Persistent infection of a novel Picornavirus in the microplanarian Stenostomum leucops (Catenulida).

Persistent infection of a novel Picornavirus in the microplanarian Stenostomum leucops (Catenulida). | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Persistent infection of a novel Picornavirus in the microplanarian Stenostomum leucops (Catenulida). Marcos Trindade Rosa, Gabriel da Luz Wallau, Elgion L S Loreto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4390948/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Nov, 2024 Read the published version in Archives of Virology → Version 1 posted 4 You are reading this latest preprint version Abstract We present the genome sequence, organization and evidence of persistence of a new picornavirus infecting the flatworm Stenostomum leucops . The complete genome sequence belongs to a virus with a positive single-stranded RNA genome encoding three open reading frames (ORFs) flanked by untranslated regions and polyadenylated termination. The ORFs encode conserved protein motifs typical of the picornavirus superfamily. Phylogenetic analyses confirm membership of this viral superfamily, with closely related viral species found in Biomphalaria (Mollusca) in France and a virus detected in metagenomic analyses of water sources from the USA, suggesting widespread distribution. RT-PCR analysis revealed that this virus persists in a laboratory-grown worm isolate for at least five years, indicating persistent infection. However, no deleterious effects were observed in the infected worms, suggesting a commensalistic or mutualistic relationship between the virus and the worms. Picornavirus flatworm virus ssRNA virus RdRP gene Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Viruses are the most abundant and diverse biological entities on Earth and have been found in virtually all host species studied to date, although understanding of the virosphere is still very limited (Paez-Espino et al. 2016; Shi et al. 2016). Characterizing and classifying this enormous diversity has been a difficult task. Originally, this task was limited to viruses that could be isolated in culture cells and induce cell death or alterations. In recent decades, however, high-throughput sequencing technologies have enabled an enormous expansion of knowledge about the viral component of the microbiome, the so-called viromes, which are based on the high-resolution viral sequences recovery (Wolf et al., 2018). Despite increasing efforts to characterize the virome of different organisms and environments, our current understanding is biased for chordates, arthropods and plants. Platyhelminthes, a phylum composed of free-living and parasitic flatworms, is a poorly studied group in terms of its virome, even though several studies have described viruses on certain species of planarians, Schistosoma and Taenia (Jones MK and Whittington 1992; Justine and Bonami 1993; Noury-Sraïria et al 1995; Crespo-González et al 2008; Rebrikov et al, 2002). More recently, comprehensive studies analyzing platyhelminth genomes produced by NSG have found more than a hundred viruses infecting 50 species of free-living and 65 species of parasitic flatworms, including viruses belonging to the Picornavirales, Jingchuvirales, Maretellivirales, Flaviviridae, Nyamiviridae, Ghabrivirales, Rhabdoviridae and Bunyavirales families (Dheilly et al, 2022; Zhang et al 2023). To our knowledge, no studies have been conducted to characterize viruses of the Catenulida, which are a more basal group of platyhelminthes (Telford et al., 2003; Wallberg et al., 2007). Catenulida are small free-living flatworms ranging in size from 0.5 to 2.5 mm that reproduce asexually by paratomy, a process in which the anterior body structures form in the mid-body regions, resulting in segmented organisms (Rosa et al., 2015). We have maintained a clonal culture of S. leucops (Catenulida) for more than a decade. This strain has a flexible relationship with its microbiome that may be associated with phenotypic plasticity (Rosa and Loreto 2023) and is capable of expressing genes found in its microbiome or eDNA (Rosa and Loreto 2019). In the present study, we characterized a complete genome of a (+) ssRNA virus from the Picornaviridae family that is maintained for at least five years in a S. leucops colony, suggesting persistent and long-term infection. Phylogenetic analysis revealed that this virus is a novel virus closely related to the virus found in Biomphalaria . Based on the long-term association of this virus with S. leucops and the apparent lack of deleterious host effects, it is likely that this virus and its host engage in a mutualistic or commensalistic interaction. Stenostomum leucops specimens were collected in 2009 in a pond in Santa Maria, Brazil (53°17′W; 29°28′S). An isoline was first established and the worms have been kept in reconstituted water at 28°C and fed with milk powder, following the details described by Rosa et al. (2015). To gain deeper insights into this non-model species, we performed a transcriptomic characterization. Total RNA was isolated using Trizol (Invitrogen®) according to the manufacturer's guidelines. RNA quality was checked using a Nanodrop spectrophotometer (LabTech, USA) and RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies, USA). For library preparation, mRNA was enriched using the Dynabeads® mRNA purification kit (Invitrogen) and library preparation was performed using the Total Ion RNA-Seq v2 kit (Thermo Fisher Scientific). Sequencing was performed with the IonTorrent S5 sequencer (Thermo Fisher Scientific) using the Ion 540 TM Kit-OT2 and the Ion 540 TM Chip. Viral