Dsrna-Seq Reveals Novel RNA Virus and Virus-Like Putative Complete Genome Sequences from Hymeniacidon Sp. Sponge

Home , Cardoreovirus, Cryspovirus, Oryzavirus, Partitiviridae, Seadornavirus

Microbes Environ. Vol. 35, No. 2, 2020 https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME19132 dsRNA-seq Reveals Novel RNA Virus and Virus-Like Putative Complete Genome Sequences from Hymeniacidon sp. Sponge Syun-ichi Urayama1,2,3*, Yoshihiro Takaki4, Daisuke Hagiwara2,3, and Takuro Nunoura1 1Research Center for Bioscience and Nanoscience (CeBN), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15 Natsushima-cho, Yokosuka, Kanagawa 237–0061, Japan; 2Laboratory of Fungal Interaction and Molecular Biology (donated by IFO), Department of Life and Environmental Sciences, University of Tsukuba, 1–1–1 Tennodai, Tsukuba, Ibaraki, 305– 8577, Japan; 3Microbiology Research Center for Sustainability (MiCS), University of Tsukuba, 1–1–1 Tennodai, Tsukuba, Ibaraki, 305–8577, Japan; and 4Super-cutting-edge Grand and Advanced Research (SUGAR) Program, JAMSTEC, 2–15 Natsushima-cho, Yokosuka, Kanagawa 237–0061, Japan (Received October 17, 2019—Accepted January 17, 2020—Published online February 28, 2020)

Invertebrates are a source of previously unknown RNA viruses that fill gaps in the viral phylogenetic tree. Although limited information is currently available on RNA viral diversity in the marine sponge, a primordial multicellular animal that belongs to the phylum Porifera, the marine sponge is one of the well-studied holobiont systems. In the present study, we elucidated the putative complete genome sequences of five novel RNA viruses from Hymeniacidon sponge using a combination of double-stranded RNA sequencing, called fragmented and primer ligated dsRNA sequencing, and a conventional transcriptome method targeting single-stranded RNA. We identified highly diverged RNA-dependent RNA polymerase sequences, including a potential novel RNA viral lineage, in the sponge and three viruses presumed to infect sponge cells.

