Complete genome of a novel narnavirus with inverted complementary termini from the rice blast fungus Magnaporthe oryzae isolate NJ471

Complete genome of a novel narnavirus with inverted complementary termini from the rice blast fungus Magnaporthe oryzae isolate NJ471 | 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 Complete genome of a novel narnavirus with inverted complementary termini from the rice blast fungus Magnaporthe oryzae isolate NJ471 Cong Li, YuXin Wu, XinYi Li, Qingchao Deng, JiaTao Xie This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6299189/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jun, 2025 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract A novel single-stranded (+ ss) RNA mycovirus, designated as Magnaporthe oryzae narnavirus 2 (MoNV2), was identified in the rice blast fungus Magnaporthe oryzae isolate NJ471. MoNV2 has a genomic RNA fragment of 3,086 nucleotides, which contains a single open reading frame (ORF) that is predicted to encode an RNA-dependent RNA polymerase (RdRp). Genome sequence comparisons and phylogenetic analysis suggested that MoNV2 is a new member of the genus narnavirus in the family Narnaviridae . The 5' and 3' terminal sequences of the genomic RNA of MoNV2 have inverted complementarity and potentially form a panhandle structure, which is very rare in RNA viruses. Figures Figure 1 Figure 2 Introduction Mycoviruses (or fungal viruses) have been described in all major fungal groups, their genome type can be either DNA (minority) or RNA (majority), and the hosts can be filamentous fungi, yeasts and oomycetes [1-5]. In 1962, Hollins first discovered a mycovirus in Agaricus bisporus [6]. Since then, the number of mycoviruses discoveries has skyrocketed with the rapid advances in high-throughput sequencing technology, which has not only increased our understanding of the diversity of mycoviruses in the kingdom of fungi, which has also increased our understanding of the origin and evolution of viruses[7-9]. While most mycoviruses induce not observable phenotypic alterations in their fungal host, there are some studies that have identified exceptions where these mycoviruses significantly alter the biology of the host. Certain mycoviruses can alter colony morphology, regulate fungal growth rates, suppress spore production, or confer hypovirulence. These unique interactions not only advance our understanding of fungal-virus coevolution but also position mycoviruses as emerging candidates for biocontrol applications [5, 10, 11]. Rice blast is a serious threat to the yield of grain crops rice. The filamentous fungus Magnaporthe oryzae (teleomorph; Herbert) Barr (anamorph: Pyricularia oryzae ) is the pathogen of rice blast [12]. So far, various mycoviruses have been identified in M. oryza , all of which are RNA viruses. Viruses with a genome type of double-stranded RNA（dsRNA）include Magnaporthe oryzae chrysovirus 1 (MoCV1) of the family Chrysoviridae [13-15], Magnaporthe oryzae partitivirus 1, 2 and 4 (MoPV1, MoPV2 and MoPV4) of the Partitiviridae family [16-18], Magnaporthe oryzae virus 1, 2 and 3 (MoV1, 2 and 3) of the family Totiviridae [19] and Magnaporthe oryzae polymycovirus 1 (MoPmV1) in the family Polymycoviridae [20]. Viruses with a genome type of positive-sense single-stranded RNA (+ssRNA) include Magnaporthe oryzae virus A (MoVA) of the family Tombusviridae [21] , Magnaporthe oryzae narnavirus 1 of the family Narnaviridae [22], Magnaporthe oryzae ourmia-like virus 1 and 4 (MOLV1 and MOLV4), Pyricularia oryzae ourmia-like viruses 1, 2, and 3 (PoOLV1, PoOLV2, and PoOLV3), and Magnaporthe oryzae botourmiavirus 5, 6, 7, 9, 10 (MoBV5, MoBV6, MoBV7, MoBV9 and MoBV10) [23-27] of the Botourmiaviridae family. There is only one virus with the genome type of negative-stranded（-ssRNA）has been identified in M. oryzae at present, which is Magnaporthe oryzae mymonavirus 1 (MoMNV1) of the family Mymonaviridae [28] . The family Narnaviridae comprises viruses with positive-sense single-stranded RNA (+ssRNA) genomes phylogenetically related to the families Mitoviridae , Botourmiaviridae , and Fiersviridae , these virus families together forming the phylum Lenarviriota . Meanwhile, the family Narnaviridae of viruses possesses the simplest genome of all RNA viruses, with their genome sizes ranging from 2.3-3.6 kb, encoding only an open reading frame (ORF) with an RNA-dependent RNA polymerase (RdRp) domain, and no viral particles have been detected [29]. In addition, some narnaviruses contain a reverse open reading frame (rORF) that is about the same size as the open reading frame(ORF) encoded by the positive strand of the genome[30]. Currently, there is only one genus of narnavirus in the family Narnaviridae , represented by the yeast-infecting