sequences were also searched for in the genome sequences. For Oxford Nanopore long-read sequencing, DNA from approximately 30,000 worms was extracted using the protocol described by Sassi et al. (2005). Sequencing was performed on a PromethION-Oxford Nanopore platform from GenOne, RJ, Brazil. The genome and RNAseq sequences were deposited in GenBank (BioProject PRJNA1037460; accession SAMN38172808 and the S_leucopus_picornavirus sequence was deposited with acession PP818844). For Illumina sequencing, genomic DNA was isolated from a pool of 100 individuals according to a previously described protocol (Oliveira et al 2009). A genomic DNA library was prepared from 200 ng of DNA using the Illumina TruSeq Nano DNA Kit. For sequencing, a MiSeq Reagent Kit v3 (600 cycles) was used to generate paired-end reads. Sequencing was performed using an Illumina MiSeq platform from Unidade de Genômica Computacional Darcy Fontoura de Almeida/LNCC/Brazil (BAM accession number: SAMN12500763). To analyse the presence of the picornavirus genome found by RNAseq analysis, RT-PCR was performed. RNA was isolated using Quick-Zol (Ludwig Biotecnologia/Brazil), quantified using a QuBit 3.0 fluorometer (Thermo Fisher Scientific) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer's recommendations. Two primer pairs were used: 1) F_Ls_pic_sit1: CACTGGTGCCAAAATCTG; R_Ls_pic_sit1: GTAGCTGGACGATACATAC; 2) F_Ls_pic_sit2: CGCACTTGGCATAGTACAC; R_Ls_pic_sit2: GATGTCAGGTTCGACTGAC; amplifying a 468bp and 386 bp fragment, respectively. Viral search on the assembled transcriptome was performed using a pipeline described by Silva et al (2023). Briefly, pairwise alignment using Blastp from DIAMOND (Buchfink et al., 2015) was performed using the assembled transcripts against the viral database from NCBI (protein database NCB--txid10239- using the E-Value of 1E-3 and using the more-sensitive parameter. After was removed false positives matches running a blast with the contigs that passed the threshold blasting it against the NCBI non-redundant protein bank. The sequences that came back as viral positive and among the top 5 searches were used for the next step. Only viral transcripts larger than 600 bp were used to obtain the taxonomy information and to carry out the phylogenetic reconstruction. The complete virus sequence and the RdRp amino acid sequence of the viruses were used for separate phylogenetic analysis together with the other Picornavirus found in Platyhelminthes described by Dheilly et al, (2022). To these sequences we also added some sequences showing high similarity to S. leucops virus found in the NR NCBI database. The accession number of the sequences used can be found at the Supplementary table 1). The sequences were aligned using Clustal W and ML phylogeny was reconstructed with IQ-Tree (Minh et al, 2020 ) using a substitution model: GTR+F, and 10000 bootstrap replicates. For estimates of Evolutionary Divergence between virus sequences, the number of base differences per site between sequences are conducted in MEGA X (Kumar et al, 2018) using gamma distribution. and bootstrap procedure (1000 replicates). Analysis of the transcriptome of S. leucops revealed the complete sequence of a novel RNA virus. A search for a similar sequence in the genome of S. leucops revealed that the viral sequence is not present in the genome, indicating that it is a bona fide RNA virus. Blast searches in the NCBI nr and virus databank showed that the most similar sequences correspond to polyprotein YP_009342320.1 [ Biomphalaria virus 2] and polyprotein AVA16916.1 [Pittsburgh sewage-associated virus 1], with an overall nucleotide divergence between these viruses of approximately 0.35 to 0.38, depending on the region of the virus (Table 1). These are (+) ssRNA viruses from the Picornaviridae family. The complete sequence of S. leucops p icornavirus has 8548 bp with three ORFs (Fig. 1). The first ORF encodes a polyprotein ranging from nucleotide 766 to 4425. This polyprotein corresponds to an RNA helicase, a peptidase C3 and a peptidase C3G. The second ORF extends from nucleotide 4401 to 7358 and encodes an RNA-dependent RNA polymerase ( RdRp ) and an rhv-like capsid protein. There is an overlap of 24 base pairs on the two ORFs. The third ORF expands from 4372 to 8412 nucleotides and encodes a CRPV capsid protein that belongs to a family of proteins found in positive stranded ssRNA. In addition, there is a 16 bp overlap between the second and third ORFs Since there is a five-year gap between the time when the RNA extraction for transcriptome analysis was performed and the virome analyses using this data, we decided to develop a PCR assay to perform RT-PCR analysis in the S. leucops culture to see if the virus is still found in the S. leucops colony and has been maintained over this time period. As can be seen in Figure 2, the amplicons were produced at the expected size. We can show that this virus has been maintained for at least 5 years in the S. leucops isolates cultured in the laboratory. Phylogenetic analyses were performed using the nucleotide sequence of the virus and the amino acid sequence of the viral gene RdRp , which is the most conserved sequence in these viral genomes. As can be seen in Figures 3 and 4, the topology of the trees is very similar. The sequences used gave rise to two main clades, which we refer to as clades A and B. Clade A includes several viruses present in flatworms and from other sources such as Pittsburgh sewage virus, Biophalaria virus and a water virus identified in a metagenomic analysis. S. leucops picornavirus_1 is grouped with these viruses from other sources. Group B includes