Key words: RNA virus, metagenome, dsRNA, sponge

The marine sponge is a primordial multicellular animal derived, and identifies novel viral small RNAs in a mollusc that belongs to the phylum Porifera. Sponges are recog‐ and a brown alga. bioRxiv doi: https://doi.org/ nized as hosts of diverse symbiotic lives (Taylor et al., 10.1101/166488). 2007), including bacteria, archaea, fungi, protists, and To access RNA viromes, conventional total RNA-seq viruses (Webster and Thomas, 2016), and the relationships (single-stranded RNA [ssRNA]-seq) is the most commonly between the host sponge and symbionts vary from mutual‐ used method to detect RNA viral sequences in diverse envi‐ ism to parasitism (Webster and Taylor, 2012; Thomas et al., ronments and organisms, such as oceanic waters (Steward et 2016). To understand the ecosystems associated with al., 2013; Culley et al., 2014), lake waters (Djikeng et al., sponges, sponges have been described as “holobionts,” 2009), seafloor sediments (Engelhardt et al., 2015), inverte‐ which are defined as assemblages of multiple species that brates (Shi et al., 2016), and vertebrates (Shi et al., 2018). In form ecological units (Webster and Taylor, 2012). Sponge- the latter two studies, deep RNA sequencing was performed bacteria/-archaea interactions have been extensively exam‐ on total RNAs from invertebrates and vertebrates and thou‐ ined (Schmitt et al., 2012; Thomas et al., 2016; Pita et al., sands of RNA viruses that appeared to infect them were 2018); however, the impact of viruses on sponge holobionts identified (Shi et al., 2016; 2018). However, this method remains unclear. Among the limited number of virus studies does not have the capacity to obtain complete genome on sponge holobionts, the presence of diverse virus-like par‐ sequences from RNA viruses because of the principle of ticles in sponge cells and symbiont compartments has been cDNA synthesis. The rapid amplification of the cDNA end reported (Vacelet and Gallissian, 1978; Pascelli et al., 2018). (RACE) or alternative methods have been conducted to Furthermore, metagenomic analyses suggested the domi‐ obtain complete RNA viral genomes/segments, including nance of bacteriophage lineages in DNA viromes associated terminal sequences, (Roossinck et al., 2010; Shi et al., with reef sponge holobionts (Butina et al., 2015; Laffy et 2016). In contrast, double-stranded RNA (dsRNA) al., 2016; Laffy et al., 2018). To the best of our sequencing has the advantage of the superior enrichment of knowledge, one metatranscriptome study on a sponge RNA viral genomes over the conventional RNA sequencing holobiont characterized the presence of the partial genome method (Roossinck et al., 2010). Long cellular dsRNA con‐ sequences of RNA viruses related to the families sists of dsRNA virus genomes and replicative intermediates Narnaviridae, Dicistroviridae, Partitiviridae, of ssRNA viruses (Morris and Dodds, 1979). Fragmented Picobirnaviridae, Picornaviridae, Tombusviridae, and primer-ligated dsRNA sequencing (FLDS), a method to Nodaviridae, and Hepeviridae as well as unclassified obtain the full-length sequence of dsRNA including terminal viruses (Waldron, et al. [2018] Metagenomic sequencing sequences, was recently developed (Koyama et al., 2016; suggests arthropod antiviral RNA interference is highly Urayama et al., 2016; 2018b; Fukasawa et al., 2019; Higashiura et al., 2019). The feature of this method is to * Corresponding author. E-mail: [email protected]; retrieve the sequences from terminal regions that are needed Tel: +81–29–853–6636; Fax: +81–29–853–4605. to reconstruct segmented RNA virus genomes based on the 2 / 11 Urayama et al. conserved sequences of terminal regions in each genome tool, each contig was screened under the following conditions: at segment (Urayama et al., 2018a). In the present study, we least 3× average coverage and 500 bp in length. If >10 reads applied the FLDS method in combination with ssRNA-seq defined the same terminal position of a certain contig, the position was recognized as a terminal end. Contigs for which both ends to reveal sponge RNA viromes. were identified as termini were defined as potential genome seg‐ ments. RNA viral genes in potential genome segments and contigs Materials and Methods were identified based on sequence similarities to known RNA viral proteins in the NCBI non-redundant (nr) database using BLASTX Sample collection (Camacho et al., 2009) with an e-value≤1×10–5. Sequences that Sponges on tidal rocks in Tokyo Bay (35.3405°N, 139.6396°E) matched a known RNA-dependent RNA polymerase (RdRp) gene were sampled in April 2014 and 2015. After washing with distilled by BLASTX with an e-value≤1×10–5 were collected from RNA water, samples were stored at –80°C until further analyses. virus contigs and segments. The presence of the RdRp domain (PF00680, PF00978, and PF02123) was confirmed using Extraction and purification of dsRNA and ssRNA PfamScan (Chojnacki et al., 2017) with an e-value≤1×10–5 when a Sponge samples were disrupted in liquid nitrogen in a mortar. sequence had significant similarity (e-value≤1×10–5) to an RNA Total nucleic acids were manually extracted from approximately viral polyprotein or hypothetical protein. Nucleotide sequences 1 g of sponge powder. dsRNA was purified using a combination of encoding the RdRp gene were clustered at 90% identity using the cellulose column method (Urayama et al., 2015) and an enzy‐ VSEARCH (Rognes et al., 2016). The centroid sequence of each matic treatment, as described previously (Urayama et al., 2018a). cluster was defined as a representative sequence. We also identi‐ Some of the powder was processed to obtain total RNA using the fied the signature amino acid residues conserved in the RdRp TRIzol Plus RNA Purification Kit (Invitrogen) according to the domain (Venkataraman et al., 2018) in the ORFs of potential manufacturer’s protocol. Total RNA was treated with DNase I genome segments that had no ORF with significant similarities (e- (Invitrogen) and further purified using the RNA Clean and Con‐ value≤1×10–5) to sequences in the nr database. Quality-based var‐ centrator-5 Kit (Zymo Research). iant detection was performed using CLC Genomics Workbench ver. 11.0 (CLC Bio). dsRNA sequencing library construction Raw sequencing reads from ssRNA-seq were processed as Nuclease-treated dsRNA was fragmented by ultrasound at 4°C described previously (Urayama et al., 2016). Small subunit (SSU) in Snap-Cap microTUBEs (Covaris) using Covaris S220 (settings: rRNA sequences were reconstructed from ssRNA-seq reads with run time 35 s, peak power 140.0 W, duty factor 2.0%, and 200 EMIRGE (Miller et al., 2011). cycles burst–1) (Covaris) (Urayama et al., 2018a). After the purifi‐ cation of fragmented dsRNAs using the Zymoclean Gel RNA Phylogenetic analyses Recovery Kit (Zymo Research), double-stranded cDNA was syn‐ The deduced amino acid sequences of the putative RdRp genes thesized as described previously (Urayama et al., 2018a). Briefly, a obtained in the present study and their relatives in the NCBI nr ssDNA adapter was ligated to the 3′-ends of fragmented dsRNA database were aligned using MUSCLE (Edgar, 2004) in MEGA5 and cDNA was synthesized using the SMARTer RACE 5′/3′ Kit (Tamura et al., 2011). To exclude ambiguous positions, the align‐ (Takara Bio) with an oligonucleotide primer that had a comple‐ ment was trimmed by trimAl (option: -gt 1) (Capella-Gutiérrez et mentary sequence to the adapter sequence. After amplification, al., 2009). Phylogenetic analyses were conducted using RAxML cDNA was quantified using the Qubit dsDNA HS Kit (Thermo (Stamatakis, 2014). Bootstrap tests were conducted with 1,000 Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technol‐ samplings. The model of amino acid substitution for each phylog‐ ogies). eny was selected by Aminosan (Tanabe, 2011), as judged by Akaike’s information criterion (Sugiura, 1978), i.e., rtREV+F+G ssRNA sequencing library construction for Partitiviridae, LG+F for Reoviridae, LG+F+G for Double-stranded cDNA was synthesized from 2 μg of total RNA Picobirnaviridae, and rtREV+F+G for Dicistroviridae. Tree‐ with the Prime Script Double Strand cDNA Synthesis Kit (Takara Graph2 (Stöver and Müller, 2010) and FigTree (Rambaut, 2014) Bio) using random primers (9-mers). The resultant cDNA was were used to illustrate the resulting phylogenies. quantified using the Qubit dsDNA HS Kit (Thermo Fisher Scien‐ tific) and Agilent 2100 Bioanalyzer (Agilent Technologies). Accession numbers The sequences obtained in the present study are available in the Illumina sequencing GenBank database repository (accession nos. DDBJ: Double-stranded cDNAs were fragmented using Covaris S220 BKZT01000001–BKZT01000434, BKZU01000001–BKZU01000431, (settings: run time 55 s, peak power 175.0 W, duty factor 5.0%, and LC504241–LC504250) and Short Read Archive database and 200 cycles burst–1), and fragmented cDNAs were used as tem‐ (accession nos. DDBJ: DRA008491). plates for KAPA Hyper Prep Kit Illumina platforms (Kapa Biosys‐ tems). The resultant Illumina libraries were evaluated using the Results KAPA library quantification kit (Kapa Biosystems) and applied to the Illumina MiSeq platform (Illumina) according to the manufac‐ Overview of NGS reads turer’s protocol (600-cycle kit to perform 300-bp paired-end We obtained 1.8–4.1 M high-quality reads in four RNA- sequencing). seq libraries (Fig. S1). Regarding dsRNA-seq libraries, Data processing reads associated with RNA viruses (see below) and the Raw sequencing reads for dsRNA-seq were processed as descri‐ remaining rRNA reads occupied approximately 20 and 30% bed previously (Urayama et al., 2018a). rRNA reads in dsRNA-seq of dsRNA reads, respectively. The ratio of rRNA reads in libraries were identified by SortMeRNA (Kopylova et al., 2012) dsRNA-seq libraries varied among the samples reported pre‐ and excluded from further virome analyses. In RNA virome analy‐ viously (Urayama et al., 2016; 2018a; 2018b; Decker et al., ses, reads were subjected to de novo assembly using CLC Genom‐ ics Workbench ver. 11.0 (CLC Bio) with the following parameters: 2019). Some rRNAs potentially contaminated the dsRNA a minimum contig length of 500, word value and bubble size set to fraction due to their secondary structures. Thus, the relative auto, and assemblies that were manually examined and extended amount of dsRNA to rRNA in samples may have affected using the Tablet viewer (Milne et al., 2010). Using the mapping the rRNA read ratio in dsRNA sequencing. Furthermore, the RNA Viral Genomes Associated with Sponge 3 / 11 formation of dsRNAs of sense/antisense transcripts from mated based on mapped read numbers on SSU rRNA overlapping regions may influence the results of dsRNA sequences (Miller et al., 2011). Hymeniacidon sp. SSU sequencing (Decker et al., 2019). In ssRNA-seq libraries, rRNA reads occupied >90% of all SSU rRNA reads in total RNA viruses and rRNA reads occupied approximately 0.5 ssRNA libraries from sponges obtained in both 2014 and and 80% of ssRNA reads, respectively. 2015 (Fig. 1A). The next abundant eukaryotic SSU rRNA sequence was identified as Mytilus edulis (0.8%) mussel, Diversity of cellular rRNA in sponges and the abundance of other eukaryotic SSU rRNA sequen‐ To identify potentially active biomes in sponges obtained ces was lower than 0.2% of all SSU rRNA reads. No arch‐ in 2014 and 2015, SSU rRNA sequences were reconstructed aeal or bacterial SSU rRNA sequence was found from reads from total ssRNA-seq reads, and compositions were esti‐ that dominated >2% of all SSU rRNA reads.

