td></td><td>18.95</td></tr></tbody></table>
Extended Data Table 6 | Detected RBS motif

<table><thead><tr><th>Gene</th><th>Gene start</th><th>Gene end</th><th>Start codon</th><th>RBS motif</th><th>RBS spacer</th><th>GC%</th><th>Length, aa</th><th># TMD</th></tr></thead><tbody><tr><td>HSPV-H4_RNA1_a_1</td><td>196</td><td>1689</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.489</td><td>497</td><td></td></tr><tr><td>HSPV-H4_RNA1_b_1</td><td>194</td><td>1684</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.518</td><td>496</td><td></td></tr><tr><td>HSPV-H4_RNA2_a_1</td><td>194</td><td>952</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.519</td><td>252</td><td></td></tr><tr><td>HSPV-H4_RNA2_a_2</td><td>949</td><td>1557</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.53</td><td>202</td><td>2</td></tr><tr><td>HSPV-H4_RNA2_b_1</td><td>198</td><td>953</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.541</td><td>251</td><td></td></tr><tr><td>HSPV-H4_RNA2_b_2</td><td>950</td><td>1561</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.521</td><td>203</td><td>2</td></tr><tr><td>HSPV-H4_RNA2_c_1</td><td>481</td><td>1245</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.527</td><td>254</td><td></td></tr><tr><td>HSPV-H4_RNA2_c_2</td><td>1242</td><td>1856</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.532</td><td>204</td><td>2</td></tr><tr><td>HSPV-H4_RNA2_d_1</td><td>190</td><td>945</td><td>AUG</td><td>AGGAGG</td><td>5-10bp</td><td>0.525</td><td>251</td><td></td></tr><tr><td>HSPV-H4_RNA2_d_2</td><td>942</td><td>1553</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.513</td><td>203</td><td>2</td></tr><tr><td>HSPV-H5_RNA1_a_1</td><td>196</td><td>1689</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.489</td><td>497</td><td></td></tr><tr><td>HSPV-H5_RNA1_b_1</td><td>195</td><td>1685</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.52</td><td>496</td><td></td></tr><tr><td>HSPV-H5_RNA2_a_1</td><td>194</td><td>952</td><td>AUG</td><td>AGGAGG</td><td>5-10bp</td><td>0.539</td><td>252</td><td></td></tr><tr><td>HSPV-H5_RNA2_a_2</td><td>949</td><td>1560</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.521</td><td>203</td><td>2</td></tr><tr><td>HSPV-H5_RNA2_b_1</td><td>194</td><td>952</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.519</td><td>252</td><td></td></tr><tr><td>HSPV-H5_RNA2_b_2</td><td>949</td><td>1557</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.524</td><td>202</td><td>2</td></tr><tr><td>HSPV-H5_RNA2_c_1</td><td>198</td><td>953</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.532</td><td>251</td><td></td></tr><tr><td>HSPV-H5_RNA2_c_2</td><td>950</td><td>1561</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.511</td><td>203</td><td>2</td></tr><tr><td>HSPV-Y66_RNA1_a_1</td><td>467</td><td>1627</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.526</td><td>386</td><td></td></tr><tr><td>HSPV-Y66_RNA1_b_1</td><td>193</td><td>1683</td><td>AUG</td><td>AGGAGG</td><td>3-4bp</td><td>0.516</td><td>496</td><td></td></tr><tr><td>HSPV-Y66_RNA2_a_1</td><td>486</td><td>1235</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.516</td><td>249</td><td></td></tr><tr><td>HSPV-Y66_RNA2_a_2</td><td>1232</td><td>1855</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.516</td><td>207</td><td>2</td></tr><tr><td>HSPV-Y66_RNA2_b_1</td><td>193</td><td>948</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.519</td><td>251</td><td></td></tr><tr><td>HSPV-Y66_RNA2_b_2</td><td>945</td><td>1556</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.493</td><td>203</td><td>2</td></tr><tr><td>HSRV_RNA1a_1</td><td>309</td><td>713</td><td>AUG</td><td>AATAA</td><td>6bp</td><td>0.407</td><td>134</td><td></td></tr><tr><td>HSRV_RNA1a_2</td><td>706</td><td>1092</td><td>AUG</td><td>None</td><td>None</td><td>0.37</td><td>128</td><td></td></tr><tr><td>HSRV_RNA1a_3</td><td>1097</td><td>3994</td><td>AUG</td><td>AATAA</td><td>15bp</td><td>0.378</td><td>965</td><td></td></tr><tr><td>HSRV_RNA1a_4</td><td>4012</td><td>5784</td><td>AUG</td><td>None</td><td>None</td><td>0.386</td><td>590</td><td></td></tr><tr><td>HSRV_RNA1b_1</td><td>379</td><td>711</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.435</td><td>110</td><td></td></tr><tr><td>HSRV_RNA1b_2</td><td>708</td><td>1100</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.369</td><td>130</td><td></td></tr><tr><td>HSRV_RNA1b_3</td><td>1103</td><td>3904</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.392</td><td>933</td><td></td></tr><tr><td>HSRV_RNA1b_4</td><td>3916</td><td>5661</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.408</td><td>581</td><td></td></tr><tr><td>HSRV_RNA1b_5</td><td>5710</td><td>5943</td><td>AUG</td><td>None</td><td>None</td><td>0.385</td><td>77</td><td>2</td></tr><tr><td>HSRV_RNA1c_1</td><td>15</td><td>755</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.328</td><td>246</td><td></td></tr><tr><td>HSRV_RNA1c_2</td><td>748</td><td>1119</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.309</td><td>123</td><td></td></tr><tr><td>HSRV_RNA1c_3</td><td>1122</td><td>4469</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.338</td><td>1115</td><td></td></tr><tr><td>HSRV_RNA1c_4</td><td>4444</td><td>6219</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.336</td><td>591</td><td></td></tr><tr><td>HSRV_RNA1c_5</td><td>6212</td><td>6481</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.333</td><td>89</td><td>3</td></tr><tr><td>HSRV_RNA1d_1</td><td>406</td><td>720</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.387</td><td>104</td><td></td></tr><tr><td>HSRV_RNA1d_2</td><td>720</td><td>1103</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.385</td><td>127</td><td></td></tr><tr><td>HSRV_RNA1d_3</td><td>1105</td><td>3900</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.393</td><td>931</td><td></td></tr><tr><td>HSRV_RNA1d_4</td><td>3915</td><td>5660</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.41</td><td>581</td><td></td></tr><tr><td>HSRV_RNA2a_1</td><td>422</td><td>910</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.429</td><td>162</td><td>2</td></tr><tr><td>HSRV_RNA2a_2</td><td>916</td><td>1527</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.363</td><td>203</td><td>4</td></tr><tr><td>HSRV_RNA2a_3</td><td>1533</td><td>2726</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.403</td><td>397</td><td>4</td></tr><tr><td>HSRV_RNA2a_4</td><td>2795</td><td>3