The full‑length genome sequence of a novel polymycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea

The full‑length genome sequence of a novel polymycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea | 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 The full‑length genome sequence of a novel polymycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea Shunpei Xie, Yanfen Wang, Mingyue Gong, Haiyan Wu, Haiqiang Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6254514/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jun, 2025 Read the published version in Archives of Virology → Version 1 posted 4 You are reading this latest preprint version Abstract Here, a novel dsRNA virus belonging to the family Polymycoviridae was identified in phytopathogenic fungal strain B. dothidea ZM200473, and tentatively named “Botryosphaeria dothidea polymycovirus 2” (BdPmV2). The genome of BdPmV2 have five genomic dsRNAs, ranging from 1224 to 2407 bp, encoding five putative open reading frames (ORFs), of which ORF1 encodes a conserved RNA-dependent RNA polymerase (RdRp) consisting of 763 amino acids (aa) with a molecular mass of 84.03 kDa, ORF3 encodes a putative methyltransferase (Met) consisting of 627 amino acids (aa) with a molecular mass of 68.41 kDa, ORF4 encodes a P-A-S-rich protein behaving as coat protein (CP) consisting of 261 amino acids (aa) with a molecular mass of 27.60 kDa. ORF2 and ORF5 encode putative proteins with unknown functions which consisting of 697 amino acids (aa) with a molecular mass of 76.49 kDa and consisting of 323 amino acids (aa) with a molecular mass of 33.92 kDa, respectively. BLASTp analysis revealed that the RdRp protein of BdPmV2 had the highest similarity 56.52% to a virus previously identified as " RNA dependent RNA polymerase of Aspergillus fumigatus polymycovirus 1". Phylogenetic analysis based on the RdRp aa sequence indicated that BdPmV2 is a new member of the family Polymycoviridae . Figures Figure 1 Figure 2 Introduction Mycoviruses (fungal viruses) are widely distributed across nearly all major groups of fungi [ 1 , 2 ]. Most mycoviruses possess double-stranded RNA (dsRNA) or positive-sense single-stranded RNA (+ ssRNA) genomes, while a smaller number have been reported with negative-sense single-stranded RNA (− ssRNA) or single-stranded DNA (ssDNA) genomes [ 3 ]. Generally, mycoviruses do not appear to significantly affect their fungal hosts; however, some mycoviruses associated with hypovirulence can reduce or even eliminate fungal pathogenicity, while simultaneously conferring heightened resistance in plants. For instance, the Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) has been utilized to transform Sclerotinia sclerotiorum into beneficial endophytes, thereby enhancing wheat yields by providing protection against Fusarium head blight (FHB) and stripe rust [ 4 , 5 ]. Similarly, Stemphylium lycopersici alternavirus 1 (SlAV1) induces hypovirulence and causes pigmentation loss by inhibiting the fungal biosynthesis of the phytotoxin Alternariol A in Stemphylium lycopersici , and the genomic integration and expression of a key SlAV1 gene in the fungal host can convert the pathogen into a biocontrol agent, enhancing plant resistance against virulent strains [ 6 ]. These findings highlight the significant potential of mycoviruses as biocontrol agents in the management of crop diseases. Botryosphaeria dothidea is the type species of Botryosphaeria (family Botryosphaeriaceae, order Botryosphaeriales) [ 7 ]. B. dothidea is a well-known pathogen affecting a variety of woody and horticultural plants, including Malus pumila Mill, Pyrus spp., Eucalyptus spp. [ 8 – 10 ]. This pathogen is known to cause leaf spot, stem canker, shoot blight and dieback in woody plants, severely impacting fruit tree yield and quality and resulting in significant economic losses [ 11 , 12 ]. The advent of high-throughput sequencing technology has led to a rapid increase in the identification and sequencing of mycovirus genomes [ 13 , 14 ]. To date, over 20 mycoviruses have been characterized and reported from the pathogenic fungus B. dothidea , four of which are known to induce hypovirulence: Botryosphaeria dothidea chrysovirus 1 (BdCV1), Botryosphaeria dothidea RNA virus 1 (BdRV1), Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmRV1-BdEW220), and Botryosphaeria dothidea botrexvirus 1 (BdBV1) [ 15 – 20 ]. In this study, we determined the complete genome sequence of a novel polymycovirus derived from B. dothidea strain ZM200473, which we have tentatively designated as “Botryosphaeria dothidea polymycovirus 2” (BdPmV2). Phylogenetic analysis indicates that BdPmV2 is closely related to members of the family Polymycoviridae . Provenance of the virus material The B. dothidea strain ZM20047 was isolated in 2020 from the diseased leaves of Quercus serrata Thunb in Nanyang City, Henan Province, China. This strain was identified as B. dothidea based on morphological characteristics and detection using the specific primers B. d-F (5´-GTCTGCATCATTCTCAGCGTGGG-3´) / B. d-R (5´-TTACCCTCAGTGTAGTGACCCTTG-3´) for B. dothidea [ 21 ]. Strain ZM200473 was cultured on potato dextrose agar (PDA) at 25° C for 3 days in the dark to observe the colony morphology, which exhibited fluffy aerial mycelia, reverse pale gray and white (Fig. 1 A). Mycelium from a pool of 75 strains of B. dothidea (including strain ZM200473), isolated from different plant disease samples in Henan Province, was sent to Novogene Bioinformatics Technology Co. Ltd. for sequencing using the Illumina method. Among the high-quality viral contigs obtained, five contigs (The contig-100_3736, contig-100_12319, contig-100_12415, contig-100_20901 and contig-100_4742) exhibited the highest similarity to Aspergillus fumigatus tetramycovirus 1 by a BLASTx search. Notably, contig-100_3736 showed greatest similarity to the RNA-dependent RNA polymerase of Aspergillus fumigatus tetramycovirus 1 (partial genome; identity, 62.40%; query coverage, 93%; GenBank accession number, BCH36613.1), respectively (Supplementary File S1). Total RNA was extracted using TRIzol Reagent (Adlai, Beijing, China), and cDNA was synthesized was performed with a SuperScript III First-Strand Synthesis System Kit (Vazyme, Nanjing, China). Specific primers were designed based on the five contig sequences, and the presence of the virus in the host (strain ZM200473) was confirmed by RT-PCR. DsRNA was extracted from the mycelium using a previously described column separation method with minor modifications [ 22 ]. A series of dsRNA segments of approximately 1.0–3.0 kbp in size was visualized on a 1.0% agarose gel (Fig. 1 B), excised, and purified using a gel extraction kit (Vazyme, Shanghai, China) according to the manufacturer’s instructions. The dsRNA was subsequently digested with DNase I and S1 nuclease (Takara, Dalian, China) to eliminate DNA and ssRNA. The treated dsRNA was then electrophoresed in a 1.0% (w/v) agarose gel stained with Goldview and photographed on a UV transilluminator. Six dsRNA fragments with estimated sizes between 1.0 and 3.0 kbp were isolated, five of which corresponded to the genome of BdPmV2 (associated with c36267_g1_i1) (Fig. 1 B). The terminal sequences of BdPmV2 were obtained using nested primers designed from central sequences of BdPmV2, employing the ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The PCR products were purified, cloned into the pMD18-T vector (Takara, Dalian, China), and transformed into competent Escherichia coli DH5α cells (Tsingke, Beijing, China). At least three distinct clones covering the same genome region were sequenced to ensure accuracy (Sangon, Shanghai, China). The primer sets used in this study are detailed in Supplementary Table S1 . Final viral genome sequences were assembled using DNAMAN software (version 5.2.2). Potential open reading frames (ORFs) were predicted using ORF Finder ( http://www.unafold.org/mfold/applications/rna-folding-form.php ), while conserved domains within the BdPmV2 genome were identified via the Conserved Domain Database (CDD) Search ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). Sequence comparisons were conducted using the NCBI database, with multiple alignments of reference sequences performed using CLUSTALX 2.0 [ 23 ]. The resulting alignment was annotated and color-coded using GeneDoc software. A phylogenetic tree was constructed using MEGA 11 software employing the maximum likelihood (ML) method with the LG + G + I + F model, supported by a bootstrap test of 1000 replicates [ 24 ]. The resulting phylogenetic tree was further refined and illustrated using Adobe Illustrator CS6. Sequence properties The complete genomic dsRNA sequences of BdPmV2 isolated from strain ZM200473 were obtained through RT-PCR and RNAligase-mediated rapid amplification of cDNA ends (RLMRACE), which contained five full-length dsRNA segments. The sequences corresponding to dsRNAs 1–5 of BdPmV2/ZM200473 have been deposited in GenBank under accession numbers PV264823–PV264827 (Supplementary File S2). A schematic representation of the BdPmV2 genome structure, detailing the coding strands of dsRNAs 1–5, was shown in Fig. 1 C. The 5’ and 3’ termini of dsRNA1 contain untranslated regions (UTRs) of 36 nt and 79 bp, respectively, and potential stem-loop structures were predicted using RNAfold (Fig. 1 D). The 5’-UTRs of the coding strands of dsRNAs 1–5 were 37, 73, 56, 115, and 93 nt in length. The 