A novel narnavirus isolated from the edible fungus Pholiota nameko

A novel narnavirus isolated from the edible fungus Pholiota nameko | 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 A novel narnavirus isolated from the edible fungus Pholiota nameko Yingying Liu, Xiayang Zhou, Yang Xu, Zhe Wang, Huaping Li, Pengfei Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7248914/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract A novel positive single-stranded RNA virus, tentatively named “Pholiota nameko narnavirus 1” (PnNV1), has been identified in an edible fungus Pholiota nameko strain N1. The complete genome of PnNV1 is 2254 nucleotides in length, containing a single large open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp) of 682 amino acids (aa) with a molecular mass of 77.81 kDa. blastp analysis indicated that PnNV1 exhibited the highest similarity (30.80%) to Rhizoctonia solani narnavirus 18. Sequence alignments and phylogenetic analysis based on RdRp sequences indicate that PnNV1 can be classified as a new member of the family Narnaviridae . To our knowledge, this is the first mycovirus reported in the edible fungus Pholiota nameko . mycovirus Narnaviridae Pleurotus nameko narnavirus PnNV1 Figures Figure 1 Figure 2 Introduction Mycoviruses (fungal viruses) are viruses that infect fungi and are capable of replicating and multiplying within their hosts [ 1 ]. Mycoviruses are ubiquitous across nearly all fungal kingdoms [ 2 ]. In 1962, the first fungal virus was discovered in an abnormally growing edible fungus Agaricus bisporus by Hollings, marking the beginning of fungal virology research [ 3 ]. With the advent of next-generation sequencing (NGS) technologies, an increasing number of mycoviruses have been identified [ 4 , 5 ]. Studies have shown that most mycoviruses do not cause significant external symptoms in their hosts [ 6 , 7 ]. However, a small number of mycoviruses can attenuate the pathogenicity of their hosts, offering potential for biocontrol applications [ 5 , 8 ]. In edible fungi, mycoviruses often cause severe diseases with symptoms including delayed fruiting body formation, deformation of fruiting bodies, shortened stipes, thin mushroom caps, mycelium degeneration, and reduced yields [ 9 , 10 ]. Currently, over 80 mycoviruses have been identified in edible fungi, with over 31 having complete genome sequences. To date, only RNA genome types have been identified in edible fungi. Mycoviruses in edible fungi with dsRNA genomes are found in the families Partitiviridae and Totiviridae ; those with (+ ss) RNA genomes are found in eight families: Barnaviridae , Botourmiaviridae , Deltaflexiviridae , Endornaviridae , Fusariviridae , Hypoviridae , Narnaviridae and T ymoviridae ; and those with (-)ssRNA genomes are found in the families Mymonaviridae and Phenuiviridae [ 11 ]. To date, edible fungal viruses have been report in more than 24 species, including Agaricus bisporus [ 12 ], Agrocybe aegerita [ 13 ], Armillaria species[ 14 ], Auricularia heimuer [ 15 ], Boletus edulis [ 16 ], Bondarzewia berkeleyi [ 17 ], Clitocybe odora [ 18 ], Cordyceps chanhua [ 19 ], Flammulina velutipes [ 20 ], Grifola frondosa [ 21 ], Gyromitra esculenta [ 22 ], Hebeloma mesophaeum [ 23 ], Lactarius tabidus [ 24 ], Lentinula edodes [ 25 ], Leucocybe candicans [ 26 ], Morchella esculenta [ 27 ], Neurospora intermedia [ 28 ], Picoa juniperi [ 29 ], Pleurotus citrinopileatus [ 10 ], Pleurotus eryngii [ 30 ], Pleurotus ostreatus [ 31 ], Tuber aestivum [ 32 ], Ustilago maydis [ 33 ], Volvariella volvace [ 34 ], However, to our knowledge, no fungal viruses have been reported in Pholiota nameko . Pholiota nameko , also known as the phosphate umbrella, is an edible mushroom with medicinal properties[ 35 ]. It is widely cultivated in China for food and traditional medicine [ 36 ]. Rich in protein, carbohydrates, fiber, vitamins, and unsaturated fatty acids, P. nameko exhibits various biological activities, including anti-inflammatory, anti-hyperlipidemia, and antitumor effects [ 36 , 37 ]. However, the impact of viral infections on the quality and yield of this fungus has not yet been investigated. Members of the viral family Narnaviridae have the simplest viral genome structure [ 38 , 39 ]. They lack a protein capsid and are not associated with virus particles other than lipid vesicles. Their genome contains a single large ORF encoding only an RNA-dependent RNA polymerase (RdRp) to direct their replication [ 38 , 39 ]. Narnaviruses are widely distributed in fungi, plants, oomycetes, mosquitoes, arthropods, algae, trypanosomatids, and potentially apicomplexans [ 8 , 40 , 41 ].Only two species have been approved by the ICTV in this family: Saccharomyces 20S RNA narnavirus and Saccharomyces 23S RNA narnavirus [ 42 ]. Both viruses have small genomes and possess short terminal inverted repeats (5’-GGGGC and GCCCC-3’) [ 39 ]. In this study, we discovered a novel + ssRNA