604</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.383</td><td>269</td><td></td></tr><tr><td>HSRV_RNA2a_5</td><td>3606</td><td>4199</td><td>AUG</td><td>None</td><td>None</td><td>0.37</td><td>197</td><td></td></tr><tr><td>HSRV_RNA2a_6</td><td>4207</td><td>4338</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.364</td><td>43</td><td></td></tr><tr><td>HSRV_RNA2b_1</td><td>197</td><td>784</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.415</td><td>195</td><td>2</td></tr><tr><td>HSRV_RNA2b_2</td><td>860</td><td>1471</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.373</td><td>203</td><td>2</td></tr><tr><td>HSRV_RNA2b_3</td><td>1478</td><td>2653</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.429</td><td>391</td><td>6</td></tr><tr><td>HSRV_RNA2b_4</td><td>2724</td><td>3440</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.411</td><td>238</td><td></td></tr><tr><td>HSRV_RNA2b_5</td><td>3478</td><td>4230</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.393</td><td>250</td><td></td></tr><tr><td>HSRV_RNA2c_1</td><td>397</td><td>903</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.448</td><td>168</td><td>2</td></tr><tr><td>HSRV_RNA2c_2</td><td>903</td><td>1520</td><td>AUG</td><td>AGGAGG</td><td>3-4bp</td><td>0.401</td><td>205</td><td>4</td></tr><tr><td>HSRV_RNA2c_3</td><td>1526</td><td>2719</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.424</td><td>397</td><td>7</td></tr><tr><td>HSRV_RNA2c_4</td><td>2775</td><td>3584</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.41</td><td>269</td><td></td></tr><tr><td>HSRV_RNA2c_5</td><td>3629</td><td>4324</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.399</td><td>231</td><td></td></tr><tr><td>HSRV_RNA2d_1</td><td>447</td><td>950</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.433</td><td>167</td><td>2</td></tr><tr><td>HSRV_RNA2d_2</td><td>947</td><td>1564</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.39</td><td>205</td><td>2</td></tr><tr><td>HSRV_RNA2d_3</td><td>1569</td><td>2762</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.427</td><td>397</td><td>6</td></tr><tr><td>HSRV_RNA2d_4</td><td>2822</td><td>3628</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.416</td><td>268</td><td></td></tr><tr><td>HSRV_RNA2d_5</td><td>3661</td><td>4338</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.409</td><td>225</td><td></td></tr><tr><td>HSRV_RNA2a*_a_1</td><td>264</td><td>674</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.45</td><td>136</td><td>2</td></tr><tr><td>HSRV_RNA2a*_a_2</td><td>667</td><td>1440</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.376</td><td>257</td><td>2</td></tr><tr><td>HSRV_RNA2a*_a_3</td><td>1445</td><td>2773</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.402</td><td>442</td><td>5</td></tr><tr><td>HSRV_RNA2a*_a_4</td><td>2825</td><td>3364</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.361</td><td>179</td><td></td></tr><tr><td>HSRV_RNA2a*_a_5</td><td>3378</td><td>4520</td><td>AUG</td><td>None</td><td>None</td><td>0.392</td><td>380</td><td></td></tr><tr><td>HSRV_RNA2b_1</td><td>225</td><td>755</td><td>AUG</td><td>None</td><td>None</td><td>0.392</td><td>176</td><td>2</td></tr><tr><td>HSRV_RNA2b_2</td><td>757</td><td>1515</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.406</td><td>252</td><td>2</td></tr><tr><td>HSRV_RNA2b_3</td><td>1520</td><td>2827</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.388</td><td>435</td><td>5</td></tr><tr><td>HSRV_RNA2b_4</td><td>2830</td><td>2955</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.389</td><td>41</td><td></td></tr><tr><td>HSRV_RNA2b_5</td><td>2912</td><td>3523</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.395</td><td>203</td><td></td></tr><tr><td>HSRV_RNA2b_6</td><td>3535</td><td>3690</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.353</td><td>51</td><td></td></tr><tr><td>HSRV_RNA2b_7</td><td>3687</td><td>4190</td><td>AUG</td><td>None</td><td>None</td><td>0.401</td><td>167</td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_1</td><td>59</td><td>1042</td><td>UUG</td><td>None</td><td>None</td><td>0.467</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_2</td><td>1177</td><td>1428</td><td>AUG</td><td>AGGA</td><td>5-10bp</td><td>0.508</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_3</td><td>1428</td><td>2864</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.495</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_4</td><td>2903</td><td>4564</td><td>AUG</td><td>None</td><td>None</td><td>0.486</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_5</td><td>4561</td><td>4746</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.462</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_6</td><td>4824</td><td>5153</td><td>AUG</td><td>GGAGG</td><td>3-4bp</td><td>0.448</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_1</td><td>83</td><td>454</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.495</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_2</td><td>464</td><td>844</td><td>AUG</td><td>AGGA</td><td>5-10bp</td><td>0.462</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_3</td><td>844</td><td>1533</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.457</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_4</td><td>1514</td><td>2092</td><td>AUG</td><td>AGGA</td><td>5-10bp</td><td>0.489</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_5</td><td>2089</td><td>3573</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.492</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_6</td><td>3570</td><td>5294</td><td>AUG</td><td>4Base/6BMM</td><td>13-15bp</td><td>0.482</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_1</td><td>160</td><td>330</td><td>UUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.415</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_2</td><td>330</td><td>665</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.506</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_3</td><td>687</td><td>1988</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.52</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_4</td><td>1995</td><td>3485</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.53</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_5</td><td>3466</td><td>5214</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.525</td><td></td><td></td></tr></tbody></table>
Extended Data Table 6 | Detected RBS motif

