Plant (meta)transcriptome data mining identified twenty-two putative novel taxa in the family Closteroviridae

Plant (meta)transcriptome data mining identified twenty-two putative novel taxa in the family Closteroviridae | 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 Plant (meta)transcriptome data mining identified twenty-two putative novel taxa in the family Closteroviridae V Kavi Sidharthan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7560636/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The family Closteroviridae comprises filamentous, RNA genome-containing viruses that infect plants. In the present study, public domain SRA libraries derived from plants were mined for novel closteroviral sequences, resulting in the identification of twenty-two putative novel closterovirids across twenty-one plant genera. The identified viruses were represented by eighteen coding-complete and four partial genomes. Based on genome organization, pairwise sequence identity and phylogenetic analysis, the viruses were classified in the following genera: Ampelovirus (6), Bluvavirus (1), Closterovirus (7), Olivavirus (2) and Velarivirus (2), while four other viruses may represent four novel genera within the family. Other significant findings of the study include: (i) the identification of a 3’→5’ exonuclease-like protein in ampeloviruses and olivaviruses, (ii) the identification of an ampelovirus that encodes a polyprotein containing an RNA-dependent RNA polymerase motif without employing a + 1 ribosomal frameshift, (iii) the identification of a virus with the largest known genome among closterovirids, and (iv) the identification of a monopartite crini-like virus in Musa hosts potentially representing a novel genus. Besides, expanding the known closterovirid diversity by 0.25-fold, this study provides a base for future research aimed at understanding the biology and distribution of the identified novel viruses. Closteroviridae data-driven virus discovery diversity host range novel Figures Figure 1 Figure 2 Figure 3 1. Introduction The family Closteroviridae includes filamentous, plant-infecting viruses with positive sense single-stranded RNA genomes (Fuchs et al., 2020). Members of this family are known to cause economically important plant diseases such as citrus tristeza, beet yellows, lettuce infectious yellows and grapevine leafroll (German-Retana et al., 2004 , Rybicki and Foster, 2024 ). In general, the closterovirid-incited plant diseases depict leaf discolouration symptoms, whilst a few diseases present leaf roll and stem pitting/grooving symptoms. Though the closterovirids cause systemic infection in plants, they are generally phloem-limited (Fuchs et al., 2020). Historically, characterization of beet yellows virus (BYV) led to the recognition of the earlier-accepted ‘ Closterovirus group’ by the International Committee on Taxonomy of Viruses (ICTV), the group name being derived from the Greek word ‘kloster’ referring to the thread-like virus particles. In the year 2000, the family Closteroviridae was established with two genera— Closterovirus (monopartite genome, over 1000 nm long virions) and Crinivirus (split genome, less than 1000 nm long virions with two modal lengths). Subsequent taxonomical revisions led to the formation of two new genera— Ampelovirus and Velarivirus (Martelli, 2019 ). The recent recognition of three new genera— Bluvavirus , Menthavirus and Olivavirus —by the ICTV (Sabanadzovic et al., 2021 ), makes the current number of recognized genera in the family to seven. Until 2024, the family Closteroviridae comprised 58 recognized species across genera— Ampelovirus (13), Bluvavirus (1), Closterovirus (17), Crinivirus (14), Menthavirus (1), Olivavirus (3) and Velarivirus (9) (Fuchs et al., 2020). In 2025, 31 additional species were recognized, while a species was excluded in the family (Aboughanem-Sabanadzovic et al., 2025 ). Virions of closterovirids are non-enveloped and appear as flexuous filaments (12 nm diameter x 650–2200 nm) (Fuchs et al., 2020). Their genomes consist of a monopartite (except in criniviruses) or bi-/tripartite (in criniviruses) RNA molecule(s), with lengths ranging from 13 kb to 19.3 kb (combined length in case of criniviruses), making them the largest among the plant viruses (Candresse and Fuchs, 2020 , Fuchs et al., 2020). Sequence duplication and RNA recombination mediated non-viral coding sequence acquisition (heat shock protein 70-like protein (HSP70) and protease) account for the large genome size of closterovirids. Though the number and relative position of open reading frames (ORFs) differ across genera and species in the family Closteroviridae , their genomes invariably encode a large replicase polyprotein with the conserved methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRP) motifs from ORFs 1a and 1b, typically expressed via a + 1 ribosomal frameshift (RFS). Downstream of the replicase-encoding ORFs is the conserved five-gene module coding for a small hydrophobic protein (ca. 6 kDa), the HSP70, a ca. 60 kDa protein, a major (CP) and a minor coat protein (CPm) in the 5’ to 3’ direction. Exceptions to this are the members of the genus Closterovirus that have CPm gene upstream of CP gene and subgroup II ampeloviruses that lack CPm gene. The closterovirid genome possibly has the 5’ cap but its 3’ end lacks the polyadenylated tail and the transfer RNA-like structure (Candresse and Fuchs, 2020 , Fuchs et al., 2020). Closterovirids are known to be semi-persistently transmitted by various sap-sucking hemipterans—aphids vector closteroviruses and the sole known menthavirus, mealybugs and soft scales vector ampeloviruses and whiteflies vector criniviruses. Vectors are not known for bluvavirus, olivaviruses and velariviruses (Candresse and Fuchs, 2020 , Fuchs et al., 2020, Sabanadzovic