Saccharomyces 20S RNA narnavirus (SCV20) and Saccharomyces 23S RNA narnavirus (SCV23), with the presence of RNA genomes at the 5' and 3' ends with reverse complementary five nucleotides (5'-GGGGGC-GCCCC-3'), and in the case of genetic abnormalities, such as deletions, it is able to repair itself to maintain the correct ends, which may be an important reason for the virus to be able to sustain a stable infestation in yeast [31, 32, 33]. Virus isolation and sequencing The M. oryzae strain NJ471 was isolated from a lesion of a rice neck-panicle sample in Nanjing, Jiangsu, China, in 2023 September, which showed a normal biological phenotype. High-throughput RNA sequencing revealed that M. oryzae strain NJ471 was infected with two mycoviruses. One of them was recognized as a new isolate of MoBV7, while the other was identified as a new virus, which was tentatively named as ‘Magnaporthe oryzae narnavirus 2’ (MoNV2). M. oryzae strain NJ471 was inoculated onto the surface of PDA medium covered with cellophane film and incubated in the dark at 28°C for 7 days before total RNA of the target strain was extracted using TRIzol reagent (GenStar, Beijing, China). Gene specific primers (primer sequence F, 5' - AGTTCGCGTGGTGCTTTCTA- 3' /R, 5' - GAGACACTGGCGAACCAGAA- 3') were designed based on the high-throughput sequence results of MoNV2 and used for RT-PCR, and the accuracy of the original sequence was confirmed by Sanger sequencing. The end sequences of the viral genome were determined by amplifying the 5' and 3' end cDNAs using a T4 RNA ligase-mediated method[20, 22]. Each base pair was sequenced in both orientations by sequencing three or more independent overlapping clones. To obtain the complete genome sequence of MoNV2, splicing and assembly were performed using DNAMAN version 8. The complete nucleotide sequence of MoNV2 has been deposited in the GenBank database under the accession numbers PQ871412. RNA secondary structure of the termini of MoNV2 was predicted using online software (http://www.unafold.org/). The open reading frames (ORFs) of the MoNV2 genomic sequence were predicted using the eukaryotic genetic code with the online software ((http://www.ncbi.nlm.gov/gorf/gorf.html). Conserved domains were identified using the NCBI Conserved Domain Data-base (https://www.ncbi.nlm.nih.gov/cdd). The amino acid (aa) sequence of the putative RdRp of MoNV2 was aligned with other virus RdRp sequences using the Jalview program. After aligning the sequences, a phylogenetic tree was constructed using the maximal likelihood (ML) method using the MEGA version 11 program, with the amino acid substitution model (VT+F+R8) selected by the IQ-TREE program. Sequence properties The complete genome sequence of MoNV2 was determined to be 3086 nucleotides (nt) in full length, with a GC content of 53.1% (1,638/3,086) and contained only one Open Reading Frame（ORF）encoding RNA-dependent RNA polymerase (RDRP). The open reading frame consists of 2913 nucleotides and encodes a protein of 970 amino acids (aa) with a molecular weight of 106.7 kDa. The length of the 5'- and 3'-untranslated regions (UTRs) of the MoNV2 genome is 61 nt and 111 nt, respectively (Fig. 1. A). The 5' terminal sequence (nt positions 1-43) and 3' terminal sequence (nt positions 3036-3086) of MoNV2 were predicted to be folded into potentially stable stem-loop structures with ΔG values of -9.8 and -17.60 kcal/mol, respectively (Fig. 1. B). Stem-loop structures formed in the untranslated regions of the viruses genome are important and may be associated with replication and translation, which are common among members of the families Narnaviridae and Botourmiaviridae [34]. Moreover,, the 5' (nt positions 2-15, starting from the second base) and 3' (nt positions 3072-3086, starting from the penultimate base) terminal sequences of the untranslated region of the genome of MoNV2 were completely complementary for about 13 bases, suggesting that they can form panhandle structures , with a ΔG value of -9.00 kcal/mol (Fig. 1. B). The structural feature of terminal complementarity, while observed in various RNA viral genomes, demonstrates striking rarity. Disparate instances include: (i) (-) ssRNA viruses (Lentinula edodes negative-strand virus 2 ,LeNSRV2), (ii) (+) ssRNA viruses (MoBV10, SCV20 and SCV23), and (iii) dsRNA viruses (Diadromus pulchellus reovirus, DpRV) [27,31,32]. A homology search of the GenBank database using BLASTp showed that the MoMNV2 RdRp was most closely related to the RdRps of some members of the genus narnavirus in the family narnaviridae . Such as Plasmopara viticola lesion associated narnavirus 9 (QIR_30288.1; identity, 75.4%; query coverage, 95%; e-value, 0), Aspergillus lentulus narnavirus 1 (BCH_36643.1; identity, 74.4%; query coverage, 