3’-UTRs were 79, 90, 25, 353, and 160 nt in length, respectively. The 5’ and 3’- termini of coding strands of the five dsRNAs contained conserved 12 nt sequence (GAUUAUAA···CUUG) and 8 nt sequence (CCCCCCCC), respectively, except for the 3' termini of dsRNA3 (Fig. 1 E). Sequence analysis of the full-length cDNAs for dsRNAs 1–5 revealed respective lengths of 2397, 2184, 1967, 1131, and 1060 bp, each containing a single open reading frame (ORF). The corresponding ORFs (ORF1 to ORF5) predictably encode proteins (P1–P5) of 763, 697, 627, 261, and 323 amino acids (aa) with calculated molecular masses of 84, 76, 68, 28, and 34 kDa, respectively. Proteins P1–P4 exhibit substantial amino acid identity with the corresponding proteins of Aspergillus fumigatus tetramycovirus 1 (AfuTmV-1), with identities of 56.62%, 49.28%, 55.01%, and 56.81%. In contrast, P5 shows lower similarity to the hypothetical protein of AfuTmV-1 (identity of 47.83%, query coverage of 20%, GenBank accession number BBU42083.1). Homology analysis indicates that P1 of BdPmV2 is most closely related to the RdRp encoded by AfuTmV-1, sharing 56.62% amino acid sequence identity (Accession number BBU42080, E-value 0.0). The RdRp sequence similarity of BdPmV2 to these viruses is significantly lower than the International Committee on Taxonomy of Viruses (ICTV) cutoff of ≤ 70% sequence identity, which is used to delineate species within the genus Polymycovirus ( https://ictv.global/report/chapter/polymycoviridae/polymycoviridae/polymycovirus ) (Supplementary File S2). To further assess the taxonomic status of BdPmV2, a phylogenetic tree was constructed based on the RdRp aa sequences of BdPmV2 and other selected mycoviruses within the family Polymycoviridae . The results showed that BdPmV2, Botryosphaeria dothidea RNA virus 1 (BdRV1), Aspergillus fumigatus polymycovirus 1 (AfuTmV-1), Penicillium digitatum polymycovirus 1 (PdPmV1) formed a separated clade from other family dsRNA viruses, and BdPmV2 clustered closely (92% bootstrap support) with AfuTmV-1 of the Polymycoviridae (Fig. 2 ). In the clade, BdPmV2 was alone from AfuTmV-1, BdRV1, and PdPmV1[ 17 , 25 , 26 ]. BdRV1 conferred hypovirulence to its host and could be transmitted through conidia and hyphae contact [ 17 ]. Recent studies have shown that polymycoviruses are encapsidated in filamentous virions composed of P-A-S-rich coat proteins (CPs) in CcFV1, BdRV1, PcsPmV1, and PhcPmVs. Moreover, PcsPmV1 infection may enhance the growth and virulence of its fungal hosts [ 27 ]. Hence, further research is needed to evaluate how BdPmV2 influences the biological control potential of B. dothidea . In conclusion, based on the genome organization, sequence comparison and phylogenetic analysis, we propose that Botryosphaeria dothidea polymycovirus 2 (BdPmV2) from B. dothidea strain ZM200473 represents a novel polymycovirus belonging to the family Polymycoviridae . Declarations Acknowledgments This work was supported by Xinjiang Apple Industry Technology System-Disease Prevention (XJLGCYJSTX04-2024-11), National Natural Science Foundation of China (32400008), Natural Science Foundation of Henan (232300420264 & 242300420481), and Henan Agricultural University High-level Talent Special Support Fund Project (30501502). Data availability The data supporting this study's findings are available in the GenBank database. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This study did not include experiments with human participants or animals performed by any of the authors. Supplementary Information The online version contains supplementary material available. References Kondo H, Botella L, Suzuki N (2022) Mycovirus diversity and evolution revealed/inferred from recent studies. 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J Virol 99:e01515–e01524. https://doi.org/10.1128/jvi.01515-24 Supplementary Files BdPmV2SupplementaryFileS1.docx BdPmV2SupplementaryFileS2.docx BdPmV2SupplementaryTableS1.xlsx Submission2934318.txt Cite Share Download PDF Status: Published Journal Publication published 17 Jun, 2025 Read the published version in Archives of Virology → Version 1 posted Reviewers agreed at journal 29 Mar, 2025 Reviewers invited by journal 29 Mar, 2025 Editor assigned by journal 27 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. 