et al., 2021 ). In recent times, plant transcriptome data have increasingly been deposited in the public-domain sequence data repositories like National Centre for Biotechnology Information (NCBI). Besides the host transcipts, these datasets may also contain sequences of plant viruses if the sampled host was infected at the time of sampling. This facilitates the comprehensive exploration of non-host fraction of transcriptome data of a wide range of plant species for sequences of novel viruses- a process known as data-driven virus discovery (DDVD), which otherwise would require the costlier Next Generation Sequencing (NGS)-based virome studies (Lauber and Seitz, 2022 , Sidharthan and Baranwal, 2024 ). DDVD studies have considerably expanded the genetic diversity and host range of several plant virus groups like alphaflexiviruses (Sravani et al., 2024 ), amalgaviruses (Nibert et al., 2016 , Sidharthan et al., 2022 ), enamoviruses and poleroviruses (Sidharthan et al., 2025 ), kitaviruses (Reddy and Sidharthan, 2024 ), rhabdoviruses (Bejerman et al., 2021 , 2023 , Bejerman and Debat, 2025 ), secoviruses (Sidharthan et al., 2023 , 2024 ), tymoviruses (Bejerman and Debat, 2022 ) and varicosaviruses (Bejerman et al., 2022 ). In our previous study, we identified the genome of a novel closterovirus in the transcriptome-assembled contigs of dwarf polish wheat (Sidharthan and Baranwal, 2022 ). Considering the vast amount of plant transcriptome data deposited in the sequence read archives (SRA) database of NCBI over the transcriptome shotgun assembly (TSA) database (Lauber and Seitz, 2022 ), we assumed that the existing plant transcriptome data in SRA database would contain sequences of novel closteroviruses and that their discovery would expand the phylogenetic diversity of the family Closteroviridae . Thus, we mined the existing plant (meta)transcriptome data in the SRA database for novel closteroviral sequences and identified twenty-two putative novel taxa in the family Closteroviridae . 2. Materials and methods 2.1. Identification of plant (meta)transcriptome libraries with reads of putative novel closteroviruses Putative novel closterovirus-positive SRA libraries were identified in the Serratus RdRP search (Edgar et al., 2022 ) using the family Closteroviridae as query with default parameters (alignment identity: ≥45%, score: ≥50). The resulting libraries derived from plants and sharing < 90% identity with known closteroviruses were only retained for further analysis. 2.2. Identification of putative novel closteroviruses from closterovirus-positive libraries and their genome recovery Plant SRA libraries tentatively identified to have reads of putative novel closteroviruses were imported into the Galaxy server ( https://usegalaxy.org/ ) (Galaxy Community, 2022) and trimmed using the Trimmomatic tool v 0.39 (Bolger et al., 2014 ) to remove adapter sequences and sequences with average quality score < 25. Trimmed libraries were de novo assembled using MEGAHIT v 1.2.9 (Li et al., 2015 ) and subjected to BLASTx analysis (evalue cutoff: 1e-5) against a database made of protein sequences of known closteroviruses (retrieved from https://www.ncbi.nlm.nih.gov/labs/virus/vssi/ ) using the NCBI BLAST + tool v 2.16.0 (Cock et al., 2015 ). The largest matching hit to an individual closteroviral genus in each library was subjected to ORF prediction using the NCBI’s ORF finder tool ( https://www.ncbi.nlm.nih.gov/orffinder/ ) to determine the presence of the conserved closteroviral ORFs, and the encoded RdRP, HSP70 and CP protein sequences were analysed through BLASTp against NCBI’s non-redundant protein sequences (nr) database. If the sequences of analysed proteins shared < 75% identity to the existing sequences in GenBank and the intact contig length corresponded to the genome length of related closterovirus, it was regarded as coding-complete genome of a putative novel closterovirus. If the coding-complete genome of a putative novel closterovirus could not be recovered, the corresponding trimmed library was de novo assembled using rnaSPAdes v 4.2.0 (Bushmanova et al., 2019 ) and/or Trinity v 2.15.1 (Grabherr et al., 2011 ) assemblers and the resulting contigs were analysed to identify the coding-complete genome. In two instances, the overlapping contigs assembled by MEGAHIT and rnaSPAdes were manually assembled to obtain the coding-complete genome. Further, if the contigs were relatively shorter and did not possess all the conserved closteroviral ORFs, they were regarded as partial genome sequences of the identified putative novel closteroviruses. In instances where more than one putative novel closterovirus-positive SRA library was available for a host species, the library with the maximum number of closteroviral reads in each independent bioproject was analysed. Genome coverage estimates were obtained using the QualiMap v 2.3 (Okonechnikov et al., 2016 ) tool after mapping the trimmed reads onto the recovered closteroviral genome sequences using the HISAT2 v 2.2.1 (Kim et al., 2015 ) tool. 2.3. Genome annotation of putative novel closteroviruses ORFs determined in the recovered genomes were annotated based on BLASTp analysis of encoded proteins against nr database and motifs predicted using the MOTIF Search tool ( https://www.genome.jp/tools/motif/ ). Molecular weight of encoded proteins was determined using the Expasy tool ( https://web.expasy.org/compute_pi/ ) while the transmembrane helices (TMHs) were predicted using the Phobius tool ( https://phobius.sbc.su.se/ ). 