mainly viruses found in Tricladida and also some viruses found in Trematoda. The sequences of Prostheceraeus (Policladida) and of Dugesia japonica and Procotyla (Tricladida) showed some differences in the positions of the phylogenetic analysis of nucleotides and proteins, sometimes outside the two main clades. Previous studies have shown that Picornaviridae are the most representative viruses in free-living flatworms (Dheilly et al, 2022). Stenostomum leucops is a free-living flatworm from the order Catenulida, a basal group of platyhelminthes (Telford et al., 2003; Wallberg et al., 2007). The new virus described here (Sleuc_Pic_1), which harbours S. leucops , also belongs to the Picornaviridae family. Although the picornaviruses of the Platyhelminthes apparently form a monophyletic group, the picornavirus of S. leucops described here belongs to a group with other picornaviruses that do not occur in flatworms but harbour Biomphalaria (Mollusca) and were found in metagenomic analyses (water and sewage). Since Catenulida is a cosmopolitan group (Larson et al. 2008) that lives in the aquatic environment, similar viruses found in the metagenomic analyses are likely to be from Catenulida. However, the presence of viruses of the same clade in Biomphalaria (Mollusca) may also indicate that this particular group of picornaviruses may be more widespread in limnic organisms. One point to emphasize is that in the genome of Sleuc_Pic_1 the ORFs show an overlap, resulting in three separate polyproteins CDSs. In general, picornaviruses have a single long ORF that encodes the polyproteins. However, there are cases where the polyproteins are encoded by multiple ORFs, similar to the virus we sequenced from S. leucops, for example, the picornavirus found in platyhelminths (accession numbers BK059711.1; BK059741.1; BK059754.1; and BK059682.1). High-throughput sequencing has significantly improved the discovery of new viruses and increased our knowledge of viromes. However, this approach little inform regarding the interactions between the virus and its hosts. S. leucops reproduces asexually, which theoretically allows the virus to be passed from cell to cell and maintained over generations. We have shown that Sleuc_Pic_1 persisted for at least five years in the worms of a laboratory colony and thus represents a persistent infection. We found no adverse effects on S. leucops associated with the presence of this virus. Similarly, Burrows et al. (2020) have shown that a double-stranded RNA (dsRNA) totivirus-like virus is maintained as a persistent infection in an asexual strain of Schmidtea mediterranea planaria that has a mutualistic or commensal relationship with its host. These examples raise the question of whether other asexual flatworms also maintain viruses with these ecological interactions. A limitation of our study is that we did not examine stressful conditions or other factors that may activate or overactivate Sleuc_Pic_1 virus to observe whether it can cause harmful infection in worms. However, these questions can be addressed in future studies. Declarations Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico/ CNPq/ Brazil and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ CAPES / Brazil. Author Contributions MTR was responsible for worm manipulation, preparation of the genome and transcriptome libraries and their assembly. All authors were responsible for designing the study, performing the search, extracting and analyzing the data, interpreting the results, updating the reference lists and writing the manuscript. Competing Interests The authors declare no competing interests. Data Availability The sequences were deposited in GenBank (NCBI), accession SAMN38172808 and SAMN12500763. Other dataset generated during the current study are available from the corresponding author on reasonable request. References Buchfink B, Xie C, Huson DH, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat. Methods 12, 59–60. https://doi.org/10.1038/nmeth.3176 Burrows JTA, Depierreux D, Nibert ML, Pearson BJ (2020) A Novel Taxon of Monosegmented Double-Stranded RNA Viruses Endemic to Triclad Flatworms. J Virol. 94(22):e00623-20. doi: 10.1128/JVI.00623-20. 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Silva AF, Machado LC, Silva LI1,Dezordi FZ, Wallau GL (2024) Highly divergent and diverse viral community infecting sylvatic mosquitoes from Northeast Brazil. bioRxiv preprint doi: https://doi.org/10.1101/2023.06.27.546706 Telford MJ, Lockyer AE, Cartwright-Finch C, Littlewood DTJ (2003) Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms. Proc. R. Soc. London B. 270, 1077–1083. doi: 10.1098/rspb.2003.2342. Wallberg A, Curini-Galletti M, Ahmadzadeh A, Jondelius U (2007) Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades. Zool. Scripta. 36, 509–523. doi.org/10.1111/j.1463-6409.2007.00295.x Wolf YI, Kazlauskas D, Iranzo J, Lucía-Sanz A, Kuhn JH, Krupovic M, Dolja VV, Koonin EV (2018) Origins and evolution of the global RNA virome. mBio 9:e02329-18. https://doi.org/10.1128/mBio.02329-18. Zhang P, Zhang Y, Cao L, Li J, Wu C, Tian M, Zhang Z, Zhang C, Zhang W, Li Y (2023) A Diverse Virome Is Identified in Parasitic Flatworms of Domestic Animals in Xinjiang, China. Microbiol Spectr.11(3):e0070223. doi: 10.1128/spectrum.00702-23. Table Table 1 - Estimates of evolutionary divergence between nucleotide sequences encoding three polyproteins of Stenostomum leucops Picornavirus 1; Biomphalaria virus 2 and Pittsburgh sewage-associated virus 1 Polyprotein_1 Polyprotein_2 Polyprotein_3 S. leucops virus X Pittsburgh virus 0.389 +/- 0.008 0.363 +/- 0.009 0.380 +/- 0.015 S.leucops virus X