virus with the proposed name “Pholiota nameko narnavirus 1” (PnNV1) in Pholiota nameko strain N1. Sequence alignments and phylogenetic analysis showed that PnNV1 can be classified as a new member of the family Narnaviridae . Materials and methods The Pholiota nameko strain N1 was obtained from the Shouguang Institute of Edible Fungi in Shandong Province, China. The strain was cultured on Potato Dextrose Agar (PDA) medium at 25°C for 7 days for dsRNA multiplication and sent to Shanghai Biotechnology Corporation for high-throughput sequencing. The sequencing data indicated the presence of a fungal virus, likely a member of the family Narnaviridae . dsRNA was extracted from fungal mycelium using the CF-11 cellulose chromatography method[ 43 ]. The dsRNA was then purified with DNase I and S1 nuclease (TaKaRa Dalian, China) to remove contaminating DNA and single-stranded RNA (ssRNA)[ 44 ]. The treated dsRNA was electrophoresed in a 1% (w/v) agarose gel. Although no bands were visible in the electrophoresis analysis, the presence of the virus was detected using virus-specific primers. The full-length cDNA sequence of the virus was cloned primarily using the ligase-mediated rapid amplification of cDNA ends (RLM-RACE) protocol described by Xie et al[ 45 ]. The complete sequence of PnNV1 was deposited in the GenBank database under the accession number OR822278. Potential RNA secondary structures of the 5' and 3' untranslated regions (UTR) of this viral genome were predicted using the online tool available at http://www.unafold.org/mfold/applications/rna-folding-form-v2.php . Protein domains searches were performed using the Conserved Domain Database (CDD) ( https://www.ncbi.nlm.nih.gov/cdd ). Multiple sequence alignment of protein sequences was conducted using DNAMAN software (Version 9). The ORF of PnNV1 was identified and translated using DNAMAN software (Version 9). Phylogenetic trees were generated using the maximum likelihood method in Molecular Evolutionary Genetics Analysis (MEGA) version 10.1.8, with 1,000 bootstrap replicates[ 46 ]. Sequence properties The complete genome of PnNV1 is 2254 nucleotides in length, containing a single large ORF encoding a 682-amino-acid (aa) protein on its positive strand, with a molecular mass of 77.81 kDa (Fig. 1 ). The lengths of the 5' and 3' UTR were 50 and 152 nucleotides, with ΔG values of -18.34 kcal/mol and − 31.35 kcal/mol (Fig. S1 ), respectively. Secondary structure predictions of the 5' and 3' UTRs revealed that both contain stem-loop structures. BLASTp analysis of the RNA-dependent RNA polymerases (RdRps) showed significant similarity to Rhizoctonia solani narnavirus 18. We selected three RdRp sequences with high accuracy and similarity, along with representative species of the genus Narnavirus for the prediction of conserved motifs and multiple sequence comparisons. The results identified a total of six conserved motifs, with their exact locations shown in Fig. S2. The abbreviation of virus names, GenBank accession numbers, identity, and query coverage are as follows: Fusarium poae narnavirus 1(YP_009272902.1, 21.9% identity, 68% query coverage); Plasmopara viticola lesion associated narnavirus 18(QIR30297.1, 27.07% identity, 80% query coverage); Puccinia striiformis narnavirus 1 (QJQ50000.1, 27.26% identity, 73% query coverage); Saccharomyces 20S RNA narnavirus (NP_660178.1); Saccharomyces 23S RNA narnavirus(NP_660177.1). A molecular phylogenetic tree was constructed based on the RdRp amino acid sequences of PnNV1, other selected viruses with high similarity, and several representative members of the family Narnaviridae and Mitoviridae . The maximum likelihood tree indicates that PnNV1 can be classified as a new member of the family Narnaviridae , forming a distinct clade with Plasmopara viticola lesion associated narnavirus 18 as a sister branch of narnaviruses (Fig. 2 ). To our knowledge, a variety of different genome types of edible fungal viruses have been identified in several edible fungi species, except Pholiota nameko . A small number of viruses have been shown to have significant effects on the growth and yield of edible fungi. As an edible fungus widely cultivated in China with medicinal value, it is necessary to excavate the mycovirus resources in the Pholiota nameko and conduct in-depth research. Declarations Funding This research was supported by the National Natural Science Foundation of China (32202381), Guangdong Basic and Applied Basic Research Foundation (2024A1515010080), and Guangzhou Science and Technology Planning Project (SL2022A04J01161). Compliance with ethical standards Conflict of interest All authors declare that they have no conflicts of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. References Xie J, Jiang D (2014) New Insights into Mycoviruses and Exploration for the Biological Control of Crop Fungal Diseases. Annual Review of Phytopathology 52:45-68 Hough B, Steenkamp E, Wingfield B, Read D (2023) Fungal Viruses Unveiled: A Comprehensive Review of Mycoviruses. Viruses 15 Hollings M (1964) Viruses Associated with A Die-Back Disease of Cultivated Mushroom. Nature Kondo H, Botella L, Suzuki N (2022) Mycovirus Diversity and Evolution Revealed/Inferred from Recent Studies. Annual Review of Phytopathology 60:307-336 Wang S, Zhang J, Nzabanita C, Zhang M, Nie J, Guo L (2022) Fungal Virus, FgHV1-Encoded p20 Suppresses RNA Silencing through Single-Strand Small RNA Binding. 