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-6254514","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435704896,"identity":"874f5b8c-7355-4bb6-948b-1e64b7dc2503","order_by":0,"name":"Shunpei Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIie3PsWrDMBCAYZkDZTmjVcJ9CJWAQyCkr2JT8FQCJVAyCgxauzpv0alrFA6aJcQP0C6h0NnBa4fagUJbiJKxgz403HA/hxgLgv8Ijq8fwLlGT1AIc3HC8321KK5U5S461MPhELc00Sbzr+sNUHtv32ZiwFIZ2xo1c1FzuDudqJIXydJ+zFXJCqnsK47AgFo+n04EYAqxpfyJ2Iu87pKxcRxiT8JBtG2frCiyMrc71C7zJ90VlhyvAIB2W3c+6f6SJrijuSQe7c3iFlW1Lr1/0TW9t/hAM/FYN/SppzdClOvm4Em+ZT/myJzf/5MEQRAEv30BcLJPvsMM9lIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0009-9079-7829","institution":"Henan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Shunpei","middleName":"","lastName":"Xie","suffix":""},{"id":435704897,"identity":"540be062-edc2-4cd2-872d-f019d475ae78","order_by":1,"name":"Yanfen Wang","email":"","orcid":"","institution":"Henan Institute of Technology: Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanfen","middleName":"","lastName":"Wang","suffix":""},{"id":435704898,"identity":"eec7d6e4-9f84-4e3c-b955-60b323d6fdea","order_by":2,"name":"Mingyue Gong","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mingyue","middleName":"","lastName":"Gong","suffix":""},{"id":435704899,"identity":"9482aeac-2ad8-43f3-a7db-4846ccd3ca73","order_by":3,"name":"Haiyan Wu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Wu","suffix":""},{"id":435704900,"identity":"2e8e9298-7f3f-496c-9b27-44b27e67f8a4","order_by":4,"name":"Haiqiang Li","email":"","orcid":"","institution":"Xinjiang Academy of Animal Science","correspondingAuthor":false,"prefix":"","firstName":"Haiqiang","middleName":"","lastName":"Li","suffix":""},{"id":435704901,"identity":"f243bba8-955d-4f2c-a071-4a44f9e073ec","order_by":5,"name":"Qinzhou Ma","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qinzhou","middleName":"","lastName":"Ma","suffix":""},{"id":435704902,"identity":"77a509f9-47cf-4599-b138-6e1899a77651","order_by":6,"name":"Yashuang Guo","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yashuang","middleName":"","lastName":"Guo","suffix":""},{"id":435704903,"identity":"817297ec-db7a-4f3f-8b45-54afb4c69192","order_by":7,"name":"Yuehua Geng","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuehua","middleName":"","lastName":"Geng","suffix":""},{"id":435704904,"identity":"ce93c1a4-b8e7-471c-982b-2d984f9ce351","order_by":8,"name":"Bingshan Liu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bingshan","middleName":"","lastName":"Liu","suffix":""},{"id":435704905,"identity":"30fb7929-315f-4280-91ae-ef4aba1e73ee","order_by":9,"name":"Meng Zhang","email":"","orcid":"https://orcid.org/0000-0003-1941-1136","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-18 15:12:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6254514/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6254514/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-025-06330-5","type":"published","date":"2025-06-17T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80906050,"identity":"98a21e51-9a7e-4fe7-8436-036ed86bb2fa","added_by":"auto","created_at":"2025-04-18 14:43:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1079802,"visible":true,"origin":"","legend":"\u003cp\u003eColony morphology, electrophoretic analysis, genomic characteristics, secondary structural projections and conserved sequences of the 5’ and 3’-UTRs. A: The colony morphology of \u003cem\u003eB. dothidea\u003c/em\u003e strains ZM200473. The left photo shows the obverse side of morphology, while the right is the reverse side of the corresponding morphology. B: The double-stranded RNA (dsRNA), resolved by electrophoresis in a 1.0% agarose gel. Lane M, DL5000 DNA marker; Lanes N represent dsRNA extracted from strains ZM200473. Lanes D and N represent dsRNA from ZM200473 treated with DNase I and S1 nuclease, respectively. C: Schematic representation of the genome organization of BdPmV2, with key nucleotides and amino acids indicated. The open reading frames (ORF1–ORF5) and the untranslated regions (UTRs) are indicated by a pale blue box and double black lines, respectively. D: The putative secondary structures of the 5’ and 3’ termini UTRs of BdPmV2. E: Conserved sequences of the 5’ and 3’ termini UTRs of the dsRNAs of BdPmV2 and BdRV1. Black, grey, and light grey backgrounds indicate denote nucleotide identity no less than 100%, 80%, and 60%, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6254514/v1/651d4bf57464850f28454d51.jpeg"},{"id":80906045,"identity":"df8f5057-a49f-48c6-967f-77da90989c75","added_by":"auto","created_at":"2025-04-18 14:43:30","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1663222,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular phylogeny of BdPmV2. Phylogenetic tree constructed based on the RdRp sequence of BdPmV2 and those of selected members of the families \u003cem\u003eChrysoviridae\u003c/em\u003e, \u003cem\u003eTotiviridae\u003c/em\u003e, \u003cem\u003ePolymycovirida\u003c/em\u003e, \u003cem\u003ePartitiviridae\u003c/em\u003e, \u003cem\u003eAmalgamaviridae\u003c/em\u003e \u003cem\u003eReoviridae\u003c/em\u003e and Unclassified dsRNA viruses using the maximum-likelihood (ML) method in MEGA 11 with 1000 bootstrap replicates. BdPmV2 is highlighted in red and marked with a five-pointed star. Bootstrap values larger than 60% are shown. The scale bar represents a genetic distance of 0.1.