Biomphalaria virus 0.372 +/- 0.008 0.354 +/- 0.009 0.382 +/- 0.015 Pittsburgh virus X Biomphalaria virus 0.227 +/- 0.007 0.236 +/- 0.008 0.234 +/- 0.013 The number of base differences per site between sequences are shown followed by the standard error estimates obtained by a bootstrap procedure (1000 replicates). The rate variation among sites was modeled with a gamma distribution. There were a total of 3870 positions to polyprotein1, 2961 to polyprotein2 and 1075 to polyprotein3 in three dataset. Evolutionary analyses were conducted in MEGA X Supplementary Files GenBankaccessionPP818844.pdf GenBank.txt Cite Share Download PDF Status: Published Journal Publication published 15 Nov, 2024 Read the published version in Archives of Virology → Version 1 posted Reviewers agreed at journal 06 Jun, 2024 Reviewers invited by journal 22 May, 2024 First submitted to journal 21 May, 2024 Editorial decision: Major Revision 14 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4390948","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":305702479,"identity":"edb100eb-7ef9-483b-9d2d-ead7ee683ba7","order_by":0,"name":"Marcos Trindade Rosa","email":"","orcid":"","institution":"Universidade Federal de Santa Maria","correspondingAuthor":false,"prefix":"","firstName":"Marcos","middleName":"Trindade","lastName":"Rosa","suffix":""},{"id":305702480,"identity":"fb76cf6e-ce91-4d88-99cf-cbd65a92e991","order_by":1,"name":"Gabriel da Luz Wallau","email":"","orcid":"","institution":"Fundação Oswaldo Cruz: Fundacao Oswaldo Cruz","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"da Luz","lastName":"Wallau","suffix":""},{"id":305702481,"identity":"4f2b0495-1333-47a6-82ad-ccbffc3df5ed","order_by":2,"name":"Elgion L S Loreto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIie2SPwrCMBSHXym0yytZUxS9QjoreJUWQVdHB5FKoV3EUSyKxyiOLQFduji7VLxAewMj4p/F1FEwHySPhHy8X0gAFIpfxARIRWneV2MgYjbkin5XUAzNhxxs/xsFnooWfqEQ3Tym1Q6QNPihqLactpepcRlLFDvAURbngPZi4M3ihFN2ck0nlyiMo8utEJDl6ARWwqes4Rq3dB/pvRRSBdZGBFvXKEw300cXLbB8TuFUo1COkMUhRXtuOPFqPxR38SJHppAoupRV2G0R1IuynHREsP7+LFPEi7Bbs/cdTS6IH1PUHFAoFIq/5wq0oUi31kxhgQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7586-8168","institution":"Universidade Federal de Santa Maria","correspondingAuthor":true,"prefix":"","firstName":"Elgion","middleName":"L S","lastName":"Loreto","suffix":""}],"badges":[],"createdAt":"2024-05-08 17:54:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4390948/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4390948/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-024-06175-4","type":"published","date":"2024-11-15T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57714133,"identity":"8dd43ce3-3c9e-4f61-8f7f-a2d5f4d1130e","added_by":"auto","created_at":"2024-06-04 16:31:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) Organization of the genome of Stenostomum leucops picornavirus 1.\u003c/strong\u003eThe ORFs are indicated with the nucleotide position for the beginning and end of each ORF. ORF2 overlaps with ORF1 and ORF3. The size of the UTRs regions are indicated. B) The polyprotein domains encoded by the ORFs, with the start and end of each protein indicated.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4390948/v1/fa460a6c0c26f359349feda7.jpg"},{"id":57714137,"identity":"28a9e88b-ccae-421e-8fdf-68ac209fb0e2","added_by":"auto","created_at":"2024-06-04 16:31:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":271937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRT-PCR for S_leuc_Pv1. \u003c/strong\u003eLd= DNA Kb ladder; 1) Negative control for ORF1 amplification; 2) ORF1 amplification (386 pb); 3) Negative control for ORF2 amplification; 4) ORF2 amplification (468 pb);\u003c/p\u003e\n\u003cp\u003e5) Negative control for tubuline transcript; 2) Tubuline transcript amplification (187 pb). In the negative control, the cDNA was not included in the PCR reaction. The size of some ladders is indicated on the right.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4390948/v1/ab5fa8ec86fec9bf781306a9.jpg"},{"id":57714136,"identity":"349c011b-62f8-4cea-9d12-0239aa7041c6","added_by":"auto","created_at":"2024-06-04 16:31:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":326259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum likelihood phylogeny reconstructed from the nucleotide sequence of the viral genome.\u003c/strong\u003e The phylogeny was done with IQ-Tree using the substitution model: GTR+F, and bootstrap with 10,000 replicates. The number in the OTUs is the GenBank accession number, followed by genus name and order. The numbers in the branches are bootstrap values.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4390948/v1/04a912c003597b149809c94b.jpg"},{"id":57714135,"identity":"f44ca8ee-6403-44f0-9d72-2b5cde0bf2f6","added_by":"auto","created_at":"2024-06-04 16:31:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":412413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum likelihood phylogeny using the amino acid sequence of the viral gene RdRp. \u003c/strong\u003eThe phylogeny was done using IQ-Tree using the substitution model: GTR+F, and bootstrap with 10,000 replicates. The number in the OTUs is the GenBank accession number, followed by genus name and order. The blue circle represents the bootstrap values.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4390948/v1/a3b7d4252a1232085d10564b.jpg"},{"id":69274983,"identity":"6e35da35-fbae-4842-a2ef-867d53682c44","added_by":"auto","created_at":"2024-11-18 16:42:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1589897,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4390948/v1/cd868611-ffc3-4e09-a821-5590c506ad03.pdf"},{"id":57714138,"identity":"4a4cc057-e3e5-4c99-88ad-9431b568562b","added_by":"auto","created_at":"2024-06-04 16:31:28","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":64082,"visible":true,"origin":"","legend":"","description":"","filename":"GenBankaccessionPP818844.