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Molecular Biology and Evolution 35:1547-1549 Supplementary Files SI.docx Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Archives of Virology → Version 1 posted Reviewers agreed at journal 11 Aug, 2025 Reviewers invited by journal 11 Aug, 2025 Editor assigned by journal 04 Aug, 2025 First submitted to journal 03 Aug, 2025 Editorial decision: Major Revision 01 Aug, 2025 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-7248914","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498693101,"identity":"e66cb946-d5b6-49ba-be38-d5b3a0ddad2e","order_by":0,"name":"Yingying Liu","email":"","orcid":"","institution":"South China Agricultural University College of Plant Protection","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Liu","suffix":""},{"id":498693102,"identity":"7919f21d-00c5-459d-b639-912b29c03306","order_by":1,"name":"Xiayang Zhou","email":"","orcid":"","institution":"South China Agricultural University College of Plant Protection","correspondingAuthor":false,"prefix":"","firstName":"Xiayang","middleName":"","lastName":"Zhou","suffix":""},{"id":498693103,"identity":"e6412aba-44a2-4a47-9562-61b1b0a72fe3","order_by":2,"name":"Yang Xu","email":"","orcid":"","institution":"South China Agricultural University College of Plant Protection","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Xu","suffix":""},{"id":498693104,"identity":"c49878dd-8bd0-4d9f-865c-d2c1859dab7e","order_by":3,"name":"Zhe Wang","email":"","orcid":"","institution":"South China Agricultural University College of Plant Protection","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Wang","suffix":""},{"id":498693105,"identity":"92ce377d-0656-49bd-89a0-063032611902","order_by":4,"name":"Huaping Li","email":"","orcid":"","institution":"South China Agricultural University College of Plant Protection","correspondingAuthor":false,"prefix":"","firstName":"Huaping","middleName":"","lastName":"Li","suffix":""},{"id":498693106,"identity":"8f061a87-8890-4778-bb9e-2c2d0fc389e2","order_by":5,"name":"Pengfei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACPmYwJcfAxt4AFTpAQAsbRIsxAxsPTClBLQxQLQwSCcRqYec9+Ljgl4Ecn+Tzhx9+tjHI8d1IYPxcgNdhfMnGM/sMjNmkc4wle9sYjCVvJDBLz8CrhcdMmrfnT2KbdA4bA28bQ+KGGwlAQfxazH/z9hjUt0kef8b4t42hnhgtZsw8PwwS2CQYzJiBtiQYENbClyzN22Bg2MaTYywtc07CcOaZh83S+LTw8589+Jnnj4G8fPvxhx/flNnI8x1PBorg0cLAAJRlbIPzJICYsQGvBrAWhj8E1IyCUTAKRsHIBgCiCj6pKX9tcQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7076-7886","institution":"South China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-30 05:53:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7248914/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7248914/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-025-06462-8","type":"published","date":"2025-12-18T15:58:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89371644,"identity":"6fe91d51-d403-4757-8225-5daf54031fed","added_by":"auto","created_at":"2025-08-19 10:15:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11291,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the PnNV1 genome structure. The single line and the black rectangular frame represent untranslated regions (UTRs) and open reading frames (ORFs), respectively.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7248914/v1/44e7a6b207b6bc0f4bcdad79.jpg"},{"id":89372160,"identity":"496bb625-5528-41ac-8426-ac7b770ce103","added_by":"auto","created_at":"2025-08-19 10:23:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154521,"visible":true,"origin":"","legend":"\u003cp\u003eBased on the RdRp amino acid sequences, phylogenetic trees were generated using the maximum likelihood method in Molecular Evolutionary Genetics Analysis (MEGA) version 10.1.8, with 1,000 bootstrap replicates.