<table><thead><tr><th>Gene</th><th>Gene start</th><th>Gene end</th><th>Start codon</th><th>RBS motif</th><th>RBS spacer</th><th>GC%</th><th>Length, aa</th><th># TMD</th></tr></thead><tbody><tr><td>HSPV-H4_RNA1_a_1</td><td>196</td><td>1689</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.489</td><td>497</td><td></td></tr><tr><td>HSPV-H4_RNA1_b_1</td><td>194</td><td>1684</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.518</td><td>496</td><td></td></tr><tr><td>HSPV-H4_RNA2_a_1</td><td>194</td><td>952</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.519</td><td>252</td><td></td></tr><tr><td>HSPV-H4_RNA2_a_2</td><td>949</td><td>1557</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.53</td><td>202</td><td>2</td></tr><tr><td>HSPV-H4_RNA2_b_1</td><td>198</td><td>953</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.541</td><td>251</td><td></td></tr><tr><td>HSPV-H4_RNA2_b_2</td><td>950</td><td>1561</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.521</td><td>203</td><td>2</td></tr><tr><td>HSPV-H4_RNA2_c_1</td><td>481</td><td>1245</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.527</td><td>254</td><td></td></tr><tr><td>HSPV-H4_RNA2_c_2</td><td>1242</td><td>1856</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.532</td><td>204</td><td>2</td></tr><tr><td>HSPV-H4_RNA2_d_1</td><td>190</td><td>945</td><td>AUG</td><td>AGGAGG</td><td>5-10bp</td><td>0.525</td><td>251</td><td></td></tr><tr><td>HSPV-H4_RNA2_d_2</td><td>942</td><td>1553</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.513</td><td>203</td><td>2</td></tr><tr><td>HSPV-H5_RNA1_a_1</td><td>196</td><td>1689</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.489</td><td>497</td><td></td></tr><tr><td>HSPV-H5_RNA1_b_1</td><td>195</td><td>1685</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.52</td><td>496</td><td></td></tr><tr><td>HSPV-H5_RNA2_a_1</td><td>194</td><td>952</td><td>AUG</td><td>AGGAGG</td><td>5-10bp</td><td>0.539</td><td>252</td><td></td></tr><tr><td>HSPV-H5_RNA2_a_2</td><td>949</td><td>1560</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.521</td><td>203</td><td>2</td></tr><tr><td>HSPV-H5_RNA2_b_1</td><td>194</td><td>952</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.519</td><td>252</td><td></td></tr><tr><td>HSPV-H5_RNA2_b_2</td><td>949</td><td>1557</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.524</td><td>202</td><td>2</td></tr><tr><td>HSPV-H5_RNA2_c_1</td><td>198</td><td>953</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.532</td><td>251</td><td></td></tr><tr><td>HSPV-H5_RNA2_c_2</td><td>950</td><td>1561</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.511</td><td>203</td><td>2</td></tr><tr><td>HSPV-Y66_RNA1_a_1</td><td>467</td><td>1627</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.526</td><td>386</td><td></td></tr><tr><td>HSPV-Y66_RNA1_b_1</td><td>193</td><td>1683</td><td>AUG</td><td>AGGAGG</td><td>3-4bp</td><td>0.516</td><td>496</td><td></td></tr><tr><td>HSPV-Y66_RNA2_a_1</td><td>486</td><td>1235</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.516</td><td>249</td><td></td></tr><tr><td>HSPV-Y66_RNA2_a_2</td><td>1232</td><td>1855</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.516</td><td>207</td><td>2</td></tr><tr><td>HSPV-Y66_RNA2_b_1</td><td>193</td><td>948</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.519</td><td>251</td><td></td></tr><tr><td>HSPV-Y66_RNA2_b_2</td><td>945</td><td>1556</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.493</td><td>203</td><td>2</td></tr><tr><td>HSRV_RNA1a_1</td><td>309</td><td>713</td><td>AUG</td><td>AATAA</td><td>6bp</td><td>0.407</td><td>134</td><td></td></tr><tr><td>HSRV_RNA1a_2</td><td>706</td><td>1092</td><td>AUG</td><td>None</td><td>None</td><td>0.37</td><td>128</td><td></td></tr><tr><td>HSRV_RNA1a_3</td><td>1097</td><td>3994</td><td>AUG</td><td>AATAA</td><td>15bp</td><td>0.378</td><td>965</td><td></td></tr><tr><td>HSRV_RNA1a_4</td><td>4012</td><td>5784</td><td>AUG</td><td>None</td><td>None</td><td>0.386</td><td>590</td><td></td></tr><tr><td>HSRV_RNA1b_1</td><td>379</td><td>711</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.435</td><td>110</td><td></td></tr><tr><td>HSRV_RNA1b_2</td><td>708</td><td>1100</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.369</td><td>130</td><