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6254514/v1/e66b1b0db6afc50cd8ffe0c8.jpeg"},{"id":85232010,"identity":"d39bc6bd-e118-4867-a193-e8066c8540d5","added_by":"auto","created_at":"2025-06-23 16:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3151716,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6254514/v1/b3000e57-baf1-430b-99b8-43c5624fee45.pdf"},{"id":80906042,"identity":"967af772-f92e-450f-b7a0-a041ae1fde5e","added_by":"auto","created_at":"2025-04-18 14:43:30","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17965,"visible":true,"origin":"","legend":"","description":"","filename":"BdPmV2SupplementaryFileS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6254514/v1/a1df4efadb5d2b9e6c49f711.docx"},{"id":80906049,"identity":"8d149642-485a-4ad8-ad0b-795d596c11d1","added_by":"auto","created_at":"2025-04-18 14:43:30","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":671904,"visible":true,"origin":"","legend":"","description":"","filename":"BdPmV2SupplementaryFileS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6254514/v1/b7374d25203c76efeaf00f90.docx"},{"id":80906043,"identity":"3d13cf03-b367-4bee-8c8f-5438702d37ed","added_by":"auto","created_at":"2025-04-18 14:43:30","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10565,"visible":true,"origin":"","legend":"","description":"","filename":"BdPmV2SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6254514/v1/dc63d4ab7431df3fd716bbe5.xlsx"},{"id":80906349,"identity":"a6165c79-0abe-48a4-bd27-7caf2581cf53","added_by":"auto","created_at":"2025-04-18 14:51:30","extension":"txt","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":24426,"visible":true,"origin":"","legend":"","description":"","filename":"Submission2934318.txt","url":"https://assets-eu.researchsquare.com/files/rs-6254514/v1/af2e1323eaf88dc86e58bd9f.txt"}],"financialInterests":"","formattedTitle":"The full‑length genome sequence of a novel polymycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycoviruses (fungal viruses) are widely distributed across nearly all major groups of fungi [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Most mycoviruses possess double-stranded RNA (dsRNA) or positive-sense single-stranded RNA (+\u0026thinsp;ssRNA) genomes, while a smaller number have been reported with negative-sense single-stranded RNA (\u0026minus;\u0026thinsp;ssRNA) or single-stranded DNA (ssDNA) genomes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Generally, mycoviruses do not appear to significantly affect their fungal hosts; however, some mycoviruses associated with hypovirulence can reduce or even eliminate fungal pathogenicity, while simultaneously conferring heightened resistance in plants. For instance, the Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) has been utilized to transform \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e into beneficial endophytes, thereby enhancing wheat yields by providing protection against Fusarium head blight (FHB) and stripe rust [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, Stemphylium lycopersici alternavirus 1 (SlAV1) induces hypovirulence and causes pigmentation loss by inhibiting the fungal biosynthesis of the phytotoxin Alternariol A in \u003cem\u003eStemphylium lycopersici\u003c/em\u003e, and the genomic integration and expression of a key SlAV1 gene in the fungal host can convert the pathogen into a biocontrol agent, enhancing plant resistance against virulent strains [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These findings highlight the significant potential of mycoviruses as biocontrol agents in the management of crop diseases.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBotryosphaeria dothidea\u003c/em\u003e is the type species of \u003cem\u003eBotryosphaeria\u003c/em\u003e (family Botryosphaeriaceae, order Botryosphaeriales) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eB. dothidea\u003c/em\u003e is a well-known pathogen affecting a variety of woody and horticultural plants, including \u003cem\u003eMalus pumila\u003c/em\u003e Mill, \u003cem\u003ePyrus\u003c/em\u003e spp., \u003cem\u003eEucalyptus\u003c/em\u003e spp. [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This pathogen is known to cause leaf spot, stem canker, shoot blight and dieback in woody plants, severely impacting fruit tree yield and quality and resulting in significant economic losses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The