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7248914/v1/0df817b901d554bec0977088.jpg"},{"id":98814022,"identity":"f31944e3-3f94-4aad-8410-1153eaaad86c","added_by":"auto","created_at":"2025-12-22 16:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":522313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7248914/v1/8babec6a-d8f3-4646-b6f3-45026067bd66.pdf"},{"id":89371651,"identity":"eceb9341-532c-4d8b-aa91-6eee0b8696ea","added_by":"auto","created_at":"2025-08-19 10:15:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":223537,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7248914/v1/db31da33802a510252089124.docx"}],"financialInterests":"","formattedTitle":"A novel narnavirus isolated from the edible fungus Pholiota nameko","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycoviruses (fungal viruses) are viruses that infect fungi and are capable of replicating and multiplying within their hosts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mycoviruses are ubiquitous across nearly all fungal kingdoms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In 1962, the first fungal virus was discovered in an abnormally growing edible fungus \u003cem\u003eAgaricus bisporus\u003c/em\u003e by Hollings, marking the beginning of fungal virology research [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. With the advent of next-generation sequencing (NGS) technologies, an increasing number of mycoviruses have been identified [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Studies have shown that most mycoviruses do not cause significant external symptoms in their hosts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, a small number of mycoviruses can attenuate the pathogenicity of their hosts, offering potential for biocontrol applications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In edible fungi, mycoviruses often cause severe diseases with symptoms including delayed fruiting body formation, deformation of fruiting bodies, shortened stipes, thin mushroom caps, mycelium degeneration, and reduced yields [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Currently, over 80 mycoviruses have been identified in edible fungi, with over 31 having complete genome sequences. To date, only RNA genome types have been identified in edible fungi. Mycoviruses in edible fungi with dsRNA genomes are found in the families \u003cem\u003ePartitiviridae\u003c/em\u003e and \u003cem\u003eTotiviridae\u003c/em\u003e; those with (+\u0026thinsp;ss) RNA genomes are found in eight families: \u003cem\u003eBarnaviridae\u003c/em\u003e, \u003cem\u003eBotourmiaviridae\u003c/em\u003e, \u003cem\u003eDeltaflexiviridae\u003c/em\u003e, \u003cem\u003eEndornaviridae\u003c/em\u003e, \u003cem\u003eFusariviridae\u003c/em\u003e,\u003cem\u003eHypoviridae\u003c/em\u003e, \u003cem\u003eNarnaviridae\u003c/em\u003e and T\u003cem\u003eymoviridae\u003c/em\u003e; and those with (-)ssRNA genomes are found in the families Mymonaviridae and Phenuiviridae [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo date, edible fungal viruses have been report in more than 24 species, including \u003cem\u003eAgaricus bisporus\u003c/em\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eAgrocybe aegerita\u003c/em\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], \u003cem\u003eArmillaria\u003c/em\u003e species[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], \u003cem\u003eAuricularia heimuer\u003c/em\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003eBoletus edulis\u003c/em\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003eBondarzewia berkeleyi\u003c/em\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003eClitocybe odora\u003c/em\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], \u003cem\u003eCordyceps chanhua\u003c/em\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], \u003cem\u003eFlammulina velutipes\u003c/em\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eGrifola frondosa\u003c/em\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], \u003cem\u003eGyromitra esculenta\u003c/em\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], \u003cem\u003eHebeloma mesophaeum\u003c/em\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003cem\u003eLactarius tabidus\u003c/em\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eLentinula edodes\u003c/em\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], \u003cem\u003eLeucocybe candicans\u003c/em\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], \u003cem\u003eMorchella esculenta\u003c/em\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], \u003cem\u003eNeurospora intermedia\u003c/em\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003ePicoa juniperi\u003c/em\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], \u003cem\u003ePleurotus citrinopileatus\u003c/em\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], \u003cem\u003ePleurotus eryngii\u003c/em\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], \u003cem\u003ePleurotus ostreatus\u003c/em\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003eTuber aestivum\u003c/em\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cem\u003eUstilago maydis\u003c/em\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], \u003cem\u003eVolvariella volvace\u003c/em\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], However, to our knowledge, no fungal viruses have been reported in \u003cem\u003ePholiota nameko\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePholiota nameko\u003c/em\u003e, also known as the phosphate umbrella, is an edible mushroom with medicinal properties[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It is widely cultivated in China for food and traditional medicine [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Rich in protein, carbohydrates, fiber, vitamins, and unsaturated fatty acids, \u003cem\u003eP. nameko\u003c/em\u003e exhibits various biological activities, including anti-inflammatory, anti-hyperlipidemia, and antitumor effects [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, the impact of viral infections on the quality and yield of this fungus has not yet been investigated.