2.4. Phylogenetic and sequence identity analyses Protein sequences of RdRP, HSP70 and CP genes of identified and recognized closteroviruses were aligned using the MUSCLE tool implemented in MEGA7 v 7.0.26 (Kumar et al., 2016 ). Best-fit models were identified for the individual protein alignment using the ‘Find Best DNA/Protein Models’ tool available in MEGA7. Using the determined best-fit model, maximum-likelihood (ML) tree was constructed for each protein in MEGA7 with 100 bootstrap replicates. Pairwise sequence identity values were obtained using the Sequence Demarcation Tool v 1.2 (Muhire et al., 2014 ). 3. Results 3.1. Identification of putative novel members of the family Closteroviridae in plant (meta)transcriptomes In total, sequences of 22 putative novel closterovirids were identified in 21 plant genera belonging to 19 plant families. Of these, 15 are dicot families, 3 are monocot families and 1 is a gymnosperm family. In each of the family Asteraceae, Betulaceae and Juglandaceae, two putative novel closterovirids were identified. Coding-complete genomes of 18 identified novel closterovirids were obtained while for the remaining 4 viruses, only partial genome sequences were recovered. Sequences of three viruses were identified in two independent (meta)transcriptome libraries of the same plant species/genus. Mean depth of identified viral genome sequences ranged from 8.1x to 2,325.8x across libraries (Table 1). 3.2. Identification of putative novel ampeloviruses in plant (meta)transcriptomes Genomes of six putative novel ampeloviruses—black pepper ampelovirus 1 (BPAV1), Leucadendron ampelovirus 1 (LeuAV1), Matucana ampelovirus 1 (MatAV1), oak ampelovirus 1 (OaAV1), pecan ampelovirus 1 (PecAV1), turmeric ampelovirus 1 (TurAV1)—were identified in transcriptomes of six plant species (5 dicots and 1 monocot), of which five are coding-complete (except LeuAV1) and one is partial (LeuAV1) (Table 1). In case of OaAV1, coding-complete genome of two isolates sharing 88% sequence identity were obtained from two independent bioprojects of oak. Genome organization of BPAV1 and TurAV1 were similar and contained seven ORFs. ORFs 1a and 1b, possibly via a + 1 RFS, encode a replicase polyprotein with viral MTR (PF01660), HEL (PF01443) and RdRP (PF00978) motifs. ORF2 encodes a putative transmembrane protein (5–6 kDa) with no predicted motif (BPAV1)/ MP p6 (PF06716) motif. ORFs 3, 4 and 5 encodes HSP70 (59–60 kDa), heat shock protein 90 homolog (HSP90h) (61 kDa) and CP (28–36 kDa), respectively with HSP70 (PF00012), HSP90h (PF03225) and closterovirus CP (PF01785) motif while ORF6 encodes a 24 kDa hypothetical protein with no predicted motif. The encoded proteins of both the viruses, except the transmembrane protein of BPAV1, shared a maximum of 67% identity (at maximum query coverage) with the corresponding sequences of known ampeloviruses in BLAST analysis (Table S1). Genomes of LeuAV1, MatAV1, OaAV1 and PecAV1 invariably encode replicase polyprotein (from ORFs 1a and 1b via possible + 1 RFS, except OaAV1 where ORF1 directly encodes replicase), a small transmembrane MP-like protein (5–6 kDa), HSP70 (59–61 kDa), HSP90h (54–58 kDa, excluding the partial sequence of LeuAV1), CP (32–39 kDa) and CPm (55–61 kDa) in the order except the reverse orientation of CP and CPm ORFs in MatAV. Replicase encoded by MatAV1 contains an additional 2OG-Fe(II) oxygenase superfamily motif (PF13532) in between MET and HEL motifs. BLASTp analysis of these encoded proteins revealed a maximum of 72% identity with the corresponding sequences of known ampeloviruses. Additionally, each of these viral genomes contain ORFs encoding hypothetical proteins: two ORFs in LeuAV1 (downstream of the CPm ORF) and MatAV1 (one between the CPm and CP ORFs, and the other downstream of the CP ORF); four ORFs in OaAV (downstream of CPm ORF); and six ORFs in PecAV1 (one between ORF1b and MP-like ORF, and the remaining ORFs located downstream of the CPm ORF). This includes those encoding a 31 kDa thaumatin-like protein (PF00314) and a 25 kDa 3'→5' exonuclease-like protein (PF01612) of PecAV1, and a papain family cysteine protease (PF00112) of OaAV1 isolates (Table S1). Pairwise sequence identity matrix revealed 38.9–69.2% amino acid sequence identity of RdRP, HSP70 and CP encoded by the identified novel ampeloviruses with that of known members (Figs. S1–S3, Table S2). Phylogenetic analysis based on RdRP, HSP70 and CP sequences grouped LeuAV1, MatAV1, OaAV1 and PecAV1 with subgroup I ampeloviruses, while BPAV1 and TurAV1 grouped with subgroup II ampeloviruses (Figs. 1–3). 3.3. Identification of a putative novel bluvaviruses in plant (meta)transcriptomes Genome (18.3 kb) of a novel bluvavirus—Rhododendron bluvavirus 1 (RhBlV1)—was identified in the transcriptome of Rhododendron arboretum and contained twelve ORFs. ORFs 1a and 1b, possibly via + 1 RFS, encode replicase with viral protease (PF05533), MTR, HEL and RdRP motifs. ORFs downstream to ORF1b encode a 11 kDa hypothetical protein, 7 kDa putative transmembrane protein, HSP70 (64 kDa), HSP90h (61 kDa), CP (32 kDa) and five hypothetical proteins (23 kDa, 24 kDa, 7 kDa, 8kDa and 9 kDa) in the order. BLASTp analysis revealed the maximum identity of 57% of any encoded RhBlV1 protein with the corresponding protein of blueberry virus A (BVA) (Table S1). Sequence identity matrix revealed 32.4–56.6% amino acid sequence identity of RdRP, HSP70 and CP encoded by RhBlV1 with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic analysis grouped RhBlV1 with BVA in RdRP, HSP70 and CP-based trees (Figs. 1–3). 