95%; e-value, 0), Dracophyllum associated narna-like virus 31 (WPR17236.1; identity, 75.0%; query coverage, 95%; e-value, 0), Erysiphe necator associated narnavirus 2 (QHD64825.1; identity, 67.2%; query coverage, 95%; e-value, 0), XiangYun narna-levi-like virus 11 (UUG74244.1; identity, 58.9%; query coverage, 95%; e-value, 0) and Botryosphaeria dothidea narnavirus 3 (QQD86177.1; identity, 55.9%; query coverage, 92%; e-value, 0), were most closely related to RdRP. It is noteworthy that the Blastp alignment results showed the highest homology to a partial genomic sequence of MoNV2 (GenBank accession number: LC553714.1; identity, 99.4 %; query coverage, 94%; e-value, 0), which was uploaded by Urayama et al. in June 2020. This sequence was obtained through FLDS (fungal long-read sequencing)-based high-throughput screening, but its accuracy and completeness have not been determined. In addition, a Conserved Domain Database (CDD) search and multiple amino acid sequence comparisons confirmed that the MoNV2-encoded protein contains eight conserved motifs characteristic of (+)ssRNA viral RDRPs(Fig. 2. A), and that RdRps contain a typical ‘GDD’ motif (motif VI, which is not present in MoMNV1 and FpNV2 are absent), which is highly conserved in almost all viruses RdRps [35]. To determine the phylogenetic relationship of MoNV2, we constructed a phylogenetic tree based on the rdrp-aa sequences of MoNV2 with members of the families Narnaviridae , Botourmiaviridae , Fiersviridae , and Mitoviridae using the MEGA version 11 software using the maximal likelihood (ML) method. The evolutionary tree results clearly demonstrate that MoNV2 clusters with other Narnaviridae in a large branch that remains distinct from members of the families Botourmiaviridae , Fiersviridae and Mitoviridae (Fig. 2, B). In conclusion, we report the first complete genomic sequence of MoNV2. Comparative genomic analysis and phylogenetic analysis indicate that MoNV2 represents a novel member within the family Narnaviridae , and also that the genomic RNA of MoNV2 has rare inverted complementary termini. (QBA55490.1)；FpNV2, Fusarium poae narnavirus 2 (YP 009272903.1); MoNV1, Magnaporthe oryzae narnavirus virus 1 (MN480844.1); MoNV2, Magnaporthe oryzae narnavirus virus 2 (PQ871412). Asterisks, colons, and dots represent identical, conserved, and semi-conserved amino acid residues, respectively. Asterisks, colons, and dots represent identical, conserved, and semi-conserved amino acid residues, respectively. (B). Phylogenetic analysis based on the RDRP sequences of MoNV2 and related viruses of the families Narnaviridae ， Botourmiaviridae , Fiersviridae, and Mitoviridae . The phylogenetic tree was constructed by the maximal likelihood (ML) method using the program MEGA 11, with 1,000 bootstrap replicates. This tree was rooted with four viruses of Fiersviridae . The position of MoNV2 is indicated by a red star, the other one (MoNV1) previously identified full-length sequnenced viruses in the family Narnaviridae found in M. oryzae are indicated by blue stars. Only p values for the approximate likelihood ratios (SH test) of >0.5(50%) are indicated. Scale barscorrespond to 0.5 amino acid substitution per site. Sequence accession numbers are given for each sequence. Declarations Author contributions Cong Li : Writing – original draft, Validation, Methodology, Investigation, Data curation, Conceptualization. Yuxin Wu : Writing – review & editing, Data curation, Methodology, Investigation,. Xinyi Li : Methodology, Formal analysis. Jiatao Xie : Resources, Investigation, Project administration, Data curation, Funding acquisition. Qingchao Deng : Resources, Investigation, Project administration, Data curation, Funding acquisition. Funding This thesis is supported by the State Key Laboratory of Agricultural Microbiology (No. AMLKF202209) and the National Natural Science Foundation of China (NFSC No. 31201474). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data availability The datasets generated and/or analyzed in the current study are available from the corresponding author on reasonable request. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest All authors declare that they have no conflict of interest. References Ghabrial SA, Suzuki N (2009) Viruses of plant pathogenic fungi[J]. 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Curr Top Microbiol Immunol 320(1):137–156. https://doi.org/ 10.1007/978-3-540-75157-1_7 Supplementary Files Submission2914140.txt SupplementaryFigure1.pdf SupplementaryFigure2.pdf SupplementaryFigure3.pdf SupplementaryFigure4.pdf Cite Share Download PDF Status: Published Journal Publication published 08 Jun, 2025 Read the published version in Archives of Virology → Version 1 posted Editorial decision: Minor Revision 21 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Editor assigned by journal 28 Mar, 2025 First submitted to journal 25 Mar, 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. 