(A) 7 2014 48

2015 19 14 0% 20% 40% 60% 80% 100%

Hymeniacidon sp. Other Eukaryotes Bacteria Archaea

(B)

4000 0.0083 % of read numbers in non-rRNA reads 0.0032 3000 0.003 0.0026

2000 0.002 Coverage

1000 0.001

0 0

SRV SpiRVSrRV 2014-3 2014-42014-5 2014-62014-22014-8 2014-9 2014-1 SpaRV2014-14 2014-17 2014-212014-18 2014-222014-16 2014-102014-11

6000 0.003 0.0025 Coverage 4000 0.002 0.0014

2000 0.001

0 0

SRV SdRV 015-46015-112015-9SpiRV015-41015-122015-3015-10015-492015-7015-17015-15015-35015-48015-24015-45015-262015-8015-28SrRV015-19015-14015-44015-37015-38015-52015-18015-25015-36 SpaRV 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Fig. 1. Summary of dsRNA-seq and ssRNA-seq analyses. (A) Relative abundance of sequence reads mapped on SSU rRNA sequences constructed by EMIRGE. Numbers indicate OTU. (B, C) The average coverage (left axis) and relative abundance (right axis) of RdRp-encoding RNA viral genome segments (Table S1) estimated by the mapping of dsRNA-seq and ssRNA-seq data, respectively, from sponge samples collected in 2014 (B) and 2015 (C). Open bars indicate reads mapped on segments encoding CDS with no significant similarity (e-value≤1×10–5) to known viral genes. Items on the horizontal axis represent the IDs of RdRp-encoding full-length sequences (Table S1) or virus names. 4 / 11 Urayama et al.