3.4. Identification of putative novel closteroviruses in plant (meta)transcriptomes Coding-complete genomes (15.4–18.3 kb), encoding nine to eleven ORFs, of seven novel closteroviruses—Alternanthera closterovirus (AltCV1), Artemisia closterovirus 1 (ArtCV1), Cerastium closterovirus 1 (CerCV1), Chrysanthemum closterovirus 1 (ChrCV1), cowslip closterovirus 1 (CwCV1), Euphorbia closterovirus 1 (EuCV1) and thyme closterovirus 1 (ThCV1)—were identified in transcriptomes of seven dicot plants (Table 1). All the identified novel closteroviral genomes encode replicase (with viral preotease, MTR, HEL and RdRP motifs) from ORFs 1a and 1b possibly via + 1 RFS, a small transmembrane protein with MP p6 motif (5–7 kDa), HSP70 (64–67 kDa), HSP90h (61–62 kDa), CPm (24–26 kDa), CP (22–25 kDa) and a protein with RNA silencing suppressor motif(s) (PF11757, PF11479; 20–26 kDa) in the order. Besides, the genomes contain a common ORF encoding a hypothetical protein (17–21 kDa) in between CP and silencing suppressor-encoding ORFs. ArtCV1, ChrCV1, CwCV1 and EuCV1 genomes contain an additional hypothetical protein (31–33 kDa)-encoding ORF in between ORF1b and ORF encoding MP-like protein, while ChrCV1 genome also possesses an ORF, encoding a CP-like protein (48 kDa), immediately downstream of ORF1b. BLASTp analysis revealed that the maximum sequence identity of any protein encoded by the identified novel closteroviruses to the corresponding protein of known closteroviruses was 80% (Table S1). While RdRP encoded by identified novel closteroviruses shared 67.7–84.5% amino acid sequence identities with that of known and identified closterovirids, the encoded HSP70 and CP shared 31.5–71.8% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic trees constructed based on RdRP, HSP70 and CP sequences grouped the identified seven novel closteroviruses with known closteroviruses (Figs. 1–3). 3.5. Identification of putative novel olivaviruses in plant (meta)transcriptomes A coding-complete (18.2 kb) and a 5’ end partial genome (13.1 kb), each containing fourteen ORFs, were identified in transcriptomes of mango and pecan, respectively, representing two novel olivaviruses: mango olivavirus 1 (MgOV1) and pecan olivavirus 1 (PecOV1). ORFs encoding replicase (from ORFs 1a and 1b), a putative transmembrane protein (5–7 kDa), HSP70 (66–67 kDa), HSP90h (57–60 kDa), CP (25–36 kDa), thaumatin-like protein (PF00314; 26–28 kDa) and a 3’→5’ exonuclease-like protein (PF01612; 25–26 kDa) were determined in both the genomes, though their order differed. The remaining ORFs of variable lengths encoded hypothetical proteins (8–38 kDa) with no predicted motif. BLASTp analysis of proteins encoded by novel olivaviruses revealed a maximum of 50% identity to the corresponding sequence of known olivaviruses (Table S1). Sequence identity matrix revealed 31.4–55.9% amino acid sequence identity of RdRP, HSP70 and CP encoded by MgOV1 and PecOV1 with that of known closterovirids (Figs. S1–S3, Table S2). MgOV1 and PecOV1 grouped with olivaviruses in RdRP, HSP70 and CP-based phylogenetic trees (Figs. 1–3). 3.6. Identification of putative novel velariviruses in plant (meta)transcriptomes Coding-complete genomes (16.6–17.0 kDa) of two velariviruses—Camellia velarivirus 1 (CamVV1) and hazelnut velarivirus 1 (HzVV1)—were identified in transcriptomes of Camellia chekiangoleosa and hazelnut, respectively. CamVV1 genome contains nine ORFs, while thirteen ORFs were determined in HzVV1 genome, both encoding replicase (from ORFs 1a and 1b), a small putative transmembrane protein (4–5 kDa), HSP70 (62–63 kDa), HSP90h (61 kDa), CP (37–38 kDa) and CPm (26–73 kDa) in common. The remaining ORFs encoded hypothetical proteins of size 26–73 kDa with no predicted motif. BLASTp analysis revealed the 61% maximum sequence identity of any protein encoded by the novel velariviruses to the known velariviruses (Table S1). Sequence identity matrix revealed 35.5–62.1% amino acid sequence identity of RdRP, HSP70 and CP encoded by CamVV1 and HzVV1 with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic analysis based on RdRP, HSP70 and CP grouped CamVV1 and HzVV1 with known velariviruses (Figs. 1–3). 3.7. Identification of other putative novel viruses potentially representing novel closteroviral taxa in plant (meta)transcriptomes Coding-complete clostero-like viral genome was identified in transcriptomes of Betula pendula and Musa textilis , while partial clostero-like viral genome was identified in Agave tequilana , Musa hybrid cultivar and Picea abies . Genome (22.1 kb) of virus identified in Betula , named tentatively as birch clostero-like virus 1 (BiClV1), contained fourteen ORFs encoding replicase (from ORFs 1a and 1b with possible + 1 RFS), a small putative transmembrane protein (6 kDa), HSP70 (67 kDa), HSP90h (59 kDa), CP (35 kDa) and eight hypothetical proteins (9–46 kDa). Additionally, the replicase of BiClV1 contained the 2OG-Fe(II) oxygenase superfamily motif at the N-terminal region before the endopeptidase motif. A reverse transcriptase(RTase)-like motif (PF13456) was determined in the hypothetical protein (22 kDa) encoded by the last but one ORF from the 5’ end, while the proteins encoded by the last two ORFs shared sequence similarities (28–32% at 75–92% query coverage) with plant proteins. BLASTp analysis also revealed the maximum 43% sequence identity of any BiClV1 protein with that of known closterovirids (Table S1). Sequence identity matrix revealed 28.1–44.6% amino acid sequence identity of RdRP, HSP70 and CP encoded by BiClV1 with that of known closterovirids (Figs. S1–S3, Table S2). Genomes of the virus identified in Musa hosts, named tentatively as abaca clostero-like virus 1 (AbaClV1), shared 81% nucleotide sequence identity with each other and contained ten ORFs. These ORFs encode replicase (possibly via + 1 RFS from ORFs 1a and 1b), a small putative transmembrane protein (5 kDa), HSP70 (62 kDa), HSP90h (60 kDa), CPm (48 kDa), CP (32 kDa), a 24 kDa hypothetical protein, a thaumatin-like protein (31 kDa) and a 20 kDa hypothetical protein. RdRP sequence of AbaClV1 shared a maximum of 50% identity with that of known criniviruses in BLASTp analysis, which was the highest sequence identity of any AbaClV1 protein with known members (Table S1). RdRP, HSP70 and CP encoded by AbaClV1 isolates shared 30.7–52.7% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Five