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4390948/v1/cea8b79f12c43cfcf3be7113.pdf"},{"id":57714134,"identity":"540dd9c5-a703-4b9d-9dcc-7b528832b78c","added_by":"auto","created_at":"2024-06-04 16:31:27","extension":"txt","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":19023,"visible":true,"origin":"","legend":"","description":"","filename":"GenBank.txt","url":"https://assets-eu.researchsquare.com/files/rs-4390948/v1/cd63730dbceee032ba2bb705.txt"}],"financialInterests":"","formattedTitle":"Persistent infection of a novel Picornavirus in the microplanarian Stenostomum leucops (Catenulida).","fulltext":[{"header":"Full Text","content":"\u003cp\u003eViruses are the most abundant and diverse biological entities on Earth and have been found in virtually all host species studied to date, although understanding of the virosphere is still very limited (Paez-Espino et al. 2016; Shi et al. 2016). Characterizing and classifying this enormous diversity has been a difficult task. Originally, this task was limited to viruses that could be isolated in culture cells and induce cell death or alterations. In recent decades, however, high-throughput sequencing technologies have enabled an enormous expansion of knowledge about the viral component of the microbiome, the so-called viromes, which are based on the high-resolution viral sequences recovery (Wolf et al., 2018).\u003c/p\u003e\n\u003cp\u003eDespite increasing efforts to characterize the virome of different organisms and environments, our current understanding is biased for chordates, arthropods and plants. Platyhelminthes, a phylum composed of free-living and parasitic flatworms, is a poorly studied group in terms of its virome, even though several studies have described viruses on certain species of planarians, \u003cem\u003eSchistosoma\u003c/em\u003e and \u003cem\u003eTaenia\u003c/em\u003e (Jones MK and Whittington 1992; Justine and Bonami 1993; Noury-Sra\u0026iuml;ria et al 1995; Crespo-Gonz\u0026aacute;lez et al 2008; Rebrikov et al, 2002). More recently, comprehensive studies analyzing platyhelminth genomes produced by NSG have found more than a hundred viruses infecting 50 species of free-living and 65 species of parasitic flatworms, including viruses belonging to the Picornavirales, Jingchuvirales, Maretellivirales, Flaviviridae, Nyamiviridae, Ghabrivirales, Rhabdoviridae and Bunyavirales families (Dheilly et al, 2022; Zhang et al 2023).\u003c/p\u003e\n\u003cp\u003eTo our knowledge, no studies have been conducted to characterize viruses of the Catenulida, which are a more basal group of platyhelminthes (Telford et al., 2003; Wallberg et al., 2007). Catenulida are small free-living flatworms ranging in size from 0.5 to 2.5 mm that reproduce asexually by paratomy, a process in which the anterior body structures form in the mid-body regions, resulting in segmented organisms (Rosa et al., 2015). We have maintained a clonal culture of \u003cem\u003eS. leucops\u003c/em\u003e (Catenulida) for more than a decade. This strain has a flexible relationship with its microbiome that may be associated with phenotypic plasticity (Rosa and Loreto 2023) and is capable of expressing genes found in its microbiome or eDNA (Rosa and Loreto 2019).\u003c/p\u003e\n\u003cp\u003eIn the present study, we characterized a complete genome of a (+) ssRNA virus from the Picornaviridae family that is maintained for at least five years in a \u003cem\u003eS. leucops\u003c/em\u003e colony, suggesting persistent and long-term infection. Phylogenetic analysis revealed that this virus is a novel virus closely related to the virus found in \u003cem\u003eBiomphalaria\u003c/em\u003e. Based on the long-term association of this virus with \u003cem\u003eS. leucops\u0026nbsp;\u003c/em\u003eand the apparent lack of deleterious host effects, it is likely that this virus and its host engage in a mutualistic or commensalistic interaction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStenostomum leucops\u0026nbsp;\u003c/em\u003especimens were collected in 2009 in a pond in Santa Maria, Brazil (53\u0026deg;17\u0026prime;W; 29\u0026deg;28\u0026prime;S). An isoline was first established and the worms\u0026nbsp;have been kept in reconstituted water at\u0026nbsp;28\u0026deg;C and fed with milk powder, following the details described by Rosa et al. (2015).\u003c/p\u003e\n\u003cp\u003eTo gain deeper insights into this non-model species, we performed a transcriptomic characterization. Total RNA was isolated using Trizol (Invitrogen\u0026reg;) according to the manufacturer\u0026apos;s guidelines. RNA quality was checked using a Nanodrop spectrophotometer (LabTech, USA) and RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies, USA). For library preparation, mRNA was enriched using the Dynabeads\u0026reg; mRNA purification kit (Invitrogen) and library preparation was performed using the Total Ion RNA-Seq v2 kit (Thermo Fisher Scientific). Sequencing was performed with the IonTorrent S5 sequencer (Thermo Fisher Scientific) using the Ion 540 TM Kit-OT2 and the Ion 540 TM Chip.\u003c/p\u003e\n\u003cp\u003eViral sequences were also searched for in the genome sequences. For Oxford Nanopore long-read sequencing, DNA from approximately 30,000 worms was extracted using the protocol described by Sassi et al. (2005). Sequencing was performed on a PromethION-Oxford Nanopore platform from GenOne, RJ, Brazil. The genome and RNAseq sequences were deposited in GenBank (BioProject PRJNA1037460; accession SAMN38172808 and the \u003cem\u003eS_leucopus_picornavirus\u003c/em\u003e sequence was deposited with acession PP818844).