advent of high-throughput sequencing technology has led to a rapid increase in the identification and sequencing of mycovirus genomes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To date, over 20 mycoviruses have been characterized and reported from the pathogenic fungus \u003cem\u003eB. dothidea\u003c/em\u003e, four of which are known to induce hypovirulence: Botryosphaeria dothidea chrysovirus 1 (BdCV1), Botryosphaeria dothidea RNA virus 1 (BdRV1), Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmRV1-BdEW220), and Botryosphaeria dothidea botrexvirus 1 (BdBV1) [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we determined the complete genome sequence of a novel polymycovirus derived from \u003cem\u003eB. dothidea\u003c/em\u003e strain ZM200473, which we have tentatively designated as \u0026ldquo;Botryosphaeria dothidea polymycovirus 2\u0026rdquo; (BdPmV2). Phylogenetic analysis indicates that BdPmV2 is closely related to members of the family \u003cem\u003ePolymycoviridae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Provenance of the virus material","content":"\u003cp\u003eThe \u003cem\u003eB. dothidea\u003c/em\u003e strain ZM20047 was isolated in 2020 from the diseased leaves of \u003cem\u003eQuercus serrata\u003c/em\u003e Thunb in Nanyang City, Henan Province, China. This strain was identified as \u003cem\u003eB. dothidea\u003c/em\u003e based on morphological characteristics and detection using the specific primers B. d-F (5\u0026acute;-GTCTGCATCATTCTCAGCGTGGG-3\u0026acute;) / B. d-R (5\u0026acute;-TTACCCTCAGTGTAGTGACCCTTG-3\u0026acute;) for \u003cem\u003eB. dothidea\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Strain ZM200473 was cultured on potato dextrose agar (PDA) at 25\u0026deg; C for 3 days in the dark to observe the colony morphology, which exhibited fluffy aerial mycelia, reverse pale gray and white (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMycelium from a pool of 75 strains of \u003cem\u003eB. dothidea\u003c/em\u003e (including strain ZM200473), isolated from different plant disease samples in Henan Province, was sent to Novogene Bioinformatics Technology Co. Ltd. for sequencing using the Illumina method. Among the high-quality viral contigs obtained, five contigs (The contig-100_3736, contig-100_12319, contig-100_12415, contig-100_20901 and contig-100_4742) exhibited the highest similarity to Aspergillus fumigatus tetramycovirus 1 by a BLASTx search. Notably, contig-100_3736 showed greatest similarity to the RNA-dependent RNA polymerase of Aspergillus fumigatus tetramycovirus 1 (partial genome; identity, 62.40%; query coverage, 93%; GenBank accession number, BCH36613.1), respectively (Supplementary File S1). Total RNA was extracted using TRIzol Reagent (Adlai, Beijing, China), and cDNA was synthesized was performed with a SuperScript III First-Strand Synthesis System Kit (Vazyme, Nanjing, China). Specific primers were designed based on the five contig sequences, and the presence of the virus in the host (strain ZM200473) was confirmed by RT-PCR. DsRNA was extracted from the mycelium using a previously described column separation method with minor modifications [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A series of dsRNA segments of approximately 1.0\u0026ndash;3.0 kbp in size was visualized on a 1.0% agarose gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), excised, and purified using a gel extraction kit (Vazyme, Shanghai, China) according to the manufacturer\u0026rsquo;s instructions. The dsRNA was subsequently digested with DNase I and S1 nuclease (Takara, Dalian, China) to eliminate DNA and ssRNA. The treated dsRNA was then electrophoresed in a 1.0% (w/v) agarose gel stained with Goldview and photographed on a UV transilluminator. Six dsRNA fragments with estimated sizes between 1.0 and 3.0 kbp were isolated, five of which corresponded to the genome of BdPmV2 (associated with c36267_g1_i1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The terminal sequences of BdPmV2 were obtained using nested primers designed from central sequences of BdPmV2, employing the ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The PCR products were purified, cloned into the pMD18-T vector (Takara, Dalian, China), and transformed into competent \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α cells (Tsingke, Beijing, China). At least three distinct clones covering the same genome region were sequenced to ensure accuracy (Sangon, Shanghai, China). The primer sets used in this study are detailed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFinal viral genome sequences were assembled using DNAMAN software (version 5.2.2). Potential open reading frames (ORFs) were predicted using ORF Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.unafold.org/mfold/applications/rna-folding-form.php\u003c/span\u003e\u003cspan address=\"http://www.unafold.org/mfold/applications/rna-folding-form.