ments (Fig. 1B and C), we focused on the five dominant Sponge-associated non-retro RNA virome viral genome segments that represented high average cover‐ The FLDS analysis (dsRNA-seq) succeeded in enriching age (>1,000×) in each dsRNA-seq library. The five seg‐ viral reads and revealed RNA viromes associated with ments occupied more than 50% of the total average sponges collected in 2014 and 2015, while RNA virus reads coverage of RdRp-encoding segments and contigs (253 and were rare in the ssRNA-seq analysis, except for reads 233 OTU in 2014 and 2015, respectively). Following the related to Dicistroviridae in 2015 samples (Table S1). We identification of major RdRp sequences in dsRNA-seq libra‐ obtained 253 and 233 sequences encoding RdRp genes from ries, we reconstructed RNA genome sets based on the simi‐ the 2014 and 2015 samples, respectively (Table S1). These larity of terminal sequences among segments (Fig. 3 and sequences included viral sequences related to 9 dsRNA S3). These five genome sets were considered to be “putative virus families and 3 positive-sense ssRNA virus families complete” because they had very high coverage (>1000×), based on a RdRp sequence similarity analysis, while 11 and and, thus, there was a low chance of missing other frag‐ 33 families were previously established in dsRNA and ments of their genomes. Reconstructed viral genomes were positive-sense ssRNA viruses, respectively (King et al., also supported by the following parameters, in addition to 2012). We also found 78 sequences encoding RdRp genes the similarity of terminal sequences. The difference in GC% belonging to unclassified RNA viral lineages based on the among sequences within a group sharing similar terminal taxonomic linage of BLASTX top hit sequences (Table S1), sequences was <5%. Each ORF did not show amino acid and 7.7% of RdRp reads in dsRNA libraries were mapped to sequence similarity (>50% similarity) to any ORFs encoded these segments and contigs. Viral sequences related to fami‐ by other potential genome segments in a single group. We lies recognized as animal viruses, such as Birnaviridae and named these RNA viral genomes harboring ORFs that pre‐ Dicistroviridae, were also identified from sponge RNA sented significant similarity (e-value≤1×10–5) with known viromes. After the clustering of RdRp domain sequences, viral sequences after the names of the most closely related 167 representative sequences (>1.5 kb, <90% identity) were virus family (Table S2 and see below); Sponge-associated identified and defined as operational taxonomic units dicistro-like RNA virus (SdRV, accession no. (OTUs). Based on the taxonomic lineage of BLASTX top BKZU01000024), Sponge-associated partiti-like RNA virus hit sequences, the richness of OTUs was analyzed and many (SpaRV, accession no. BKZT01000015), Sponge-associated viral OTUs were suggested to be closely related with (or reo-like RNA virus (SrRV, accession no. BKZT01000005), belonging to) Totiviridae, Reoviridae, and Partitivirdae Sponge-associated picobirna-like RNA virus (SpiRV, acces‐ (Fig. 2). The majority of OTUs in unclassified groups sion no. BKZT01000016), and Sponge-associated RNA showed low amino acid sequence identity to the best hit virus (SRV, accession no. LC504241–50). sequence (Fig. S2). All of the top hit sequences in “unclassi‐ No ORF of SRV showed significant similarity (e- fied RNA viruses” were RdRp identified from Bryopsis value≤1×10–5) with sequences in the nr database. However, cinicola (Chlorophyta) (Koga et al., 2003). In “unclassified an ORF of SRV RNA2 showed insignificant similarity (e- dsRNA viruses” and “unclassified viruses”, several OTUs value=0.01) to the RT_like super family (cl02808) in the showed close sequence similarities to RdRp sequences iden‐ CDD search. Thus, we manually identified the signature tified from the Diatom colony (Urayama et al., 2016). Dom‐ amino acid residues conserved in the RdRp domain inance in the coverage of these dsRNA viruses was also (Venkataraman et al., 2018) including the “GDD” sequence confirmed based on the read abundance in dsRNA libraries in the ORF (Fig. S4). Accordingly, we concluded that the (Table S1). genome segment set corresponded to RNA viral genomes that belonged to a previously unknown viral family. Major putative complete genomes To estimate the potential host of these RNA viruses iden‐ We obtained 118 and 180 full-length potential viral tified in the dsRNA-seq analysis, we evaluated the abun‐ genome segments from 2014 and 2015 samples, respec‐ dance of these viral sequences in ssRNA-seq according to tively. Among RdRp-encoding potential viral genome seg‐ the previously proposed definition that viral reads sharing

unclassified RNA viruses ShiM-2016 Totiviridae unclassified viruses Partitiviridae unclassified RNA viruses Reoviridae unclassified dsRNA viruses 167 OTU Cystoviridae Narnaviridae Endornaviridae Picobirnaviridae Dicistroviridae Birnaviridae Chrysoviridae

Fig. 2. Overview of the RNA virome identified from the sponge. Taxonomic composition of RdRp-based OTUs identified in the present study. RNA Viral Genomes Associated with Sponge 5 / 11