\u003c/p\u003e\u003cp\u003eMembers of the viral family \u003cem\u003eNarnaviridae\u003c/em\u003e have the simplest viral genome structure [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. They lack a protein capsid and are not associated with virus particles other than lipid vesicles. Their genome contains a single large ORF encoding only an RNA-dependent RNA polymerase (RdRp) to direct their replication [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Narnaviruses are widely distributed in fungi, plants, oomycetes, mosquitoes, arthropods, algae, trypanosomatids, and potentially apicomplexans [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].Only two species have been approved by the ICTV in this family: Saccharomyces 20S RNA narnavirus and Saccharomyces 23S RNA narnavirus [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Both viruses have small genomes and possess short terminal inverted repeats (5\u0026rsquo;-GGGGC and GCCCC-3\u0026rsquo;) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we discovered a novel\u0026thinsp;+\u0026thinsp;ssRNA virus with the proposed name \u0026ldquo;Pholiota nameko narnavirus 1\u0026rdquo; (PnNV1) in \u003cem\u003ePholiota nameko\u003c/em\u003e strain N1. Sequence alignments and phylogenetic analysis showed that PnNV1 can be classified as a new member of the family \u003cem\u003eNarnaviridae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe \u003cem\u003ePholiota nameko\u003c/em\u003e strain N1 was obtained from the Shouguang Institute of Edible Fungi in Shandong Province, China. The strain was cultured on Potato Dextrose Agar (PDA) medium at 25°C for 7 days for dsRNA multiplication and sent to Shanghai Biotechnology Corporation for high-throughput sequencing. The sequencing data indicated the presence of a fungal virus, likely a member of the family \u003cem\u003eNarnaviridae\u003c/em\u003e. dsRNA was extracted from fungal mycelium using the CF-11 cellulose chromatography method[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The dsRNA was then purified with DNase I and S1 nuclease (TaKaRa Dalian, China) to remove contaminating DNA and single-stranded RNA (ssRNA)[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The treated dsRNA was electrophoresed in a 1% (w/v) agarose gel. Although no bands were visible in the electrophoresis analysis, the presence of the virus was detected using virus-specific primers. The full-length cDNA sequence of the virus was cloned primarily using the ligase-mediated rapid amplification of cDNA ends (RLM-RACE) protocol described by Xie et al[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The complete sequence of PnNV1 was deposited in the GenBank database under the accession number OR822278.\u003c/p\u003e\u003cp\u003ePotential RNA secondary structures of the 5' and 3' untranslated regions (UTR) of this viral genome were predicted using the online tool available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.unafold.org/mfold/applications/rna-folding-form-v2.php\u003c/span\u003e\u003cspan address=\"http://www.unafold.org/mfold/applications/rna-folding-form-v2.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Protein domains searches were performed using the Conserved Domain Database (CDD) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignment of protein sequences was conducted using DNAMAN software (Version 9). The ORF of PnNV1 was identified and translated using DNAMAN software (Version 9). Phylogenetic trees were generated using the maximum likelihood method in Molecular Evolutionary Genetics Analysis (MEGA) version 10.1.8, with 1,000 bootstrap replicates[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e"},{"header":"Sequence properties","content":"\u003cp\u003eThe complete genome of PnNV1 is 2254 nucleotides in length, containing a single large ORF encoding a 682-amino-acid (aa) protein on its positive strand, with a molecular mass of 77.81 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The lengths of the 5' and 3' UTR were 50 and 152 nucleotides, with ΔG values of -18.34 kcal/mol and − 31.35 kcal/mol (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively. Secondary structure predictions of the 5' and 3' UTRs revealed that both contain stem-loop structures. BLASTp analysis of the RNA-dependent RNA polymerases (RdRps) showed significant similarity to Rhizoctonia solani narnavirus 18. We selected three RdRp