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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-6299189","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436716816,"identity":"c832a29a-c29a-415f-baa8-a691693e7ab1","order_by":0,"name":"Cong Li","email":"","orcid":"","institution":"State Key Laboratory of Agricultural Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Li","suffix":""},{"id":436716817,"identity":"85edd88d-601e-42e6-bb17-02310f6380fb","order_by":1,"name":"YuXin Wu","email":"","orcid":"","institution":"Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"YuXin","middleName":"","lastName":"Wu","suffix":""},{"id":436716818,"identity":"c71bcc77-89c3-4fe5-a61b-da392f7646cd","order_by":2,"name":"XinYi Li","email":"","orcid":"","institution":"Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"XinYi","middleName":"","lastName":"Li","suffix":""},{"id":436716819,"identity":"65e1b78a-a6b3-46c3-a749-4053ca49ec12","order_by":3,"name":"Qingchao Deng","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8230-3272","institution":"State Key Laboratory of Agricultural Microbiology","correspondingAuthor":true,"prefix":"","firstName":"Qingchao","middleName":"","lastName":"Deng","suffix":""},{"id":436716820,"identity":"063d928d-00a3-49a3-baf3-ba5dbafd222b","order_by":4,"name":"JiaTao Xie","email":"","orcid":"https://orcid.org/0000-0003-1961-0338","institution":"State Key Laboratory of Agricultural Microbiology","correspondingAuthor":false,"prefix":"","firstName":"JiaTao","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-03-25 02:19:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6299189/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6299189/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-025-06337-y","type":"published","date":"2025-06-08T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81052586,"identity":"b1469130-f16f-4ef3-acc1-edf03b53b2eb","added_by":"auto","created_at":"2025-04-21 16:34:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A).\u003c/strong\u003eSchematic representation of the genome of MoNV2. (B). Potential stem-loop structures for the MoNV2 5'and 3'-terminal sequences, and potential panhandle structures formed by the reverse complementary sequences of the 5' and 3'-terminal sequences. Short lines in different colors indicate hydrogen bonds between different base pairs (red, G-C pairs; purple, A-U pairs; green, G-U pairs)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/860e3a7baa902046e4705d18.jpg"},{"id":81053173,"identity":"2472373f-9b44-4575-b8d0-2698ed1f5d50","added_by":"auto","created_at":"2025-04-21 16:42:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A).\u003c/strong\u003e Multiple alignments of amino acid sequences of the RdRps of MoNV2 and other eight members of the family \u003cem\u003eNarnaviridae\u003c/em\u003e. The eight conserved motifs of RdRp are represented by the Roman numerals I through VIII on the black line. Virus names are abbreviated as follows: SCV20, Saccharomyces 20S RNA narnavirus (P25328.1); SCV23, Saccharomyces 23S RNA narnavirus (Q07048.2); BlNV1, Blechomonas luni narnavirus 1 (YP 009553634.1); CvNV1, Coquillettidia venezuelensis narnavirus 1 (QBA55488.1); AgNV, Aedes_angustivittatus_narnavirus (QBA55490.1)；FpNV2, Fusarium poae narnavirus 2 (YP 009272903.1); MoNV1, Magnaporthe oryzae narnavirus virus 1 (MN480844.1); MoNV2, Magnaporthe oryzae narnavirus virus 2 (PQ871412). Asterisks, colons, and dots represent identical, conserved, and semi-conserved amino acid residues, respectively. Asterisks, colons, and dots represent identical, conserved, and semi-conserved amino acid residues, respectively. \u003cstrong\u003e(B). \u003c/strong\u003ePhylogenetic analysis based on the RDRP sequences of MoNV2 and related viruses of the families \u003cem\u003eNarnaviridae\u003c/em\u003e，\u003cem\u003eBotourmiaviridae\u003c/em\u003e,\u003cem\u003e Fiersviridae, \u003c/em\u003eand \u003cem\u003eMitoviridae\u003c/em\u003e. The phylogenetic tree was constructed by the maximal likelihood (ML) method using the program MEGA 11, with 1,000 bootstrap replicates. This tree was rooted with four viruses of \u003cem\u003eFiersviridae\u003c/em\u003e. The position of MoNV2 is indicated by a red star, the other one (MoNV1) previously identified full-length sequnenced viruses in the family \u003cem\u003eNarnaviridae\u003c/em\u003e found in \u003cem\u003eM. oryzae\u003c/em\u003e are indicated by blue stars. Only \u003cem\u003ep \u003c/em\u003evalues for the approximate likelihood ratios (SH test) of \u0026gt;0.5(50%) are indicated. Scale barscorrespond to 0.5 amino acid substitution per site. Sequence accession numbers are given for each sequence.