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), while conserved domains within the BdPmV2 genome were identified via the Conserved Domain Database (CDD) Search (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Sequence comparisons were conducted using the NCBI database, with multiple alignments of reference sequences performed using CLUSTALX 2.0 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The resulting alignment was annotated and color-coded using GeneDoc software. A phylogenetic tree was constructed using MEGA 11 software employing the maximum likelihood (ML) method with the LG\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;F model, supported by a bootstrap test of 1000 replicates [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The resulting phylogenetic tree was further refined and illustrated using Adobe Illustrator CS6.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSequence properties\u003c/h2\u003e \u003cp\u003eThe complete genomic dsRNA sequences of BdPmV2 isolated from strain ZM200473 were obtained through RT-PCR and RNAligase-mediated rapid amplification of cDNA ends (RLMRACE), which contained five full-length dsRNA segments. The sequences corresponding to dsRNAs 1\u0026ndash;5 of BdPmV2/ZM200473 have been deposited in GenBank under accession numbers PV264823\u0026ndash;PV264827 (Supplementary File S2). A schematic representation of the BdPmV2 genome structure, detailing the coding strands of dsRNAs 1\u0026ndash;5, was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. The 5\u0026rsquo; and 3\u0026rsquo; termini of dsRNA1 contain untranslated regions (UTRs) of 36 nt and 79 bp, respectively, and potential stem-loop structures were predicted using RNAfold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The 5\u0026rsquo;-UTRs of the coding strands of dsRNAs 1\u0026ndash;5 were 37, 73, 56, 115, and 93 nt in length. The 3\u0026rsquo;-UTRs were 79, 90, 25, 353, and 160 nt in length, respectively. The 5\u0026rsquo; and 3\u0026rsquo;- termini of coding strands of the five dsRNAs contained conserved 12 nt sequence (GAUUAUAA\u0026middot;\u0026middot;\u0026middot;CUUG) and 8 nt sequence (CCCCCCCC), respectively, except for the 3' termini of dsRNA3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Sequence analysis of the full-length cDNAs for dsRNAs 1\u0026ndash;5 revealed respective lengths of 2397, 2184, 1967, 1131, and 1060 bp, each containing a single open reading frame (ORF). The corresponding ORFs (ORF1 to ORF5) predictably encode proteins (P1\u0026ndash;P5) of 763, 697, 627, 261, and 323 amino acids (aa) with calculated molecular masses of 84, 76, 68, 28, and 34 kDa, respectively. Proteins P1\u0026ndash;P4 exhibit substantial amino acid identity with the corresponding proteins of Aspergillus fumigatus tetramycovirus 1 (AfuTmV-1), with identities of 56.62%, 49.28%, 55.01%, and 56.81%. In contrast, P5 shows lower similarity to the hypothetical protein of AfuTmV-1 (identity of 47.83%, query coverage of 20%, GenBank accession number BBU42083.1).\u003c/p\u003e \u003cp\u003eHomology analysis indicates that P1 of BdPmV2 is most closely related to the RdRp encoded by AfuTmV-1, sharing 56.62% amino acid sequence identity (Accession number BBU42080, E-value 0.0). The RdRp sequence similarity of BdPmV2 to these viruses is significantly lower than the International Committee on Taxonomy of Viruses (ICTV) cutoff of \u0026le;\u0026thinsp;70% sequence identity, which is used to delineate species within the genus \u003cem\u003ePolymycovirus\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ictv.global/report/chapter/polymycoviridae/polymycoviridae/polymycovirus\u003c/span\u003e\u003cspan address=\"https://ictv.global/report/chapter/polymycoviridae/polymycoviridae/polymycovirus\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Supplementary File S2). To further assess the taxonomic status of BdPmV2, a phylogenetic tree was constructed based on the RdRp aa sequences of BdPmV2 and other selected mycoviruses within the family \u003cem\u003ePolymycoviridae\u003c/em\u003e. The results showed that BdPmV2, Botryosphaeria dothidea RNA virus 1 (BdRV1), Aspergillus fumigatus polymycovirus 1 (AfuTmV-1), Penicillium digitatum polymycovirus 1 (PdPmV1) formed a separated clade from other family dsRNA viruses, and BdPmV2 clustered closely (92% bootstrap support) with AfuTmV-1 of the \u003cem\u003ePolymycoviridae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the clade, BdPmV2 was alone from AfuTmV-1, BdRV1, and