sequences with high accuracy and similarity, along with representative species of the genus Narnavirus for the prediction of conserved motifs and multiple sequence comparisons. The results identified a total of six conserved motifs, with their exact locations shown in Fig. S2. The abbreviation of virus names, GenBank accession numbers, identity, and query coverage are as follows: Fusarium poae narnavirus 1(YP_009272902.1, 21.9% identity, 68% query coverage); Plasmopara viticola lesion associated narnavirus 18(QIR30297.1, 27.07% identity, 80% query coverage); Puccinia striiformis narnavirus 1 (QJQ50000.1, 27.26% identity, 73% query coverage); Saccharomyces 20S RNA narnavirus (NP_660178.1); Saccharomyces 23S RNA narnavirus(NP_660177.1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA molecular phylogenetic tree was constructed based on the RdRp amino acid sequences of PnNV1, other selected viruses with high similarity, and several representative members of the family \u003cem\u003eNarnaviridae\u003c/em\u003e and \u003cem\u003eMitoviridae\u003c/em\u003e. The maximum likelihood tree indicates that PnNV1 can be classified as a new member of the family \u003cem\u003eNarnaviridae\u003c/em\u003e, forming a distinct clade with Plasmopara viticola lesion associated narnavirus 18 as a sister branch of narnaviruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To our knowledge, a variety of different genome types of edible fungal viruses have been identified in several edible fungi species, except \u003cem\u003ePholiota nameko\u003c/em\u003e. A small number of viruses have been shown to have significant effects on the growth and yield of edible fungi. As an edible fungus widely cultivated in China with medicinal value, it is necessary to excavate the mycovirus resources in the \u003cem\u003ePholiota nameko\u003c/em\u003e and conduct in-depth research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was supported by the National Natural Science Foundation of China (32202381), Guangdong Basic and Applied Basic Research Foundation (2024A1515010080), and Guangzhou Science and Technology Planning Project (SL2022A04J01161).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e All authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXie J, Jiang D (2014) New Insights into Mycoviruses and Exploration for the Biological Control of Crop Fungal Diseases. 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Molecular Biology and Evolution 35:1547-1549\u003c/li\u003e\n\u003c/ol\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":false,"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":"mycovirus, Narnaviridae, Pleurotus nameko, narnavirus, PnNV1","lastPublishedDoi":"10.21203/rs.3.rs-7248914/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7248914/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel positive single-stranded RNA virus, tentatively named \u0026ldquo;Pholiota nameko narnavirus 1\u0026rdquo; (PnNV1), has been identified in an edible fungus \u003cem\u003ePholiota nameko\u003c/em\u003e strain N1. The complete genome of PnNV1 is 2254 nucleotides in length, containing a single large open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp) of 682 amino acids (aa) with a molecular mass of 77.81 kDa. blastp analysis indicated that PnNV1 exhibited the highest similarity (30.80%) to Rhizoctonia solani narnavirus 18. Sequence alignments and phylogenetic analysis based on RdRp sequences indicate that PnNV1 can be classified as a new member of the family \u003cem\u003eNarnaviridae\u003c/em\u003e. To our knowledge, this is the first mycovirus reported in the edible fungus \u003cem\u003ePholiota nameko\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"A novel narnavirus isolated from the edible fungus Pholiota nameko","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 10:15:10","doi":"10.21203/rs.3.rs-7248914/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-11T11:02:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-11T10:16:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-04T10:19:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-08-03T23:15:20+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2025-08-01T11:22:44+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":"d19fafb9-d891-48ca-940b-dba69b89e8a3","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:03:19+00:00","versionOfRecord":{"articleIdentity":"rs-7248914","link":"https://doi.org/10.1007/s00705-025-06462-8","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2025-12-18 15:58:34","publishedOnDateReadable":"December 18th, 2025"},"versionCreatedAt":"2025-08-19 10:15:10","video":"","vorDoi":"10.1007/s00705-025-06462-8","vorDoiUrl":"https://doi.org/10.1007/s00705-025-06462-8","workflowStages":[]},"version":"v1","identity":"rs-7248914","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7248914","identity":"rs-7248914","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}