Group 1: Sponge-associated dicistrovirus-like RNA virus (SdRV) reads. Thus, we concluded that Hymeniacidon sp. appeared RNA_helicase RNA_dep_RNAP to be a host of SdRV, SpaRV, and SRV. The read abundances 1.47e-29 1.12e-87 of SpiRV and SrRV putative genomes were one to two orders of magnitude smaller than that of SdRV in ssRNA- Group 2: Sponge-associated partitivirus-like RNA virus (SpaRV) seq data, and were only detected from either of the samples. RNA1 RT_like super family 8.80e-20 Therefore, we were unable to define their host based on this RNA2 definition. It is important to note that dsRNA viruses (SpaRV, SpiRV, and SrRV) showed lower read abundance Group 3: Sponge-associated reovirus-like RNA virus (SrRV) than the ssRNA virus (SdRV) in ssRNA-seq data. RNA1 Orbi_VP1 super family 1.10e-57 RNA2 SdRV RNA3 Orbi_VP4 super family 3.30e-16 Among reconstructed viral genomes, only SdRV pre‐ RNA4 RNA5 sented significant similarity with the subject sequence Cale‐ RNA6 donia beadlet anemone dicistro-like virus 2 isolate I38 RNA7 (accession no. MF189973, publication status: unpublished) RNA8 (e-value=0.0, 71% identity) in the BLASTN analysis; its potential host Caledonia belongs to the phylum Cnidaria, Group 4: Sponge-associated picobirnavirus-like RNA virus (SpiRV) not Porifera. The SdRV genome consists of one segment RNA1 with two ORFs. The BLASTP search also revealed that the RNA2 RT_like super family 7.43e-15 most similar sequence to SdRV RdRp was putative ORF1 of RNA3 Caledonia beadlet anemone dicistro-like virus 2 isolate I38 (accession no. ASM93984) (e-value=0.0, 74% identity). A Group 5: Sponge-associated RNA virus (SRV) phylogenetic analysis of the RdRp sequence indicated that SdRV belonged to the positive-stranded ssRNA virus family RNA1 Dicistroviridae (Fig. 4). The arrangement of ORFs on the RNA2 RT_like super family 0.01 SdRV genome was similar to those on known viruses in RNA3 Dicistroviridae (Fig. S5). These results suggest that SdRV is RNA4 a new ssRNA virus that belongs to Dicistroviridae. RNA5 RNA6 SpaRV RNA7 RNA8 In the BLASTP analysis for RdRp in the SpaRV genome, RNA9 the most similar sequence in the nr database was Hubei RNA10 partiti-like virus 53 (related to viruses in Partitiviridae) (e- 1,000 nt value=3×10–43, 32% identity) found in the ssRNA-seq of invertebrates (Shi et al., 2016). The genome of SpaRV con‐ Fig. 3. Organization of reconstructed RNA viral or virus-like genomes. Values indicate the e-value with the viral top hit domain by a sists of two segments with one ORF each and the genome CD search. structure is conserved in known partitiviruses. Although the RdRp sequence showed significant similarity (e- value≤1×10–5) with sequences in Partitiviridae, SpaRV was >0.1% in non-rRNA reads in conventional RNA sequencing not classified into the established genus of Partitiviridae by may be infected with the sampled organism (Shi et al., the phylogenetic analysis (Fig. 5) (Nibert et al., 2014). 2016). Among the five major viral genomes, SdRV showed Therefore, SpaRV represents a novel genus in the family the highest read abundance, accounting for 2.7% of non- Partitiviridae. Viruses in Partitiviridae have been isolated ribosomal reads in the ssRNA-seq library (Fig. 1C). The from culturable organisms, such as plants, fungi, and proto‐ full-length sequence of SpaRV was obtained in 2014 and zoa (Nibert et al., 2014). In addition, their relatives have 2015 samples and only one base substitution was observed been detected in both vertebrates and invertebrates in tran‐ between sequences obtained from the two samplings. The scriptomic analyses (Liu et al., 2012; Shi et al., 2016); how‐ read abundance in non-ribosomal reads was approximately ever, no partitivirus has been isolated from animals (Nibert 0.0025% in both ssRNA-seq libraries. SRV was also et al., 2014). detected in 2014 and 2015 samples and occupied more than 0.008% in both ssRNA-seq libraries. Between the 2014 and SrRV, SpiRV, and SRV 2015 SRV genomes, there were only 54 positions of a The sequence that was the most similar to RdRp in SrRV single-nucleotide variant and 90% (49/54) of them were in the BLASTP analysis was Chuzan virus (genus identified as transitions. Therefore, SpaRV and SRV Orbivirus) (e-value=2×10–14, 25% identity); however, a phy‐ appeared to have infected the same or closely related organ‐ logenetic analysis of RdRp for Reoviridae revealed that isms (host) within sponges obtained in both 2014 and 2015. SrRV was not positioned into the genus Orbivirus (Fig. 6). In contrast, in both sponges, eukaryotes other than In addition, in the BLASTX analysis, an ORF of segment 3 Hymeniacidon sponge did not dominate more than 0.0025% showed significant similarity with VP3 (Orbivirus VP4 core of the SSU rRNA composition, with the exception of M. protein) of Wad Medani virus (genus Orbivirus) (e- edulis in 2015 samples, which shared 0.8% of SSU rRNA value=2×10–14, 25% identity), while other segments showed 6 / 11 Urayama et al.