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/98d278c25c65cd423abf1c01.jpg"},{"id":84242746,"identity":"a41b498c-889d-44ab-b810-734c03ccf86e","added_by":"auto","created_at":"2025-06-09 16:11:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":813451,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/f03b4273-0d09-43c2-ac91-376dc98053f2.pdf"},{"id":81052590,"identity":"fb9a5b7c-3997-4762-9cf7-053347104d91","added_by":"auto","created_at":"2025-04-21 16:34:22","extension":"txt","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":7060,"visible":true,"origin":"","legend":"","description":"","filename":"Submission2914140.txt","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/1f61a6aed603a0355e078e48.txt"},{"id":81053176,"identity":"afe50998-25c8-4d1d-b168-9fceefc89ac7","added_by":"auto","created_at":"2025-04-21 16:42:23","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1773095,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/a6fa52eaca1282c4608bcbe7.pdf"},{"id":81052592,"identity":"11e4ceed-e6fc-4c45-8725-3672cfdbf819","added_by":"auto","created_at":"2025-04-21 16:34:23","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1483412,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/b026ca2bb824846536ec000f.pdf"},{"id":81053178,"identity":"f1527a2c-9abb-4439-a9c3-e99419941ba5","added_by":"auto","created_at":"2025-04-21 16:42:23","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1540995,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/dd987e89a7ea65b41a831632.pdf"},{"id":81053174,"identity":"00e515b8-68c0-4c9e-83ec-8d2d186d7bbc","added_by":"auto","created_at":"2025-04-21 16:42:23","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1099464,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6299189/v1/5c3a81fd4d4748bbaa02e36f.pdf"}],"financialInterests":"","formattedTitle":"Complete genome of a novel narnavirus with inverted complementary termini from the rice blast fungus Magnaporthe oryzae isolate NJ471","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycoviruses (or fungal viruses) have been described in all major fungal groups, their genome type can be either DNA (minority) or RNA (majority), and the hosts can be filamentous fungi, yeasts and oomycetes [1-5]. In 1962, Hollins first discovered a mycovirus in \u003cem\u003eAgaricus bisporus\u0026nbsp;\u003c/em\u003e[6]. Since then, the number of mycoviruses discoveries has skyrocketed with the rapid advances in high-throughput sequencing technology, which has not only increased our understanding of the diversity of mycoviruses in the kingdom of fungi, which has also increased our understanding of the origin and evolution of viruses[7-9]. While most mycoviruses induce not observable phenotypic alterations in their fungal host, there are some studies that have identified exceptions where these mycoviruses significantly alter the biology of the host. Certain mycoviruses can alter colony morphology, regulate fungal growth rates, suppress spore production, or confer hypovirulence. These unique interactions not only advance our understanding of fungal-virus coevolution but also position mycoviruses as emerging candidates for biocontrol applications [5, 10, 11].\u003c/p\u003e\n\u003cp\u003eRice blast is a serious threat to the yield of grain crops rice. The filamentous fungus\u0026nbsp;\u003cem\u003eMagnaporthe oryzae\u003c/em\u003e (teleomorph; Herbert) Barr (anamorph: \u003cem\u003ePyricularia oryzae\u003c/em\u003e)\u0026nbsp;is the pathogen of rice blast [12]. So far, various mycoviruses have been identified in \u003cem\u003eM. oryza\u003c/em\u003e, all of which are RNA viruses. Viruses with a genome type of double-stranded RNA（dsRNA）include Magnaporthe oryzae chrysovirus 1 (MoCV1) of the family \u003cem\u003eChrysoviridae\u003c/em\u003e [13-15], Magnaporthe oryzae partitivirus 1, 2 and 4 (MoPV1, MoPV2 and MoPV4) of the \u003cem\u003ePartitiviridae\u003c/em\u003e family [16-18], Magnaporthe oryzae virus 1, 2 and 3 (MoV1, 2 and 3) of the family \u003cem\u003eTotiviridae\u003c/em\u003e [19] and Magnaporthe oryzae polymycovirus 1 (MoPmV1) in the family \u003cem\u003ePolymycoviridae\u003c/em\u003e\u0026nbsp; [20]. Viruses with a genome type of positive-sense\u0026nbsp;single-stranded\u0026nbsp;RNA (+ssRNA) include Magnaporthe oryzae virus A (MoVA) of the family \u003cem\u003eTombusviridae\u003c/em\u003e[21] , Magnaporthe oryzae narnavirus 1 of the family \u003cem\u003eNarnaviridae\u003c/em\u003e [22], Magnaporthe oryzae ourmia-like virus 1 and 4 (MOLV1 and MOLV4), Pyricularia oryzae ourmia-like viruses 1, 2, and 3 (PoOLV1, PoOLV2, and PoOLV3), and Magnaporthe oryzae botourmiavirus 5, 6, 7, 9, 10 (MoBV5, MoBV6, MoBV7, MoBV9 and MoBV10) [23-27] of the \u003cem\u003eBotourmiaviridae\u003c/em\u003e family. There is only one virus with the genome type of negative-stranded（-ssRNA）has been identified in \u003cem\u003eM. oryzae\u003c/em\u003e at present, which is Magnaporthe oryzae mymonavirus 1 (MoMNV1) of the family \u003cem\u003eMymonaviridae\u003c/em\u003e [28]\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe family \u003cem\u003eNarnaviridae\u003c/em\u003e comprises viruses with positive-sense single-stranded RNA (+ssRNA) genomes phylogenetically related to the families \u003cem\u003eMitoviridae\u003c/em\u003e, \u003cem\u003eBotourmiaviridae\u003c/em\u003e, and \u003cem\u003eFiersviridae\u003c/em\u003e, these virus families together forming the phylum \u003cem\u003eLenarviriota\u003c/em\u003e. Meanwhile, the family\u003cem\u003e\u0026nbsp;Narnaviridae\u003c/em\u003e of viruses possesses the simplest genome of all RNA viruses, with their genome sizes ranging from 2.3-3.6 kb, encoding only an open reading frame (ORF) with an RNA-dependent RNA polymerase (RdRp) domain, and no viral particles have been detected\u0026nbsp;[29]. In addition, some \u003cem\u003enarnaviruses\u003c/em\u003e contain a reverse open reading frame (rORF) that is about the same size as the open reading frame(ORF) encoded by the positive strand of the genome[30].\u0026nbsp;Currently, there is only one genus of \u003cem\u003enarnavirus\u003c/em\u003e in the family \u003cem\u003eNarnaviridae\u003c/em\u003e, represented by the yeast-infecting Saccharomyces 20S RNA narnavirus (SCV20) and Saccharomyces 23S RNA narnavirus (SCV23), with the presence of RNA genomes at the 5'\u0026nbsp;and 3'\u0026nbsp;ends with reverse complementary five nucleotides (5'-GGGGGC-GCCCC-3'), and in the case of genetic abnormalities, such as deletions, it is able to repair itself to maintain the correct ends, which may be an important reason for the virus to be able to sustain a stable infestation in yeast\u0026nbsp;[31, 32, 33].\u003c/p\u003e\n\n\n\n\n"},{"header":"Virus isolation and sequencing","content":"\u003cp\u003eThe \u003cem\u003eM. oryzae\u003c/em\u003e strain NJ471 was isolated from a lesion of a rice neck-panicle sample in Nanjing, Jiangsu, China, in 2023 September, which showed a normal biological phenotype. High-throughput RNA sequencing revealed that \u003cem\u003eM. oryzae\u003c/em\u003e strain NJ471 was infected with two mycoviruses. One of them was recognized as a new isolate of MoBV7, while the other was identified as a new virus, which was tentatively named as ‘Magnaporthe oryzae narnavirus 2’ (MoNV2).\u003c/p\u003e\u003cp\u003e\u003cem\u003eM. oryzae\u003c/em\u003e strain NJ471 was inoculated onto the surface of PDA medium covered with cellophane film and incubated in the dark at 28°C for 7 days before total RNA of the target strain was extracted using TRIzol reagent (GenStar, Beijing, China). Gene specific primers (primer sequence F, 5'\u0026nbsp;- AGTTCGCGTGGTGCTTTCTA- 3' /R, 5' - GAGACACTGGCGAACCAGAA- 3') were designed based on the high-throughput sequence results of MoNV2 and used for RT-PCR, and the accuracy of the original sequence was confirmed by Sanger sequencing. The end sequences of the viral genome were determined by amplifying the 5' and 3' end cDNAs using a T4 RNA ligase-mediated method[20, 22].\u0026nbsp;Each base pair was sequenced in both orientations\u0026nbsp;by sequencing three or more independent overlapping clones. To obtain the complete genome sequence of MoNV2, splicing and assembly were performed using DNAMAN version 8. The complete nucleotide sequence of MoNV2 has been deposited in the GenBank database under the accession numbers PQ871412.\u003c/p\u003e\u003cp\u003eRNA secondary structure of the termini of MoNV2 was predicted using online software (http://www.unafold.org/). The open reading frames (ORFs) of the MoNV2 genomic sequence were predicted using the eukaryotic genetic code with the online software ((http://www.ncbi.nlm.gov/gorf/gorf.html). Conserved domains were identified using the NCBI Conserved Domain Data-base (https://www.ncbi.nlm.nih.gov/cdd). The amino acid (aa) sequence of the putative RdRp of MoNV2 was aligned with other virus RdRp sequences using the Jalview program. After aligning the sequences, a phylogenetic tree was constructed using the maximal likelihood (ML) method using the MEGA version 11 program, with the amino acid substitution model (VT+F+R8) selected by the IQ-TREE program.\u003c/p\u003e"},{"header":"Sequence properties","content":"\u003cp\u003eThe complete genome sequence of MoNV2 was determined to be 3086 nucleotides (nt) in full length, with a GC content of 53.1% (1,638/3,086) and contained only one Open Reading Frame（ORF）encoding RNA-dependent RNA polymerase (RDRP). The open reading frame consists of 2913 nucleotides and encodes a protein of 970 amino acids (aa) with a molecular weight of 106.7 kDa. The length of the 5'- and 3'-untranslated regions (UTRs) of the MoNV2 genome is 61 nt and 111 nt, respectively\u0026nbsp;(Fig. 1. A).