td></td></tr><tr><td>HSRV_RNA1b_3</td><td>1103</td><td>3904</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.392</td><td>933</td><td></td></tr><tr><td>HSRV_RNA1b_4</td><td>3916</td><td>5661</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.408</td><td>581</td><td></td></tr><tr><td>HSRV_RNA1b_5</td><td>5710</td><td>5943</td><td>AUG</td><td>None</td><td>None</td><td>0.385</td><td>77</td><td>2</td></tr><tr><td>HSRV_RNA1c_1</td><td>15</td><td>755</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.328</td><td>246</td><td></td></tr><tr><td>HSRV_RNA1c_2</td><td>748</td><td>1119</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.309</td><td>123</td><td></td></tr><tr><td>HSRV_RNA1c_3</td><td>1122</td><td>4469</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.338</td><td>1115</td><td></td></tr><tr><td>HSRV_RNA1c_4</td><td>4444</td><td>6219</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.336</td><td>591</td><td></td></tr><tr><td>HSRV_RNA1c_5</td><td>6212</td><td>6481</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.333</td><td>89</td><td></td></tr><tr><td>HSRV_RNA1d_1</td><td>406</td><td>720</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.387</td><td>104</td><td></td></tr><tr><td>HSRV_RNA1d_2</td><td>720</td><td>1103</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.385</td><td>127</td><td></td></tr><tr><td>HSRV_RNA1d_3</td><td>1105</td><td>3900</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.393</td><td>931</td><td></td></tr><tr><td>HSRV_RNA1d_4</td><td>3915</td><td>5660</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.41</td><td>581</td><td></td></tr><tr><td>HSRV_RNA2a_1</td><td>422</td><td>910</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.429</td><td>162</td><td>2</td></tr><tr><td>HSRV_RNA2a_2</td><td>916</td><td>1527</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.363</td><td>203</td><td>4</td></tr><tr><td>HSRV_RNA2a_3</td><td>1533</td><td>2726</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.403</td><td>397</td><td>4</td></tr><tr><td>HSRV_RNA2a_4</td><td>2795</td><td>3604</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.383</td><td>269</td><td></td></tr><tr><td>HSRV_RNA2a_5</td><td>3606</td><td>4199</td><td>AUG</td><td>None</td><td>None</td><td>0.37</td><td>197</td><td></td></tr><tr><td>HSRV_RNA2a_6</td><td>4207</td><td>4338</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.364</td><td>43</td><td></td></tr><tr><td>HSRV_RNA2b_1</td><td>197</td><td>784</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.415</td><td>195</td><td>2</td></tr><tr><td>HSRV_RNA2b_2</td><td>860</td><td>1471</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.373</td><td>203</td><td>2</td></tr><tr><td>HSRV_RNA2b_3</td><td>1478</td><td>2653</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.429</td><td>391</td><td>6</td></tr><tr><td>HSRV_RNA2b_4</td><td>2724</td><td>3440</td><td>AUG</td><td>GGXGG</td><td>5-10bp</td><td>0.411</td><td>238</td><td></td></tr><tr><td>HSRV_RNA2b_5</td><td>3478</td><td>4230</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.393</td><td>250</td><td></td></tr><tr><td>HSRV_RNA2c_1</td><td>397</td><td>903</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.448</td><td>168</td><td>2</td></tr><tr><td>HSRV_RNA2c_2</td><td>903</td><td>1520</td><td>AUG</td><td>AGGAGG</td><td>3-4bp</td><td>0.401</td><td>205</td><td>4</td></tr><tr><td>HSRV_RNA2c_3</td><td>1526</td><td>2719</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.424</td><td>397</td><td>7</td></tr><tr><td>HSRV_RNA2c_4</td><td>2775</td><td>3584</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.41</td><td>269</td><td></td></tr><tr><td>HSRV_RNA2c_5</td><td>3629</td><td>4324</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.399</td><td>231</td><td></td></tr><tr><td>HSRV_RNA2d_1</td><td>447</td><td>950</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.433</td><td>167</td><td>2</td></tr><tr><td>HSRV_RNA2d_2</td><td>947</td><td>1564</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.39</td><td>205</td><td>2</td></tr><tr><td>HSRV_RNA2d_3</td><td>1569</td><td>2762</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.427</td><td>397</td><td>6</td></tr><tr><td>HSRV_RNA2d_4</td><td>2822</td><td>3628</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