partial contigs (2.1–10.3 kb) representing genomes of a novel clostero-like virus, named as Agave clostero-like virus 1 (AgClV1), were identified in transcriptome data of two independent bioprojects of Agave. AgClV1 contained seven ORFs, in total, across the partial contigs, encoding replicase (possibly via + 1 RFS of ORFs 1a and 1b), a small putative transmembrane protein (4 kDa), HSP70 (63 kDa), HSP90h (61 kDa), CP (28 kDa) and a hypothetical protein. The maximum sequence identity shared by any intact protein encoded by AgClV1 contigs with the corresponding sequence of known closterovirids was 53% in BLAST analysis (Table S1). RdRP, HSP70 and CP encoded by AgClV1 isolates shared 31.3–55.9% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Four partial contigs (2.8–12.9 kb) that represented the genome of a clostero-like virus, named as spruce clostero-like virus 1 (SpClV1), was identified in two related bioprojects of P. abies . Across the partial contigs, SpClV1 contained ten ORFs encoding replicase (from ORFs 1a and 1b through possible + 1 RFS), a putative transmembrane protein (7 kDa), HSP70, HSP90h, CP (28 kDa) and four hypothetical proteins (11–20 kDa). SpClV1 proteins shared a maximum of 49% identity with the corresponding sequence of known closterovirids in BLAST analysis (Table S1). RdRP, HSP70 and CP encoded by SpClV1 isolates shared 26.8–54.6% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic analysis based on RdRP, HSP70 and CP placed AbaClV1, AgClV1, BiClV1 and SpClV1 in distinct sub-clades within closterovirids. Among these, AbaClV1 and AgClV1 were related to each other and to criniviruses and velariviruses (Figs. 1–3). 4. Discussion The family Closteroviridae includes members that cause economically important diseases in cultivated plants (German-Retana et al., 2004 ; Rybicki and Foster, 2024 ). As of 2025, 87 species were recognized by the ICTV across seven genera in the family (Fuchs et al., 2020, Aboughanem-Sabanadzovic et al., 2025 ). In the present study, we identified sequences of 22 putative novel closterovirids in 21 plant genera, by mining the yet-unexplored plant (meta)transcriptome data in SRA database for novel closteroviral sequences. This discovery represents 0.25-fold expansion of the family Closteroviridae . Importantly, the first identification of a putative novel clostero-like virus—SpClV1—in a gymnosperm host not only expands the host range of closteroviruses to gymnosperms but also reiterates the vast diversity of gymnosperm virosphere (Bejerman et al., 2022 , Mifsud et al., 2022 ). Phylogenetic placement of SpClV1 in a distinct subclade within closterovirids supports the notion of divergent evolution of viruses of gymnosperms from those of angiosperms (Bejerman et al., 2022 , Sidharthan et al., 2022 , Sidharthan et al., 2024 ). TurAV1, a subgroup II ampelovirus, was identified in a host belonging to Zingiberaceae, whose members also host two other subgroup II ampelo-/ampelo-like viruses—Alpinia vein clearing virus (Larrea-Sarmiento et al., 2024 ) and ginger chlorotic fleck-associated virus 2 (Bhat et al., 2020 ). It is noteworthy that atleast six novel closterovirids were identified in tree hosts in the present study, including the two novel members of the genus Olivavirus that predominantly contains members reported from tree hosts (Read et al., 2024 ). The economically important hosts in which novel closterovirids were reported in the present study include birch, black pepper, hazelnut, mango, oak, pecan, spruce and turmeric. On the basis of sequence-based species demarcation criteria of closterovirids (i.e. <75% amino acid sequence identity based on RdRP, HSP70 and CP to known members) and phylogenetic relatedness to known members, BPAV1, LeuAV1, MatAV1, OaAV1, PecAV1 and TurAV1 are regarded as novel ampeloviruses, RhBlV1 as a novel bluvavirus, ChrCV1 and EuCV1 as novel closteroviruses, MgOV1 and PecOV1 as novel olivaviruses, and CamVV1 and HzVV1 as novel velariviruses. Though the RdRP of AltCV1, ArtCV1, CerCV1, CwCV1, and ThCV1 shared > 75% identity with RdRP of known or identified closterovirids, their HSP70 and CP sequences shared < 75% identities with that of known or identified closterovirids. It is to note that some recognized closteroviruses—carrot closterovirus 1, carrot closterovirus 2, carrot yellow leaf virus, mint virus 1, Rehmannia virus 1 and tobacco virus 1—also share > 75% RdRP sequence identity with that of other closteroviruses (Adams et al., 2014 , Kwon et al., 2018 , Schönegger et al., 2023 ). Thus, AltCV1, ArtCV1, CwCV1, and ThCV1 can be regarded as novel closteroviruses, atleast based on their HSP70 and CP sequence divergence. To our knowledge, the unusually longer BiClV1 genome (22.1 kb) is, thus far, the largest undivided RNA genome known among the closterovirids and plant viruses in general. Generally, replication of RNA viruses with larger genomes require larger replicase (Agranovsky, 2021 ). This is the case with BiClV1, which encodes a larger replicase—likely the largest among closterovirids. To note, BiClV1 genome encodes two proteins sharing sequence similarities with plant proteins, one of which contains an RTase-like domain. This domain is typically associated with a retrotransposon in plants (Marchler-Bauer et al., 2015 ). Therefore, the role of this domain in a closterovirid needs to be studied further. Although 2OG-Fe(II) oxygenase superfamily motif was determined in between MET and HEL motifs in the replicase of a known ampelovirus (Ito and Nakaune, 2016 ) and MatAV, the location of this motif in the replicase was different in BiClV1. Based on the genome organization and phylogenetic divergence, BiClV1 can be classified as a new member of a putative new genus, with tentative name Betulavirus , within the family Closteroviridae . AbaClV1, whose proteins