NP_277061_Perina_nuda_virus Iflaviridae NP_740478_Enterovirus_C 75 99 NP_740467_Foot-and-mouth_disease_virus 64 88 NP_740444_Aichi_virus_1 Picornaviridae NP_740737_Parechovirus_A 83 NP_042507_Rice_tungro_spherical_virus 95 NP_619734_Parsnip_yellow_fleck_virus 97 YP_081444_Cherry_rasp_leaf_virus 70 Secoviridae 61 NP_599086_Strawberry_mottle_virus 33 YP_001039627_Tomato_torrado_virus NP_613283_Cowpea_mosaic_virus 100 YP_053925_Arabis_mosaic_virus NP_149057_Taura syndrome virus NP_620562_Triatoma_virus 53 98 YP_009333558_Beihai_picorna-like_virus YP_009336583_Hubei_picorna-like_virus_16 37 99 YP_009336571_Hubei_diptera_virus_1 98 AMO03208_Empeyrat_virus 91 50 YP_009221981_Goose_dicistrovirus MF189973_Caledonia beadlet anemone dicistro-like virus 2 isolate I38 Dicistroviridae 100 BKZU01000024 Sponge-associated dicistrovirus-like RNA virus 66 YP_009388499_Apis_dicistrovirus 73 YP_009252204_Anopheles_C_virus 83 87 NP_044945_Drosophila_C_virus 100 AIY53985_Nilaparvata_lugens_C_virus 100AKA63263_Cricket_paralysis_virus 86 NP_647481_Cricket_paralysis_virus BDQA01000113 _Jam_Picorna-like_virus_2 97 YP_001429583_Marine_RNA_virus_JP-B 100 unassigned BDQA01000107_Jam_Picorna-like_virus_1 93 Picornavirales BAG30951_Chaetoceros_tenuissimus_RNA_virus_01 44 99 YP_001429581_Marine_RNA_virus_JP-A unassigned 89 YP_392465_Aurantiochytrium_single-stranded_RNA_virus_01 Picornavirales NP_944776_Heterosigma_akashiwo_RNA_virus Marnaviridae 0.4 Fig. 4. Maximum likelihood tree of aligned RdRp amino acid sequences from SdRV and representative members of families Dicistroviridae, Secoviridae, Picornaviridae, and Iflaviridae using 305 residues. Numbers indicate bootstrap values (%) based on 1,000 RAxML calculations. The best-fitting amino acid substitution model was [rtREV+F+G]. no significant similarity (e-value≤1×10–5) with known viral sequences. Other viral segments and contigs The most significantly similar sequence in the BLASTP Although we focused on numerically abundant viruses analysis for RdRp in SpiRV was Hubei picobirna-like virus with putative complete genome sequences in the dsRNA-seq 2. A phylogenetic analysis of RdRp suggested that SpiRV libraries, there was a RdRp-encoding contig with similar belongs to an undefined and tentatively named α clade of average coverage (1,839×) to these five genomes. The con‐ picobirnavirus-like viruses derived from marine microor‐ tig (ID: Sponge_RNAV429) was identified in 2014 samples, ganisms, diatoms, and invertebrates (Urayama et al., 2018a) and showed significant similarity (e-value=4×10–14, 24% (Fig. 7). In Picobirnaviridae, one sequence that originates identity) to known, but unclassified RdRp identified from from a sponge named Barns Ness breadcrumb sponge the chloroplasts of B. cinicola. Other RdRp-encoding seg‐ picobirna-like virus 1 (accession no. MF190026.1) was ments and contigs were lower than 1,000× average cover‐ found in the nr database, but belongs to the β clade. age. SRV was composed of 10 RNA segments with 35,412 nt In the present study, 788 partial viral genome sequences that showed no significant similarity (e-value≤1×10–5) with were identified (Table S1). To clarify the phylogenetic dis‐ sequences in the nr database in BLASTN and BLASTX tribution of these viruses, maximum likelihood trees for analyses. The RdRp sequence was too distant from all each established RNA virus family were calculated based on known RdRp sequences to identify its phylogenic position. RdRp sequences encoded in the contigs of partial or putative However, as discussed above, the genome structure of SRV complete genome segments (Fig. S6). Consistent with the resembled that of Reoviridae and the ratio of SRV reads in similarity values obtained by the BLASTX analysis (Table non-rRNA ssRNA-seq was similar to those of dsRNA S1), most viruses were phylogenetically distinct from viruses (Fig. 1B and C). When considering the maximum known RNA viral genera, and RNA viruses related to the segment number of known ssRNA viruses (viruses in Ortho‐ families Partitiviridae, Reoviridae, and Totiviridae were myxoviridae have ~8 segments), SRV may be a reo-like very diverse. novel dsRNA virus. To the best of our knowledge, the genome size of SRV is the largest among known RNA viruses. RNA Viral Genomes Associated with Sponge 7 / 11