\u003c/p\u003e\n\u003cp\u003eFor Illumina sequencing, genomic DNA was isolated from a pool of 100 individuals according to a previously described protocol (Oliveira et al 2009). A genomic DNA library was prepared from 200 ng of DNA using the Illumina TruSeq Nano DNA Kit. For sequencing, a MiSeq Reagent Kit v3 (600 cycles) was used to generate paired-end reads. Sequencing was performed using an Illumina MiSeq platform from Unidade de Gen\u0026ocirc;mica Computacional Darcy Fontoura de Almeida/LNCC/Brazil (BAM accession number: SAMN12500763).\u003c/p\u003e\n\u003cp\u003eTo analyse the presence of the picornavirus genome found by RNAseq analysis, RT-PCR was performed. RNA was isolated using Quick-Zol (Ludwig Biotecnologia/Brazil), quantified using a QuBit 3.0 fluorometer (Thermo Fisher Scientific) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer\u0026apos;s recommendations. Two primer pairs were used: 1) F_Ls_pic_sit1: CACTGGTGCCAAAATCTG; R_Ls_pic_sit1: GTAGCTGGACGATACATAC; 2) F_Ls_pic_sit2: CGCACTTGGCATAGTACAC; R_Ls_pic_sit2: GATGTCAGGTTCGACTGAC; amplifying a 468bp and 386 bp fragment, respectively.\u003c/p\u003e\n\u003cp\u003eViral search on the assembled transcriptome was performed using a pipeline described by Silva et al (2023). Briefly, pairwise alignment using Blastp from DIAMOND (Buchfink et al., 2015) was performed using the assembled transcripts against the viral database from NCBI \u0026nbsp;(protein database NCB--txid10239- using the E-Value of 1E-3 and using the more-sensitive parameter. After was removed false positives matches running a blast with the contigs that passed the threshold blasting it against the NCBI non-redundant protein bank. The sequences that came back as viral positive and among the top 5 searches were used for the next step. \u0026nbsp; Only viral transcripts larger than 600 bp were used to obtain the taxonomy information and to carry out the phylogenetic reconstruction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe complete virus sequence and the RdRp amino acid sequence of the viruses were used for separate phylogenetic analysis together with the other Picornavirus found in Platyhelminthes described by Dheilly et al, (2022). To these sequences we also added some sequences showing high similarity to \u003cem\u003eS. leucops\u003c/em\u003e virus found in the NR NCBI database. The accession number of the sequences used \u0026nbsp;can be found at the Supplementary table 1). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sequences were aligned using Clustal W and ML phylogeny was reconstructed with IQ-Tree (Minh et al, 2020 ) using a substitution model: GTR+F, and 10000 bootstrap replicates.\u003c/p\u003e\n\u003cp\u003eFor estimates of Evolutionary Divergence \u0026nbsp;between virus sequences, \u0026nbsp;the number of base differences per site between sequences are conducted in MEGA X (Kumar et al, 2018) using gamma distribution. and bootstrap procedure (1000 replicates).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Analysis of the transcriptome of \u003cem\u003eS. leucops\u003c/em\u003e revealed the complete sequence of a novel RNA virus. A search for a similar sequence in the genome of \u003cem\u003eS. leucops\u003c/em\u003e revealed that the viral sequence is not present in the genome, indicating that it is a \u003cem\u003ebona fide\u003c/em\u003e RNA virus. Blast searches in the NCBI nr and virus databank showed that the most similar sequences correspond to polyprotein YP_009342320.1 [\u003cem\u003eBiomphalaria\u003c/em\u003e virus 2] and polyprotein AVA16916.1 [Pittsburgh sewage-associated virus 1], with an overall nucleotide divergence between these viruses of approximately \u0026nbsp; 0.35 to 0.38, depending on the region of the virus (Table 1). These are (+) ssRNA viruses from the Picornaviridae family.\u003c/p\u003e\n\u003cp\u003eThe complete sequence of \u003cem\u003eS. leucops p\u003c/em\u003eicornavirus has 8548 bp with three ORFs (Fig. 1). The first ORF encodes a polyprotein ranging from nucleotide 766 to 4425. This polyprotein corresponds to an RNA helicase, a peptidase C3 and a peptidase C3G. The second ORF extends from nucleotide 4401 to 7358 and encodes an RNA-dependent RNA polymerase (\u003cem\u003eRdRp\u003c/em\u003e) and an rhv-like capsid protein. There is an overlap of 24 base pairs on the two ORFs. The third ORF expands from 4372 to 8412 nucleotides and encodes a CRPV capsid protein that belongs to a family of proteins found in positive stranded ssRNA. In addition, there is a 16 bp overlap between the second and third ORFs\u003c/p\u003e\n\u003cp\u003eSince there is a five-year gap between the time when the RNA extraction for transcriptome analysis was performed and the virome analyses using this data, we decided to develop a PCR assay to perform RT-PCR analysis in the \u003cem\u003eS. leucops\u003c/em\u003e culture to see if the virus is still found in the \u003cem\u003eS. leucops\u0026nbsp;\u003c/em\u003ecolony and has been maintained over this time period. As can be seen in Figure 2, the amplicons were produced at the expected size. We can show that this virus has been maintained for at least 5 years in the \u003cem\u003eS. leucops\u0026nbsp;\u003c/em\u003eisolates cultured in the laboratory.