.416</td><td>268</td><td></td></tr><tr><td>HSRV_RNA2d_5</td><td>3661</td><td>4338</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.409</td><td>225</td><td></td></tr><tr><td>HSRV_RNA2a*_a_1</td><td>264</td><td>674</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.45</td><td>136</td><td>2</td></tr><tr><td>HSRV_RNA2a*_a_2</td><td>667</td><td>1440</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.376</td><td>257</td><td>2</td></tr><tr><td>HSRV_RNA2a*_a_3</td><td>1445</td><td>2773</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.402</td><td>442</td><td>5</td></tr><tr><td>HSRV_RNA2a*_a_4</td><td>2825</td><td>3364</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.361</td><td>179</td><td></td></tr><tr><td>HSRV_RNA2a*_a_5</td><td>3378</td><td>4520</td><td>AUG</td><td>None</td><td>None</td><td>0.392</td><td>380</td><td></td></tr><tr><td>HSRV_RNA2b_1</td><td>225</td><td>755</td><td>AUG</td><td>None</td><td>None</td><td>0.392</td><td>176</td><td>2</td></tr><tr><td>HSRV_RNA2b_2</td><td>757</td><td>1515</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.406</td><td>252</td><td>2</td></tr><tr><td>HSRV_RNA2b_3</td><td>1520</td><td>2827</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.388</td><td>435</td><td>5</td></tr><tr><td>HSRV_RNA2b_4</td><td>2830</td><td>2955</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.389</td><td>41</td><td></td></tr><tr><td>HSRV_RNA2b_5</td><td>2912</td><td>3523</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.395</td><td>203</td><td></td></tr><tr><td>HSRV_RNA2b_6</td><td>3535</td><td>3690</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.353</td><td>51</td><td></td></tr><tr><td>HSRV_RNA2b_7</td><td>3687</td><td>4190</td><td>AUG</td><td>None</td><td>None</td><td>0.401</td><td>167</td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_1</td><td>59</td><td>1042</td><td>UUG</td><td>None</td><td>None</td><td>0.467</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_2</td><td>1177</td><td>1428</td><td>AUG</td><td>AGGA</td><td>5-10bp</td><td>0.508</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_3</td><td>1428</td><td>2864</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.495</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_4</td><td>2903</td><td>4564</td><td>AUG</td><td>None</td><td>None</td><td>0.486</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_5</td><td>4561</td><td>4746</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.462</td><td></td><td></td></tr><tr><td>Ga0169446_00510_vOTU_07046706_5662_6</td><td>4824</td><td>5153</td><td>AUG</td><td>GGAGG</td><td>3-4bp</td><td>0.448</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_1</td><td>83</td><td>454</td><td>AUG</td><td>AGGAG</td><td>5-10bp</td><td>0.495</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_2</td><td>464</td><td>844</td><td>AUG</td><td>AGGA</td><td>5-10bp</td><td>0.462</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_3</td><td>844</td><td>1533</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.457</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_4</td><td>1514</td><td>2092</td><td>AUG</td><td>AGGA</td><td>5-10bp</td><td>0.489</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_5</td><td>2089</td><td>3573</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.492</td><td></td><td></td></tr><tr><td>Ga0393213_00017_vOTU_00596427 RC_5476_6</td><td>3570</td><td>5294</td><td>AUG</td><td>4Base/6BMM</td><td>13-15bp</td><td>0.482</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_1</td><td>160</td><td>330</td><td>UUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.415</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_2</td><td>330</td><td>665</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.506</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_3</td><td>687</td><td>1988</td><td>AUG</td><td>GGAG/GAGG</td><td>5-10bp</td><td>0.52</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_4</td><td>1995</td><td>3485</td><td>AUG</td><td>GGAGG</td><td>5-10bp</td><td>0.53</td><td></td><td></td></tr><tr><td>Ga0456180_000042_vOTU_00649204 RC_5304_5</td><td>3466</td><td>5214</td><td>AUG</td><td>GGA/GAG/AGG</td><td>5-10bp</td><td>0.525</td><td></td><td></td></tr></tbody></table>
<figure><img src="image_39.png" /></figure>