PdPmV1[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBdRV1 conferred hypovirulence to its host and could be transmitted through conidia and hyphae contact [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent studies have shown that polymycoviruses are encapsidated in filamentous virions composed of P-A-S-rich coat proteins (CPs) in CcFV1, BdRV1, PcsPmV1, and PhcPmVs. Moreover, PcsPmV1 infection may enhance the growth and virulence of its fungal hosts [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Hence, further research is needed to evaluate how BdPmV2 influences the biological control potential of \u003cem\u003eB. dothidea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, based on the genome organization, sequence comparison and phylogenetic analysis, we propose that Botryosphaeria dothidea polymycovirus 2 (BdPmV2) from \u003cem\u003eB. dothidea\u003c/em\u003e strain ZM200473 represents a novel polymycovirus belonging to the family \u003cem\u003ePolymycoviridae\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Xinjiang Apple Industry Technology System-Disease Prevention (XJLGCYJSTX04-2024-11), National Natural Science Foundation of China (32400008), Natural Science Foundation of Henan (232300420264 \u0026amp; 242300420481), and Henan Agricultural University High-level Talent Special Support Fund Project (30501502).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s findings are available in the GenBank database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not include experiments with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKondo H, Botella L, Suzuki N (2022) Mycovirus diversity and evolution revealed/inferred from recent studies. 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J Virol 99:e01515\u0026ndash;e01524. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jvi.01515-24\u003c/span\u003e\u003cspan address=\"10.1128/jvi.01515-24\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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-6254514/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6254514/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHere, a novel dsRNA virus belonging to the family \u003cem\u003ePolymycoviridae\u003c/em\u003e was identified in phytopathogenic fungal strain \u003cem\u003eB. dothidea\u003c/em\u003e ZM200473, and tentatively named \u0026ldquo;Botryosphaeria dothidea polymycovirus 2\u0026rdquo; (BdPmV2). The genome of BdPmV2 have five genomic dsRNAs, ranging from 1224 to 2407 bp, encoding five putative open reading frames (ORFs), of which ORF1 encodes a conserved RNA-dependent RNA polymerase (RdRp) consisting of 763 amino acids (aa) with a molecular mass of 84.03 kDa, ORF3 encodes a putative methyltransferase (Met) consisting of 627 amino acids (aa) with a molecular mass of 68.41 kDa, ORF4 encodes a P-A-S-rich protein behaving as coat protein (CP) consisting of 261 amino acids (aa) with a molecular mass of 27.60 kDa. ORF2 and ORF5 encode putative proteins with unknown functions which consisting of 697 amino acids (aa) with a molecular mass of 76.49 kDa and consisting of 323 amino acids (aa) with a molecular mass of 33.92 kDa, respectively. BLASTp analysis revealed that the RdRp protein of BdPmV2 had the highest similarity 56.52% to a virus previously identified as \" RNA dependent RNA polymerase of Aspergillus fumigatus polymycovirus 1\". Phylogenetic analysis based on the RdRp aa sequence indicated that BdPmV2 is a new member of the family \u003cem\u003ePolymycoviridae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"The full‑length genome sequence of a novel polymycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-18 14:43:25","doi":"10.21203/rs.3.rs-6254514/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-29T13:00:57+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-29T12:08:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T07:44:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-03-25T07:38:06+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":"37b69a70-5165-4d14-9704-e1902f667cad","owner":[],"postedDate":"April 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T16:08:29+00:00","versionOfRecord":{"articleIdentity":"rs-6254514","link":"https://doi.org/10.1007/s00705-025-06330-5","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2025-06-17 15:57:39","publishedOnDateReadable":"June 17th, 2025"},"versionCreatedAt":"2025-04-18 14:43:25","video":"","vorDoi":"10.1007/s00705-025-06330-5","vorDoiUrl":"https://doi.org/10.1007/s00705-025-06330-5","workflowStages":[]},"version":"v1","identity":"rs-6254514","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6254514","identity":"rs-6254514","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}