APG78198_Beihai picobirna-like virus 11 100 AFJ79071_Otarine_picobirnavirus Picobirnaviridae BAD98236_Human picobirnavirus BKZT01000015 Sponge-associated partitivirus-like RNA virus AAA61829_Atkinsonella hypoxylon virus Porifera AAC98734_Fusarium poae virus 1 100 100 YP_392480_Rosellinia necatrix partitivirus 1-W8 Betapartitivirus 67 AGJ83767_Hop trefoil cryptic virus 2 100 ADL66905_Heterobasidion partitivirus 2 BAM78602_osellinia necatrix partitivirus 2 94 ADV15441_Heterobasidion partitivirus 1 100 AGY54938_Rhizoctonia solani dsRNA virus 2 Alphapartitivirus 77 62 ACA81389_Beet cryptic virus 1 AAX51289_Raphanus sativus cryptic virus 1 APG78249_Hubei partiti-like virus 36 87 67 APG78244_Hubei partiti-like virus 29 APG78266_Hubei diptera virus_19 71 54 99 APG78296_Sanxia water strider virus 18 APG78251_Hubei partiti-like virus 32 Arthropoda? APG78283_Hubei partiti-like virus 22 75 84 APG78323_Wuhan cricket virus 2 APG78260_Hubei partiti-like virus 19 100 BDQA01000279_Jam_Partiti-like_virus_1 AAC47805_Cryptosporidium parvum virus 1 Cryspovirus APG78229_Hubei partiti-like virus 57 70 BDQB01000299 _St73_Partiti-like_virus_2 97 100 BDQE01000199_St122_Partiti-like_virus_1 BAU79511_Diatom colony associated dsRNA virus 14 96 100 AEJ07892_Pepper cryptic virus 2 65 YP_006390091_Persimmon cryptic virus AAB27624_Beet cryptic virus 3 Deltapartitivirus 100 YP_001274391_Fragaria chiloensis cryptic virus YP_001686786_Rose cryptic virus 1 100 APT70073_Alternaria alternata partitivirus 1 AGZ84316_Botryosphaeria dothidea partitivirus 1 BDQC01000143_St79_Partiti-like_virus_2 100 AOO52902_Purpureocillium lilacinum nonsegmented virus 1 93 BDQE01000138_St122_Partiti-like_virus_2 100 100 BDQB01000238_St73_Partiti-like_virus_4 99 BDQD01000133_St97_Partiti-like_virus_2 BDQB01000236_St73_Partiti-like_virus_5 NP_659027_Gremmeniella abietina RNA virus MS1 77 100 YP_001686789_Botryotinia fuckeliana partitivirus 1 Gammapartitivirus 100 NP_116716_Discula destructiva virus 1 NP_624350_Fusarium solani virus 1

100 APG78297_Hubei partiti-like virus 53 APG78298_Hubei partiti-like virus_54 APG78299_Hubei partiti-like virus 55

Eukaryota; Alveolata; Apicomplexa Eukaryota; Viridiplantae; Streptophyta 1 Eukaryota; Opisthokonta; Fungi Eukaryota; Opisthokonta; Metazoa Fig. 5. Maximum likelihood tree of aligned RdRp amino acid sequences from SpaRV and representative members of families Picobirnaviridae and Partitiviridae using 302 residues. Numbers indicate bootstrap values (%) based on 1,000 RAxML calculations. The best-fitting amino acid substitution model was [rtREV+F+G]. Colors indicate the classification of the host organism.

retrieving entire genomic structures because of the absence Discussion of the terminal sequences of each genome segment (Ozsolak and Milos, 2011). Most contigs encoding viral genes, except Recent RNA virome analyses suggest the extent of gene for RdRp genes, must be overlooked, except when the module shuffling among diverse viral genomes (Dolja and genome sequence presents significant similarity to previ‐ Koonin, 2017). Thus, high-quality information on RNA ously reported viral genomes. For example, among the five viral genomes is essential for understanding the diversity possible RNA viral genomes from the two sponges exam‐ and evolution of RNA viruses. However, the conventional ined in the present study, it was not possible to identify metagenomic ssRNA-seq approach has a limitation in >50% of segments as RNA viral genome segments based on

8 / 11 Urayama et al.

AKP19898_African horse sickness_virus 9 sickness_virus horse AKP19898_African ALM00138_African horse sickness virus 2 virus sickness horse ALM00138_African

AAC40586_African horse sickness virus

ACI41990_African horse sickness virus

ADU57373_Equine encephalosis virus

AGZ92012_Changuinola virus AGZ92012_Changuinola

AAA88823_Bluetongue virus 2 virus AAA88823_Bluetongue

AAA87362_Bluetongue virus 11 virus AAA87362_Bluetongue AAA87363_Bluetongue virus 13 virus AAA87363_Bluetongue

ALW83178_Palyam virus

AMC39573_Chuzan virus

AAA87364_Bluetongue virus 17 virus AAA87364_Bluetongue

CAA31306_Bluetongue virus CAA31306_Bluetongue AFH41519_Pata virus AFH41519_Pata

BAA76549_Chuzan virus AHX25713_Wad Medani virus APG79108_Hubei lepidoptera virus 4

Seadornavirus ALR84811_Mangshi virus

AAF77631_Banna virus AAG34363_St Croix River virus

AAF78848_Kadipiro virus Orbivirus 97

100 62 AAT11887_Eriocheir sinensis reovirus BKZT01000005 Sponge-associated reovirus-like RNA virus 94 BAA01074_Rice dwarf virus 94