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Tablet viewer (version 1.19.09.03); phyloFlash (version 3.4); BLASTX (version 2.2.31+); Prodigal (version 2.6.3); HHpred (online server [no versions]); MEGA6.06; TMHMM (version 2.0); ColabFold 1.5.1; AlphaFold 2 through ColbFold v1.5.2; DALI (online, DaliLite.v5); ChimeraX (version 1.5); trimAl (version 1.4.rev15); RAxML (8.2.10); ProtTest (version 3.4.2); PROMALS3D; IQ-TREE (version 2.0.6); MAFFT (version 7); BLASTP (v2.9.0); HHblits (v3.3.0); ModelFinder (a part of IQ-TREE); BLASTn/p/x (2.12.0+)

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio [guidelines for submitting code & software](#) for further information.
<figure><img src="image_39.png" /></figure>

# Reporting Summary

Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our [Editorial Policies](#) and the [Editorial Policy Checklist](#).

## Statistics

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a | Confirmed

☒ ☐ The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement

☐ ☒ A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

☒ ☐ The statistical test(s) used AND whether they are one- or two-sided
*Only common tests should be described solely by name; describe more complex techniques in the Methods section.*

☒ ☐ A description of all covariates tested

☒ ☐ A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons

☒ ☐ A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)

☒ ☐ For null hypothesis testing, the test statistic (e.g. *F*, *t*, *r*) with confidence intervals, effect sizes, degrees of freedom and *P* value noted
*Give *P* values as exact values whenever suitable.*

☒ ☐ For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings

☒ ☐ For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes

☒ ☐ Estimates of effect sizes (e.g. Cohen's *d*, Pearson's *r*), indicating how they were calculated

*Our web collection on statistics for biologists contains articles on many of the points above.*

## Software and code

Policy information about [availability of computer code](#)

**Data collection** No software was used for data collection.

**Data analysis** A custom Perl pipeline script used for read clenup is available at GitHub (https://github.com/takakiy/FLDS).

The following commercial programs were used.

CLC GENOMICS WORKBENCH version 11.0 (Qiagen Japan, Tokyo, Japan); Genetyx version 14 (Genetyx, Tokyo, Japan)

The following open source programs were used.

Tablet viewer (version 1.19.09.03); phyloFlash (version 3.4); BLASTX (version 2.2.31+); Prodigal (version 2.6.3); HHpred (online server [no versions]); MEGA6.06; TMHMM (version 2.0); ColabFold 1.5.1; AlphaFold 2 through ColbFold v1.5.2; DALI (online, DaliLite.v5); ChimeraX (version 1.5); trimAl (version 1.4.rev15); RAxML (8.2.10); ProtTest (version 3.4.2); PROMALS3D; IQ-TREE (version 2.0.6); MAFFT (version 7); BLASTP (v2.9.0); HHblits (v3.3.0); ModelFinder (a part of IQ-TREE); BLASTn/p/x (2.12.0+)

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio [guidelines for submitting code & software](#) for further information.
# Data

Policy information about [availability of data](#)

All manuscripts must include a [data availability statement](#). This statement should provide the following information, where applicable:

- Accession codes, unique identifiers, or web links for publicly available datasets

- A description of any restrictions on data availability

- For clinical datasets or third party data, please ensure that the statement adheres to our [policy](#).

Datasets obtained in this study have been available in the GenBank database repository (Accession Nos. HsRV: BTCN01000001-BTCN01000010; HsPV-H4: BTCO01000001-BTCO01000006; HsPV-H5: BTCP01000001-BTCP01000005; HsPV-Y66: BTCQ01000001-BTCQ01000004; H5_contig_1: BTCR01000001; Oi_contig_1-9: BTCS01000001-BTCS01000009) and Short Read Archive database (Accession No. DRA016131). Datasets (PDB70 [mmcf_2023-10-24], Pfam [v35], UniProt-SwissProt-viral70_Nov_2021 and NCBI-CD [v3.19]) are available at http://ftp.tuebingen.mpg.de/pub/protevo/toolkit/databases/hhsuite_dbs/. Searches using the IMG/VR dataset were available only at https://img.jgi.doe.gov/cgi-bin/vr/main.cgi?section=WorkspaceBlast&page=viralform. Datasets (SILVA SSU [version 138], Neo-HMM [v1.1], and RVDB-HMM [v23.0]) are publicly available.

# Research involving human participants, their data, or biological material

Policy information about studies with [human participants or human data](#). See also policy information about [sex, gender (identity/presentation), and sexual orientation](#) and [race, ethnicity and racism](#).