shared sequence similarities with criniviruses, possessed a monopartite RNA genome, unlike the criniviruses with split genomes (Fuchs et al., 2020). Phylogenetically, AbaClV1 was related to criniviruses and velariviruses. Largely, AbaClV1 genome resembled velariviruses than criniviruses. However, it differed from velariviruses in the reverse orientation of CPm and CP ORFs and possession of an additional ORF encoding a thaumatin-like protein, similar to the ones reported in olivaviruses (Ruiz-García et al., 2020 ). Interestingly, AbaClV1 was identified in (meta)transcriptomes of two Musacaeous hosts, representing two independent bioprojects that includes a virus-indexing project on Musa germplasm (Rong et al., 2023 ). Rong et al. ( 2023 ) reported the identification of two contigs sharing sequence similarities (63% and 66%) with genome segments of lettuce chlorosis virus, a crinivirus, in a library derived from a Musa hybrid cultivar. In the same library, we identified the 5’ partial monopartite genome of AbaClV1 (14.1 kb), which corresponded to the 85% length of the AbaClV1 coding-complete genome identified in an abaca transcriptome library. Based on the genome organization and phylogenetic divergence based on RdRP and HSP70 regions, AbaClV1 can be classified as a new member of a putative new closterovirid genus, for which the tentative name Musavirus is proposed. RdRP, HSP70 and CP encoded by AgClV1 and SpClV1 shared < 56% sequence identities with known closterovirids. In phylogenetic trees, both the viruses formed distinct clades within closterovirids. Thus, these viruses may also represent potential novel taxa within the family Closteroviridae . However, their coding-complete genomes could not be recovered in the present study. Genome organization of the identified olivaviruses slightly differed from that of known olivaviruses as they possess three additional ORFs encoding hypothetical proteins in between ORF1b and the ORF encoding putative transmembrane protein. This includes the thaumatin-like protein-encoding ORF of PecOV1, like in olive yellowing leaf-associated virus, while a similar ORF was present in between HSP90h and CP ORFs in MgOV1, like in Actidinia virus 1 and persimmon virus B (Ruiz-García et al., 2020 ). In addition, an ORF encoding a protein with 3’→5’ exonuclease-like motif was determined in both the identified olivaviral genomes, though its position differed in both the viruses. Similar protein was encoded by citrus associated ampelovirus 1 (CaAV-1) (Liu et al., 2021 ). Interestingly, during the time of manuscript preparation, a genome sequence named ‘Mangifera virus 1 isolate MG (PV101221)’ deposited from India that shared 88% nucleotide sequence identity (at 94% query coverage) with MgOV1 genome identified in this study in a mango transcriptome dataset deposited from Israel, was available in the GenBank. Genome organization of Mangifera virus 1 and MgOV1 were similar, except for the three unannotated ORFs in Mangifera virus 1 and an additional ORF determined in MgOV1. Thus, the sequence named Mangifera virus 1 represents the near coding-complete genome of MgOV1, and this further validates the host of MgOV1 as mango. Typically, the closterovirids possess ORFs 1a and 1b that via a + 1 RFS encode replicase (Fuchs et al., 2020). Surprisingly, genomes of OaAV1, obtained from two independent bioprojects, contained only one ORF1 encoding a replicase with viral MET, HEL and RdRP motifs. This atypical feature was also observed in the recently reported citrus associated ampelovirus 2, a subgroup I ampelovirus (Liu et al., 2021 ) that was related to OaAV1. Taken together, these findings hint at the possible existence of an alternative strategy to + 1 RFS for the expression of RdRP gene, atleast in subgroup I ampeloviruses. Nevertheless, the absence of potential + 1 RFS in these ampeloviruses needs to be experimentally verified in further studies (Liu et al., 2021 ). Interestingly, OaAV1 isolates encode a papain family protease from a downstream ORF, a feature that is atypical of ampeloviruses. Similar to CaAV-1, PecAV1 encodes a thaumatin-like protein and a protein with 3’→5’ exonuclease-like motif (Liu et al., 2021 ). Likewise, thaumatin and 3’→5’ exonuclease motifs were determined in protein p33 and p25, respectively, of the Pistachio ampelovirus A (PAVA) (MF198462). Interestingly, in HSP70 and CP-based phylogenetic trees, CaAV-1, PAVA and PecAV1 formed a distinct group within subgroup I ampeloviruses. Thus, although typical of olivaviruses (Sabanadzovic et al., 2021 ), thaumatin-like protein is not exclusive to olivaviruses as it is encoded by a few ampeloviruses and the clostero-like virus AbaClV1. The evolutionarily distinct, animal nidovirids and plant closterovirids—both possessing the largest undivided RNA genomes in their respective domains—share strikingly similar genomic features, including the expression of 5’-proximal ORFs via ribosomal frameshifting to encode papain-like protease(s), viral MET, HEL and RdRP. Additionally, some nidovirids, as part of the ORF1b polyprotein, encode a 3’→5’ exonuclease—related to the DEDD family—that performs proofreading activity during RNA replication. However, a similar protein was not previously reported in closterovirids (Agranovsky, 2021 ). Identification of a DEDDy 3’→5’ exonuclease-like protein in three ampeloviruses and two olivaviruses in the present and an earlier study (Liu et al., 2021 ) tempts us to speculate their possible role in enhancing the fidelity of RNA replication in these viruses (Agranovsky, 2021 ). However, further experimental evidences are needed to confirm this speculation. It is noteworthy that the 3’→5’ exonuclease-like protein is encoded by a separate downstream ORF in closterovirids, unlike in nidovirids, which encode it as a part of the replication-associated