69 80 Phytoreovirus 40

Cardoreovirus 100 93 80 M97203_Rotavirus 18 CAA39085_Bovine rotavirus 100 AAC58684_Simian rotavirus 49 98 68 42 98 AAB00801_Porcine rotavirus C Rotavirus 100

Mimoreovirus 97 Aquareovirus 64 63 50 90 AAL31497_Chum salmon reovirus CS 98 90 AAM92745_GoldenAAA47255_MammalianOrthoreovirus shiner reovirus orthoreovirus 3 AAZ94042_Micromonas pusilla reovirus AAG17718_GrassAAA47245_Mammalian carp hemorrhagic virus orthoreovirus 2 43 46

98 56 60 APG79084_Beihai reo-like virus 2 MyColtivirus

100

AAM18342_Eyach virus 100 APG79142_Hubei reo-like virus 2

Mycoreovirus AAC36456_Rice ragged stunt virusYP_009333354_Wuhan heteroptera virus 3

BAC98431_Mycoreovirus 3 BAC98431_Mycoreovirus BAA08542_Nilaparvata lugens reovirus AAP45577_Cryphonectria parasitica mycoreovirus-1 AAN46860_DendrolimusAAK73087_Lymantria punctatus dispar cypovirus cypovirus 1 14

Oryzavirus Fijivirus

0.6 Cypovirus

Fig. 6. Maximum likelihood tree of aligned RdRp amino acid sequences from SrRV and representative members of the family Reoviridae using 409 residues. Numbers indicate bootstrap values (%) based on 1,000 RAxML calculations. The best-fitting amino acid substitution model was [LG +F]. Gray circles represent established genera. Black dots and green color represent OTUs derived from Urayama et al. (2018a) and Shi et al. (2016), respectively. a sequence similarity analysis when the terminal sequence known to harbor a polyA tail on their genomes (King et al., information of RNA viral segments obtained by FLDS was 2012). This difference may be explained by the methodolog‐ absent. ical bias of the FLDS method that targets cellular dsRNA, a In FLDS analyses, we were unable to identify the polyA replicative intermediate of ssRNA viruses (York and Fodor, tail of SdRV; however, most viruses in the order 2013). A previous study reported that RNA viral genome Picornavirales, including the family Dicistroviridae, are sequences related to viruses in Hypoviridae (order: unas‐ RNA Viral Genomes Associated with Sponge 9 / 11

_sponge_picobirna-like_virus_1

APG78170 APG78167 APG78169

ASM94065_Barns_Ness_breadcrumb

BAJ53293 APG78193 BAN58175 ALL29319 AFK81928 α AFK81927 ACT64131 AIY31286 AHX00960

88 AIY31287 APG78191 AIY31288

81 AIY31284 Genogroup IAFJ79071 100 ADO22678 100 47 YP_009241386 71 39 AHI59999 ALB35036 APG78188 74 AHZ46150 34 AIY31285 99 96 BAJ53291 20 YP_009333352 61 AIW53314 AGK45545 YP_009333349 100 APG78214 98 BAD98236

95 BAJ53290 APG78190 ADG56983 100 BKZT01000016picobirnavirus-like_RNA_virus Sponge-associated 100 AAG53584 100 BAJ53294 APG78268 BAU79482 100 APG78198 100 APG78271 APG78198 AIY31294 AKN50624 100 AKN50618 APG78302

APG78305 Hubei picobirna-like virus 2 100 Genogroups II and III δ

APG78210

APG78168 APG78338 0.5

APG78176

APG78315

Fig. 7. Maximum likelihood tree of aligned RdRp amino acid sequences from SpiRV and representative members of the family Picobirnaviridae using 310 residues. Numbers indicate bootstrap values (%) based on 1,000 RAxML calculations. The best-fitting amino acid substitution model was [LG+F+G]. Black dots and green color represent OTUs derived from Urayama et al. (2018b) and Shi et al. (2016), respectively. Purple color indicates unpublished deposited sequences. signed) with polyA tails were retrieved by FLDS (Urayama while RNA viromes have been overlooked in holobiont sys‐ et al., 2018a). The variance of the 3′ terminal modification tems. A high-quality list of sponge-associated RNA viruses with a polyA tail among ssRNA viral genomes retrieved by will be a fundamental knowledge base for understanding the FLDS implies variations in replication processes among activity and ecological distribution of (sponge-associated) ssRNA viruses. RNA viruses. In the present study, we assessed sponge RNA viromes using dsRNA- and ssRNA-seq and identified 167 RdRp Acknowledgements genes, which represent RNA viral OTUs. The present results revealed that RNA viruses in sponge holobionts are This research was supported in part by Grants-in-Aid for Scien‐ tific Research (Nos. 26892031 and 18H05368) from the Ministry highly diverse, similar to DNA viruses and prokaryotic pop‐ of Education, Culture, Sports, Science and Technology (MEXT) of ulations (Taylor et al., 2004; Laffy et al., 2018). The present Japan and Grants-in-Aid for Scientific Research on Innovative study suggests the importance of RNA viruses for the Areas from MEXT of Japan (Nos. 16H06429, 16K21723, and homeostasis and evolution of sponge holobiont systems, 16H06437). 10 / 11 Urayama et al.

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