**Reporting on sex and gender**

This research does not involve human participants, their data, or biological material.

**Reporting on race, ethnicity, or other socially relevant groupings**

This research does not involve human participants, their data, or biological material.

**Population characteristics**

This research does not involve human participants, their data, or biological material.

**Recruitment**

This research does not involve human participants, their data, or biological material.

**Ethics oversight**

This research does not involve human participants, their data, or biological material.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

# Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

<figure><img src="image_40.png" /></figure>

Life sciences

<figure><img src="image_41.png" /></figure>

Behavioural & social sciences

<figure><img src="image_42.png" /></figure>

Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see [nature.com/documents/nr-reporting-summary-flat.pdf](https://nature.com/documents/nr-reporting-summary-flat.pdf)

# Ecological, evolutionary & environmental sciences study design

All studies must disclose on these points even when the disclosure is negative.

**Study description**

This study collected microbes in hot spring water and performed sequencing analyses for RNA virus discovery.

**Research sample**

Microbes in hot spring water.

**Sampling strategy**

No sample-size calculations were performed.

**Data collection**

The chemical composition of hot spring water was measured by T.O. Sequencing data were obtained using Illumina Miseq platform by M.H.

**Timing and spatial scale**

Sample were collected at 09- or 10-Mar-2017 and 17- or 18-Nov-2015. Each sample was collected once.

**Data exclusions**

Data from two sampling points were not included in analyses since we could not obtain data from these two samples.

**Reproducibility**

For data analyses, all raw data is available in the GenBank database repository. Reproducibility of environmental samples and sequencing was not confirmed.

**Randomization**

No randomization was performed and no controlling for covariants is relevant to this study design.

**Blinding**

Blinding does not apply to this study since it is discovery-oriented.
# Data

Policy information about [availability of data](#)

All manuscripts must include a [data availability statement](#). This statement should provide the following information, where applicable:

- Accession codes, unique identifiers, or web links for publicly available datasets

- A description of any restrictions on data availability

- For clinical datasets or third party data, please ensure that the statement adheres to our [policy](#).

Datasets obtained in this study have been available in the GenBank database repository (Accession Nos. HsRV: BTCN01000001-BTCN01000010; HsPV-H4: BTCO01000001-BTCO01000006; HsPV-H5: BTCP01000001-BTCP01000005; HsPV-Y66: BTCQ01000001-BTCQ01000004; H5_contig_1: BTCR01000001; Oi_contig_1-9: BTCS01000001-BTCS01000009) and Short Read Archive database (Accession No. DRA016131). Datasets (PDB70 [mmcf_2023-10-24], Pfam [v35], UniProt-SwissProt-viral70_Nov_2021 and NCBI-CD [v3.19]) are available at http://ftp.tuebingen.mpg.de/pub/protevo/toolkit/databases/hhsuite_dbs/. Searches using the IMG/VR dataset were available only at https://img.jgi.doe.gov/cgi-bin/vr/main.cgi?section=WorkspaceBlast&page=viralform. Datasets (SILVA SSU [version 138], Neo-HMM [v1.1], and RVDB-HMM [v23.0]) are publicly available.

# Research involving human participants, their data, or biological material

Policy information about studies with [human participants or human data](#). See also policy information about [sex, gender (identity/presentation), and sexual orientation](#) and [race, ethnicity and racism](#).

**Reporting on sex and gender**

This research does not involve human participants, their data, or biological material.

**Reporting on race, ethnicity, or other socially relevant groupings**

This research does not involve human participants, their data, or biological material.

**Population characteristics**

This research does not involve human participants, their data, or biological material.

**Recruitment**

This research does not involve human participants, their data, or biological material.

**Ethics oversight**

This research does not involve human participants, their data, or biological material.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

# Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

<figure><img src="image_40.png" /></figure>

Life sciences

<figure><img src="image_41.png" /></figure>

Behavioural & social sciences

<figure><img src="image_42.png" /></figure>

Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see [nature.com/documents/nr-reporting-summary-flat.pdf](https://nature.com/documents/nr-reporting-summary-flat.pdf)

# Ecological, evolutionary & environmental sciences study design

All studies must disclose on these points even when the disclosure is negative.

**Study description**

This study collected microbes in hot spring water and performed sequencing analyses for RNA virus discovery.

**Research sample**

Microbes in hot spring water.

**Sampling strategy**

No sample-size calculations were performed.

**Data collection**

The chemical composition of hot spring water was measured by T.O. Sequencing data were obtained using Illumina Miseq platform by M.H.

**Timing and spatial scale**

Sample were collected at 09- or 10-Mar-2017 and 17- or 18-Nov-2015. Each sample was collected once.

**Data exclusions**

Data from two sampling points were not included in analyses since we could not obtain data from these two samples.

**Reproducibility**

For data analyses, all raw data is available in the GenBank database repository. Reproducibility of environmental samples and sequencing was not confirmed.

**Randomization**

No randomization was performed and no controlling for covariants is relevant to this study design.

**Blinding**

Blinding does not apply to this study since it is discovery-oriented.
Did the study involve field work?