\u0026nbsp;\u003c/p\u003e\u003cp\u003eThe 5' terminal sequence (nt positions 1-43) and 3' terminal sequence (nt positions 3036-3086) of MoNV2 were predicted to be folded into potentially stable stem-loop structures with ΔG values of -9.8 and -17.60 kcal/mol, respectively\u0026nbsp;(Fig. 1. B).\u0026nbsp;Stem-loop structures formed in the untranslated regions of the viruses genome are important and may be associated with replication and translation, which are common among members of the families \u003cem\u003eNarnaviridae\u003c/em\u003e and \u003cem\u003eBotourmiaviridae\u003c/em\u003e [34]. Moreover,, the 5' (nt positions 2-15, starting from the second base) and 3' (nt positions 3072-3086, starting from the penultimate base) terminal sequences of the \u0026nbsp; untranslated region of the genome of MoNV2 were completely complementary for about 13 bases, suggesting that they can form panhandle structures , with a ΔG value of -9.00 kcal/mol\u0026nbsp;(Fig. 1. B). The structural feature of terminal complementarity, while observed in various RNA viral genomes, demonstrates striking rarity. Disparate instances include: (i) (-) ssRNA viruses (Lentinula edodes negative-strand virus 2 ,LeNSRV2), (ii) (+) ssRNA viruses (MoBV10, SCV20 and SCV23), and (iii) dsRNA viruses (Diadromus pulchellus reovirus, DpRV)\u0026nbsp;[27,31,32].\u003c/p\u003e\u003cp\u003eA homology search of the GenBank database using BLASTp showed that the MoMNV2 RdRp was most closely related to the RdRps of some members of the genus \u003cem\u003enarnavirus\u0026nbsp;\u003c/em\u003ein the family \u003cem\u003enarnaviridae\u003c/em\u003e. Such as Plasmopara viticola lesion associated narnavirus 9 (QIR_30288.1; identity, 75.4%; query coverage, 95%; e-value, 0), Aspergillus lentulus narnavirus 1 (BCH_36643.1; identity, 74.4%; query coverage, 95%; e-value, 0), Dracophyllum associated narna-like virus 31 (WPR17236.1; identity, 75.0%; query coverage, 95%; e-value, 0), Erysiphe necator associated narnavirus 2 (QHD64825.1; identity, 67.2%; query coverage, 95%; e-value, 0), XiangYun narna-levi-like virus 11 (UUG74244.1; identity, 58.9%; query coverage, 95%; e-value, 0) and Botryosphaeria dothidea narnavirus 3 (QQD86177.1; identity, 55.9%; query coverage, 92%; e-value, 0), were most closely related to RdRP. It is noteworthy that the Blastp alignment results showed the highest homology to a partial genomic sequence of MoNV2 (GenBank accession number: LC553714.1; identity, 99.4 %; query coverage, 94%; e-value, 0), which was uploaded by Urayama et al. in June 2020. This sequence was obtained through FLDS (fungal long-read sequencing)-based high-throughput screening, but its accuracy and completeness have not been determined. In addition, a Conserved Domain Database (CDD) search and multiple amino acid sequence comparisons confirmed that the MoNV2-encoded protein contains eight conserved motifs characteristic of (+)ssRNA viral RDRPs(Fig. 2. A), and that RdRps contain a typical ‘GDD’ motif (motif VI, which is not present in MoMNV1 and FpNV2 are absent), which is highly conserved in almost all viruses RdRps\u0026nbsp;[35].\u0026nbsp;\u003c/p\u003e\u003cp\u003eTo determine the phylogenetic relationship of MoNV2, we constructed a phylogenetic tree based on the rdrp-aa sequences of MoNV2 with members of the families \u003cem\u003eNarnaviridae\u003c/em\u003e, \u003cem\u003eBotourmiaviridae\u003c/em\u003e, \u003cem\u003eFiersviridae\u003c/em\u003e, and \u003cem\u003eMitoviridae\u003c/em\u003e using the MEGA version 11 software using the maximal likelihood (ML) method. The evolutionary tree results clearly demonstrate that MoNV2 clusters with other \u003cem\u003eNarnaviridae\u003c/em\u003e in a large branch that remains distinct from members of the families \u003cem\u003eBotourmiaviridae\u003c/em\u003e, \u003cem\u003eFiersviridae\u003c/em\u003e and \u003cem\u003eMitoviridae\u003c/em\u003e(Fig. 2, B).\u003c/p\u003e\u003cp\u003eIn conclusion, we report the first complete genomic sequence of MoNV2. Comparative genomic analysis and phylogenetic analysis indicate that MoNV2 represents a novel member within the family \u003cem\u003eNarnaviridae\u003c/em\u003e, and also that the genomic RNA of MoNV2 has rare inverted complementary termini.\u003c/p\u003e\u003cp\u003e(QBA55490.1)；FpNV2, Fusarium poae narnavirus 2 (YP 009272903.1); MoNV1, Magnaporthe oryzae narnavirus virus 1 (MN480844.1); MoNV2, Magnaporthe oryzae narnavirus virus 2 (PQ871412). Asterisks, colons, and dots represent identical, conserved, and semi-conserved amino acid residues, respectively. Asterisks, colons, and dots represent identical, conserved, and semi-conserved amino acid residues, respectively. \u003cstrong\u003e(B).