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analyses were performed using the nucleotide sequence of the virus and the amino acid sequence of the viral gene \u003cem\u003eRdRp\u003c/em\u003e, which is the most conserved sequence in these viral genomes. As can be seen in Figures 3 and 4, the topology of the trees is very similar. The sequences used gave rise to two main clades, which we refer to as clades A and B. Clade A includes several viruses present in flatworms and from other sources such as Pittsburgh sewage virus, \u003cem\u003eBiophalaria\u003c/em\u003e virus and a water virus identified in a metagenomic analysis. \u003cem\u003eS. leucops\u003c/em\u003e picornavirus_1 is grouped with these viruses from other sources. Group B includes mainly viruses found in Tricladida and also some viruses found in Trematoda. The sequences of \u003cem\u003eProstheceraeus\u003c/em\u003e (Policladida) and of \u003cem\u003eDugesia japonica\u003c/em\u003e and \u003cem\u003eProcotyla\u003c/em\u003e (Tricladida) showed some differences in the positions of the phylogenetic analysis of nucleotides and proteins, sometimes outside the two main clades.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that Picornaviridae are the most representative viruses in free-living flatworms (Dheilly et al, 2022). \u003cem\u003eStenostomum leucops\u003c/em\u003e is a free-living flatworm from the order Catenulida, a basal group of platyhelminthes (Telford et al., 2003; Wallberg et al., 2007). The new virus described here (Sleuc_Pic_1), which harbours \u003cem\u003eS. leucops\u003c/em\u003e, also belongs to the Picornaviridae family. Although the picornaviruses of the Platyhelminthes apparently form a monophyletic group, the picornavirus of\u003cem\u003e\u0026nbsp;S. leucops\u0026nbsp;\u003c/em\u003edescribed here belongs to a group with other picornaviruses that do not occur in flatworms but harbour \u003cem\u003eBiomphalaria\u003c/em\u003e (Mollusca) and were found in metagenomic analyses (water and sewage). Since Catenulida is a cosmopolitan group (Larson et al. 2008) that lives in the aquatic environment, similar viruses found in the metagenomic analyses are likely to be from Catenulida. However, the presence of viruses of the same clade in \u003cem\u003eBiomphalaria\u003c/em\u003e (Mollusca) may also indicate that this particular group of picornaviruses may be more widespread in limnic organisms.\u003c/p\u003e\n\u003cp\u003eOne point to emphasize is that in the genome of Sleuc_Pic_1 the ORFs show an overlap, resulting in three separate polyproteins CDSs. In general, picornaviruses have a single long ORF that encodes the polyproteins. However, there are cases where the polyproteins are encoded by multiple ORFs, similar to the virus we sequenced from \u003cem\u003eS. leucops,\u0026nbsp;\u003c/em\u003efor example, the picornavirus found in platyhelminths (accession numbers BK059711.1; BK059741.1; BK059754.1; and BK059682.1).\u003c/p\u003e\n\u003cp\u003eHigh-throughput sequencing has significantly improved the discovery of new viruses and increased our knowledge of viromes. However, this approach little inform regarding the interactions between the virus and its hosts. \u003cem\u003eS. leucops\u003c/em\u003e reproduces asexually, which theoretically allows the virus to be passed from cell to cell and maintained over generations. We have shown that Sleuc_Pic_1 persisted for at least five years in the worms of a laboratory colony and thus represents a persistent infection. We found no adverse effects on \u003cem\u003eS. leucops\u003c/em\u003e associated with the presence of this virus. Similarly, Burrows et al. (2020) have shown that a double-stranded RNA (dsRNA) totivirus-like virus is maintained as a persistent infection in an asexual strain of \u003cem\u003eSchmidtea mediterranea\u003c/em\u003e planaria that has a mutualistic or commensal relationship with its host. These examples raise the question of whether other asexual flatworms also maintain viruses with these ecological interactions. A limitation of our study is that we did not examine stressful conditions or other factors that may activate or overactivate Sleuc_Pic_1 virus to observe whether it can cause harmful infection in worms. However, these questions can be addressed in future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis work was supported by\u003c/em\u003e\u0026nbsp; Conselho Nacional de Desenvolvimento Científico e Tecnológico/ CNPq/ Brazil and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ CAPES / Brazil.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTR was responsible for worm manipulation, preparation of the genome and transcriptome libraries and their assembly. All authors were responsible for designing the study, performing the search, extracting and analyzing the data, interpreting the results, updating the reference lists and writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch4\u003eData Availability\u003c/h4\u003e\n\u003cp\u003eThe sequences were deposited in GenBank (NCBI), accession SAMN38172808 and \u0026nbsp;SAMN12500763. Other dataset generated during the current study are available from the corresponding author on reasonable \u0026nbsp;request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBuchfink B, Xie C, Huson DH, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat. 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Genetics and Molecular Biology, 28(4), 734\u0026ndash;739.doi.org/10.1590/S1415-47572005000500013\u003c/li\u003e\n\u003cli\u003eShi M, Lin X-D, Tian J-H, Chen L-J, Chen X, Li C-X, Qin X-C, Li J, Cao J-P, Eden J-S, Buchmann J, Wang W, Xu J, Holmes EC, Zhang Y-Z (2016) Redefining the invertebrate RNA virosphere. Nature 540:539\u0026ndash;543. https://doi.org/10.1038/nature20167.