polyprotein (Agranovsky, 2021 ). The present study has certain limitations. As with other data-mining studies, the host assignment of the identified viruses was based solely on the metadata provided in the SRA database, due to the lack of access to the original samples for validation. Consequently, the end sequences of the coding-complete viral genomes could not be precisely determined through wet-lab based assays. Therefore, these findings should be considered preliminary until further validation (Bejerman et al., 2023 ). Nevertheless, the identification of a few novel closteroviruses in different independent libraries derived from the same or closely related hosts strengthens the evidence for their host association. In conclusion, coding-complete or partial genomes of twenty-two putative novel closterovirids were identified across a broad range of plant genera. Some of the identified viruses may represent novel genera within the family Closteroviridae , including a virus with the largest known RNA genome among closterovirids. Further studies in the respective virus-associated hosts are needed to validate the host associations and understand the distribution and biology of these novel viruses. Declarations 5. Acknowledgements The author thanks the original submitters of Next Generation Sequencing data to the NCBI SRA database whose data were analysed in this study and the NCBI SRA for making the data publicly available. The author acknowledges the support of the Group Coordinator Research and the Director, ICFRE-IFB, Hyderabad. 6. Author contributions V. Kavi Sidharthan: Conceptualization, Methodology, Formal analysis and investigation, Writing-original draft preparation, Writing - review and editing. 7. Data availability The genome sequences obtained in this study were submitted to the NCBI TPA database. In addition, they are included in the Supplementary file 3. 8. Declaration of competing interest The author declares that there is no competing interest. 9. 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Details of putative novel closteroviruses identified in plant (meta)transcriptomes available in public domain Virus name/Tentative species name Acronym Source library/ Bioproject Genome length (nt) Mean depth (x) Sample cultivar or isolate or ecotype or variety/tissue Plant Species name/ Family Data Reference Genus: Ampelovirus Black pepper ampelovirus 1/ Ampelovirus piperis BPAV1 SRR3404569/ PRJNA318916 12,080 141.11 Panniyur 1/ Leaf Piper nigrum L./ Piperaceae (dicot) Mahadevan et al., 2024 Leucadendron ampelovirus 1/ Ampelovirus leucadendri LeuAV1 ERR6130833/ PRJEB45774 5,967*/ 3,728* 11.53/8.10 Brunioides/ NA Leucadendron brunioides Meisn./ Proteaceae (dicot) Scharmann et al., 2021 Matucana ampelovirus 1/ Ampelovirus matucanae MatAV1 SRR13805638/ PRJNA705387 16,247 34.71 HBG13/ Pistil Matucana madisoniorum (Hutchison) G.D.Rowley/ Cactaceae (dicot) Ramanauskas and Igić, 2021 Oak ampelovirus 1/ Ampelovirus quercus OaAV1 SRR3636733/ PRJNA322128 17,795 2,325.80 NA/ Stem Quercus robur L./ Fagaceae (dicot) Broberg et al., 2018 ERR1354110/ PRJEB13357 16,005 31.62 NA Hearn et al., 2019 Pecan ampelovirus 1/ Ampelovirus caryae PecAV1 SRR17335006/ PRJNA792564 16,130 26.81 Oconee/ Kernel Carya illinoinensis (Wangenh.) K. Koch/ Juglandaceae (dicot) Zhang et al., 2022 Turmeric ampelovirus 1/ Ampelovirus curcumae TurAV1 SRR13594120/ PRJNA698442 12,997 157.00 IISR Prathibha/ Rhizome Curcuma longa L./ Zingiberaceae (monocot) Sheeja et al., 2022 Genus: Betulavirus Birch clostero-like virus 1/ Betulavirus betulae BiClV1 ERR3173759/ PRJEB29260 22,120 209.42 v5834/ Stem (developing phloem) Betula pendula Roth/ Betulaceae (dicot) Alonso‐Serra et al., 2019 Genus: Bluvavirus Rhododendron bluvavirus 1/ Bluvavirus rhododendri RhBlV1 SRR4449165/ PRJNA345272 18,250 44.57 Himachal Pradesh/ Flower Rhododendron arboreum Sm./ Ericaceae (dicot) Choudhary et al., 2018 Genus: Closterovirus Alternanthera closterovirus 1/ Closterovirus alternantherae AltCV1 ERR2040215/ PRJEB21674 15,852 273.45 NA/ Leaf Alternanthera brasiliana (L.) Kuntze/ Amaranthaceae (dicot) 1000 Plant Transcriptomes Initiative, Unpublished Artemisia closterovirus 1/ Closterovirus artemisiae ArtCV1 SRR15595118/ PRJNA752933 16,576 760.74 YQ7/ Root Artemisia annua L./ Asteraceae (dicot) Ma et al., 2021 Cerastium closterovirus 1/ Closterovirus cerastii CerCV1 SRR1979688/ PRJNA280277 15,402 53.85 NA/ Leaf Cerastium arvense L./ Caryophyllaceae (dicot) Brockington et al., 2015 Chrysanthemum closterovirus 1/ Closterovirus chrysanthemi ChrCV1 SRR15321559/ PRJNA751454 18,179 313.80 Ribonette/ Leaf Chrysanthemum × morifolium (Ramat.) Hemsl./ Asteraceae (dicot) Chirkov et al., 2022 Cowslip closterovirus 1/ Closterovirus primulae CwCV1 ERR5762867/ PRJEB44353 17,184 2,160.91 NA/ Flower Primula veris L./ Primulaceae (dicot) Potente et al., 2022 Euphorbia closterovirus 1/ Closterovirus euphorbiae EuCV1 SRR13511996/ PRJNA693983 16,432 1,032.09 NA/ Root Euphorbia ebracteolata Hayata/ Euphorbiaceae (dicot) Zheng et al., 2022 Thyme closterovirus 1/ Closterovirus thyme ThCV1 SRR6262814/ PRJNA417241 15,449 26.15 Tv8/ Leaf Thymus vulgaris L./ Lamiaceae (dicot) Mollion et al., 2018 Genus: Musavirus Abaca clostero-like virus 1/ Musavirus musae AbaClV1 ERR6912813/ PRJEB47952 16,509 273.94 Tangongon/ Pseudostem Musa textilis Née/ Musaceae (monocot) Ereful et al., 2022 SRR16881896/ PRJNA777477 14,134* 90.01 NA/ Leaf Musa hybrid cultivar/ Musaceae (monocot) Rong et al., 2023 Genus: Olivavirus Mango olivavirus 1/ Olivavirus mangiferae MgOV1 SRR19975612/ PRJNA855362 18,177 21.75 Shelly/ Fruit peel Mangifera indica L./ Anacardiaceae (dicot) Patel et al., 2023 Pecan olivavirus 1/ Olivavirus caryae PecOV1 SRR13411591/ PRJNA680537 13,110* 45.03 Oaxaca/ Catkin Carya illinoinensis (Wangenh.) K. Koch/ Juglandaceae (dicot) Lovell