\u0026nbsp;\u003c/strong\u003ePhylogenetic analysis based on the RDRP sequences of MoNV2 and related viruses of the families \u003cem\u003eNarnaviridae\u003c/em\u003e，\u003cem\u003eBotourmiaviridae\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Fiersviridae,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMitoviridae\u003c/em\u003e. The phylogenetic tree was constructed by the maximal likelihood (ML) method using the program MEGA 11, with 1,000 bootstrap replicates. This tree was rooted with four viruses of \u003cem\u003eFiersviridae\u003c/em\u003e. The position of MoNV2 is indicated by a red star, the other one (MoNV1) previously identified full-length sequnenced viruses in the family \u003cem\u003eNarnaviridae\u003c/em\u003e found in \u003cem\u003eM. oryzae\u003c/em\u003e are indicated by blue stars. Only \u003cem\u003ep\u0026nbsp;\u003c/em\u003evalues for the approximate likelihood ratios (SH test) of \u0026gt;0.5(50%) are indicated. Scale barscorrespond to 0.5 amino acid substitution per site. Sequence accession numbers are given for each sequence.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCong Li\u003c/strong\u003e: Writing \u0026ndash; original draft, Validation, Methodology, Investigation, Data curation, Conceptualization. \u003cstrong\u003eYuxin Wu\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Data curation, Methodology, Investigation,. \u003cstrong\u003eXinyi Li\u003c/strong\u003e: Methodology, Formal analysis.\u003cstrong\u003e\u0026nbsp;Jiatao Xie\u003c/strong\u003e: Resources, Investigation, Project administration, Data curation, Funding acquisition. \u003cstrong\u003eQingchao Deng\u003c/strong\u003e: Resources, Investigation, Project administration, Data curation, Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis thesis is supported by the State Key Laboratory of Agricultural Microbiology (No. AMLKF202209) and the National Natural Science Foundation of China (NFSC No. 31201474). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals\u003c/p\u003e\n\u003cp\u003eperformed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGhabrial SA, Suzuki N (2009) Viruses of plant pathogenic fungi[J]. 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Curr Top Microbiol Immunol 320(1):137\u0026ndash;156. https://doi.org/ 10.1007/978-3-540-75157-1_7\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":"","lastPublishedDoi":"10.21203/rs.3.rs-6299189/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6299189/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel single-stranded (+\u0026thinsp;ss) RNA mycovirus, designated as Magnaporthe oryzae narnavirus 2 (MoNV2), was identified in the rice blast fungus Magnaporthe oryzae isolate NJ471. MoNV2 has a genomic RNA fragment of 3,086 nucleotides, which contains a single open reading frame (ORF) that is predicted to encode an RNA-dependent RNA polymerase (RdRp). Genome sequence comparisons and phylogenetic analysis suggested that MoNV2 is a new member of the genus \u003cem\u003enarnavirus\u003c/em\u003e in the family \u003cem\u003eNarnaviridae\u003c/em\u003e. The 5' and 3' terminal sequences of the genomic RNA of MoNV2 have inverted complementarity and potentially form a panhandle structure, which is very rare in RNA viruses.\u003c/p\u003e","manuscriptTitle":"Complete genome of a novel narnavirus with inverted complementary termini from the rice blast fungus Magnaporthe oryzae isolate NJ471","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 16:34:18","doi":"10.21203/rs.3.rs-6299189/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2025-04-21T21:47:07+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-01T11:36:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T07:26:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-28T04:34:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-03-25T08:07:30+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":"bdce6bdd-e64d-4e55-82e3-7f0e987f3074","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:07:00+00:00","versionOfRecord":{"articleIdentity":"rs-6299189","link":"https://doi.org/10.1007/s00705-025-06337-y","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2025-06-08 15:57:36","publishedOnDateReadable":"June 8th, 2025"},"versionCreatedAt":"2025-04-21 16:34:18","video":"","vorDoi":"10.1007/s00705-025-06337-y","vorDoiUrl":"https://doi.org/10.1007/s00705-025-06337-y","workflowStages":[]},"version":"v1","identity":"rs-6299189","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6299189","identity":"rs-6299189","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}