\u003c/li\u003e\n\u003cli\u003eSilva AF, Machado LC, Silva LI1,Dezordi FZ, Wallau GL (2024) Highly divergent and diverse viral community infecting sylvatic mosquitoes from Northeast Brazil. bioRxiv preprint doi: https://doi.org/10.1101/2023.06.27.546706 \u003c/li\u003e\n\u003cli\u003eTelford MJ, Lockyer AE, Cartwright-Finch C, Littlewood DTJ (2003) Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms. Proc. R. Soc. London B. 270, 1077\u0026ndash;1083. doi: 10.1098/rspb.2003.2342.\u003c/li\u003e\n\u003cli\u003eWallberg A, Curini-Galletti M, Ahmadzadeh A, Jondelius U (2007) Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades. Zool. Scripta. 36, 509\u0026ndash;523. doi.org/10.1111/j.1463-6409.2007.00295.x\u003c/li\u003e\n\u003cli\u003eWolf YI, Kazlauskas D, Iranzo J, Luc\u0026iacute;a-Sanz A, Kuhn JH, Krupovic M, Dolja VV, Koonin EV (2018) Origins and evolution of the global RNA virome. mBio 9:e02329-18. https://doi.org/10.1128/mBio.02329-18.\u003c/li\u003e\n\u003cli\u003eZhang P, Zhang Y, Cao L, Li J, Wu C, Tian M, Zhang Z, Zhang C, Zhang W, Li Y (2023) A Diverse Virome Is Identified in Parasitic Flatworms of Domestic Animals in Xinjiang, China. Microbiol Spectr.11(3):e0070223. doi: 10.1128/spectrum.00702-23. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1 - Estimates of evolutionary divergence between nucleotide sequences encoding three polyproteins of \u003cem\u003eStenostomum leucops\u003c/em\u003e Picornavirus 1; \u003cem\u003eBiomphalaria\u003c/em\u003e virus 2 and Pittsburgh sewage-associated virus 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePolyprotein_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePolyprotein_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePolyprotein_3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. leucops\u003c/em\u003e virus X Pittsburgh virus\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.389 +/- 0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.363 +/- 0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.380 +/- 0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS.leucops\u0026nbsp;\u003c/em\u003e virus X \u003cem\u003eBiomphalaria\u003c/em\u003e virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.372 +/- 0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.354 +/- 0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.382 +/- 0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePittsburgh \u0026nbsp;virus X \u003cem\u003eBiomphalaria\u0026nbsp;\u003c/em\u003evirus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.227 +/- 0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.236 +/- 0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.234 +/- 0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe number of base differences per site \u0026nbsp;between sequences are shown followed by the standard error estimates obtained by a bootstrap procedure (1000 replicates). The rate variation among sites was modeled with a gamma distribution. There were a total of 3870 positions to polyprotein1, 2961 to polyprotein2 and 1075 to polyprotein3 in three dataset. Evolutionary analyses were conducted in MEGA X\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Picornavirus, flatworm virus, ssRNA virus, RdRP gene","lastPublishedDoi":"10.21203/rs.3.rs-4390948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4390948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe present the genome sequence, organization and evidence of persistence of a new picornavirus infecting the flatworm \u003cem\u003eStenostomum leucops\u003c/em\u003e. The complete genome sequence belongs to a virus with a positive single-stranded RNA genome encoding three open reading frames (ORFs) flanked by untranslated regions and polyadenylated termination. The ORFs encode conserved protein motifs typical of the picornavirus superfamily. Phylogenetic analyses confirm membership of this viral superfamily, with closely related viral species found in \u003cem\u003eBiomphalaria\u003c/em\u003e(Mollusca) in France and a virus detected in metagenomic analyses of water sources from the USA, suggesting widespread distribution. RT-PCR analysis revealed that this virus persists in a laboratory-grown worm isolate for at least five years, indicating persistent infection. However, no deleterious effects were observed in the infected worms, suggesting a commensalistic or mutualistic relationship between the virus and the worms.\u003c/p\u003e","manuscriptTitle":"Persistent infection of a novel Picornavirus in the microplanarian Stenostomum leucops (Catenulida).","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-04 16:31:23","doi":"10.21203/rs.3.rs-4390948/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-06-07T00:59:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-23T01:23:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2024-05-21T14:59:28+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2024-05-14T05:04:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"885df45e-eb32-4b32-a0cb-58839912b702","owner":[],"postedDate":"June 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T16:01:35+00:00","versionOfRecord":{"articleIdentity":"rs-4390948","link":"https://doi.org/10.1007/s00705-024-06175-4","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2024-11-15 15:57:21","publishedOnDateReadable":"November 15th, 2024"},"versionCreatedAt":"2024-06-04 16:31:23","video":"","vorDoi":"10.1007/s00705-024-06175-4","vorDoiUrl":"https://doi.org/10.1007/s00705-024-06175-4","workflowStages":[]},"version":"v1","identity":"rs-4390948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4390948","identity":"rs-4390948","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}