Yes

No

## Field work, collection and transport

Field conditions

The weather was sunny or cloudy.

Location

Locations of the samplings are follow;
H4: 31°54'07.5"N 130°50'06.2"E
H5: 31°54'07.5"N 130°50'06.2"E
T1-4: 31°54'37.7"N 130°49'00.6"E
Y66, Y80, Y86: 31°55'03.8"N 130°48'40.4"E
Oi: 32°44'25.3"N 130°15'48.4"E
Ob: 32°43'33.0"N 130°12'24.7"E

Access & import/export

All samples were obtained with the permission of the landowner (or official manager) and in compliance with national law. The issuer are as follow; Unzen City, Unzen Nature Conservation Bureau, Kirishima Iwasaki Hotel, NIPPON PAPER LUMBER CO. LTD. and NITTETSU MINING CO. LTD KAGOSHIMA GEOTHERMAL FACILITY.

Disturbance

Sampling was done with a minimal number of people and collected from ample spring water sources.

## Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

### Materials & experimental systems

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☑</td><td>Antibodies</td></tr><tr><td>☑</td><td>Eukaryotic cell lines</td></tr><tr><td>☑</td><td>Palaeontology and archaeology</td></tr><tr><td>☐</td><td>Animals and other organisms</td></tr><tr><td>☑</td><td>Clinical data</td></tr><tr><td>☑</td><td>Dual use research of concern</td></tr><tr><td>☑</td><td>Plants</td></tr></tbody></table>

### Methods

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☑</td><td>ChIP-seq</td></tr><tr><td>☑</td><td>Flow cytometry</td></tr><tr><td>☑</td><td>MRI-based neuroimaging</td></tr></tbody></table>

### Animals and other research organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research

Laboratory animals

This study did not involve laboratory animals.

Wild animals

This study did not involve wild animals.

Reporting on sex

This study did not involve sex information.

Field-collected samples

A total of 11 samples were collected from five hot springs regions at southern Japan, in close proximity to active volcanoes, according to the instructions of Unzen City, Unzen Nature Conservation Bureau and private companies that maintain each hot spring region. At each sampling station, approximately 10 L of hot spring water was collected in a sterilized plastic bag, and then filtered with 0.2-µm-pore-size cellulose acetate membrane filters in 47 mm diameter (Advantec, Tokyo, Japan) within 0.5-3 hours after sampling. The filters were stored at -80°C until nucleic acid extraction.

Ethics oversight

No ethical approval or guidance was required

Note that full information on the approval of the study protocol must also be provided in the manuscript.
Did the study involve field work?

Yes

No

## Field work, collection and transport

**Field conditions**

The weather was sunny or cloudy.

**Location**

Locations of the samplings are follow;

H4: 31°54'07.5"N 130°50'06.2"E

H5: 31°54'07.5"N 130°50'06.2"E

T1-4: 31°54'37.7"N 130°49'00.6"E

Y66, Y80, Y86: 31°55'03.8"N 130°48'40.4"E

Oi: 32°44'25.3"N 130°15'48.4"E

Ob: 32°43'33.0"N 130°12'24.7"E

**Access & import/export**

All samples were obtained with the permission of the landowner (or official manager) and in compliance with national law. The issuer are as follow; Unzen City, Unzen Nature Conservation Bureau, Kirishima Iwasaki Hotel, NIPPON PAPER LUMBER CO. LTD. and NITTETSU MINING CO. LTD KAGOSHIMA GEOTHERMAL FACILITY.

**Disturbance**

Sampling was done with a minimal number of people and collected from ample spring water sources.

## Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

### Materials & experimental systems

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☑</td><td>Antibodies</td></tr><tr><td>☑</td><td>Eukaryotic cell lines</td></tr><tr><td>☑</td><td>Palaeontology and archaeology</td></tr><tr><td>☐</td><td>Animals and other organisms</td></tr><tr><td>☑</td><td>Clinical data</td></tr><tr><td>☑</td><td>Dual use research of concern</td></tr><tr><td>☑</td><td>Plants</td></tr></tbody></table>

### Methods

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☑</td><td>ChIP-seq</td></tr><tr><td>☑</td><td>Flow cytometry</td></tr><tr><td>☑</td><td>MRI-based neuroimaging</td></tr></tbody></table>

### Animals and other research organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research

**Laboratory animals**

This study did not involve laboratory animals.

**Wild animals**

This study did not involve wild animals.

**Reporting on sex**

This study did not involve sex information.

**Field-collected samples**

A total of 11 samples were collected from five hot springs regions at southern Japan, in close proximity to active volcanoes, according to the instructions of Unzen City, Unzen Nature Conservation Bureau and private companies that maintain each hot spring region. At each sampling station, approximately 10 L of hot spring water was collected in a sterilized plastic bag, and then filtered with 0.2-µm-pore-size cellulose acetate membrane filters in 47 mm diameter (Advantec, Tokyo, Japan) within 0.5-3 hours after sampling. The filters were stored at -80°C until nucleic acid extraction.

**Ethics oversight**

No ethical approval or guidance was required

Note that full information on the approval of the study protocol must also be provided in the manuscript.