et al., 2021 Genus: Velarivirus Camellia velarivirus 1/ Velarivirus camelliae CamVV1 SRR10121554/ PRJNA565081 16,623 1,327.71 RISF_HS/ Endocarp Camellia chekiangoleosa Hu/ Theaceae (dicot) Yan et al., 2022 Hazelnut velarivirus 1/ Velarivirus coryli HzVV1 SRR10541224/ PRJNA591492 17,021 103.73 Dawei/ Ovule Corylus heterophylla Fisch. ex Trautv./ Betulaceae Liu et al., 2020 Unclassified members Agave clostero-like virus 1 AgClV1 SRR789742/ PRJNA193469 10,328*/2,143* 26.07/48.37 Weber azul/ NA Agave tequilana F.A.C.Weber/ Asparagaceae (monocot) Gross et al., 2013 SRR789754/ PRJNA193469 5,173* 23.76 ERR2040717/ PRJEB21674 5,072*/2,067* 28.40/13.06 NA/ Whole plant 1000 Plant Transcriptomes Initiative, Unpublished Spruce clostero-like virus 1 SpClV1 SRR11565953/ PRJNA622086 12,863*/4,674* 18.62/92.48 NA/ Root Picea abies (L.) H.Karst./ Pinaceae (gymnosperm) DOE Joint Genome Institute, 2020 SRR9030810/ PRJNA537855 6,259*/2,836* 17.70/15.16 *indicates partial genome segment length and NA indicates information not available. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7560636","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511663599,"identity":"16ccf670-558e-4934-b0af-96118463fe2d","order_by":0,"name":"V Kavi Sidharthan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACCSA+wGDAwM/AwHwAxJchWotkAwNbAojPQ5QWEABq4TEAMQhrkWw/Y3iAocBOwry95/OrGzUWPAzsh49uwKdFmifHAOiwZAmZM2e3WeccAzqMJy3tBj4tcgxpCUAtB+okJHK3GeewAbVI8Jjh18L/DKxFQkL+zTPjnH9EaJGWSD4A0SLBw/w4t40ILZIzHh84kAD0iwRPmhlzbp8EDxshv0icT2z+8OGPnYQE++HHn3O+1cnxsx8+hlcLGCRAKDZwJLERVI4EmD+QonoUjIJRMApGDgAAHnVBpoFVSdIAAAAASUVORK5CYII=","orcid":"","institution":"ICFRE-INSTITUTE OF FOREST BIODIVERSITY","correspondingAuthor":true,"prefix":"","firstName":"V","middleName":"Kavi","lastName":"Sidharthan","suffix":""}],"badges":[],"createdAt":"2025-09-08 06:26:35","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7560636/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7560636/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90856852,"identity":"2647c157-8701-4e1e-8c33-ea8c9766cab2","added_by":"auto","created_at":"2025-09-09 05:13:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":278268,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood tree showing the relationship of identified novel closterovirids (indicated by blue circles) with recognized members based on the encoded RNA-dependent RNA polymerase. The tree was constructed using the best-fit LG+G+I+F model and 100 bootstrap replicates.Bootstrap values ≥ 50% are only indicated. Accession numbers of sequences used for phylogenetic analysis are mentioned in Table S3.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7560636/v1/44c9dadec6f9411c07b08b89.jpg"},{"id":91148504,"identity":"17ff183a-d25b-4534-a860-ac2032f80340","added_by":"auto","created_at":"2025-09-12 06:44:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272569,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood tree showing the relationship of identified novel closterovirids (indicated by blue circles) with recognized members based on the encoded heat shock protein 70. The tree was constructed using the best-fit LG+G+I model and 100 bootstrap replicates.Bootstrap values ≥ 50% are only indicated. Accession numbers of sequences used for phylogenetic analysis are mentioned in Table S3.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7560636/v1/f422f0877898b6422cf1f233.jpg"},{"id":90856854,"identity":"82b4306f-5677-4ef1-bfa1-4eaf902e7c42","added_by":"auto","created_at":"2025-09-09 05:13:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":268120,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood tree showing the relationship of identified novel closterovirids (indicated by blue circles) with recognized members based on the encoded coat protein. The tree was constructed using the best-fit LG+G model and 100 bootstrap replicates. Bootstrap values ≥ 50% are only indicated. Accession numbers of sequences used for phylogenetic analysis are mentioned in Table S3.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7560636/v1/34ecebc306222fc88acab185.jpg"},{"id":91149302,"identity":"cceec219-2cc7-4f1c-b322-acaff65d3c71","added_by":"auto","created_at":"2025-09-12 06:48:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1964182,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7560636/v1/3998779a-f83d-4f7b-a11a-704f63099b47.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePlant (meta)transcriptome data mining identified twenty-two putative novel taxa in the family \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eClosteroviridae\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe family \u003cem\u003eClosteroviridae\u003c/em\u003e includes filamentous, plant-infecting viruses with positive sense single-stranded RNA genomes (Fuchs et al., 2020). Members of this family are known to cause economically important plant diseases such as citrus tristeza, beet yellows, lettuce infectious yellows and grapevine leafroll (German-Retana et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Rybicki and Foster, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In general, the closterovirid-incited plant diseases depict leaf discolouration symptoms, whilst a few diseases present leaf roll and stem pitting/grooving symptoms. Though the closterovirids cause systemic infection in plants, they are generally phloem-limited (Fuchs et al., 2020). Historically, characterization of beet yellows virus (BYV) led to the recognition of the earlier-accepted \u0026lsquo;\u003cem\u003eClosterovirus\u003c/em\u003e group\u0026rsquo; by the International Committee on Taxonomy of Viruses (ICTV), the group name being derived from the Greek word \u0026lsquo;kloster\u0026rsquo; referring to the thread-like virus particles. In the year 2000, the family \u003cem\u003eClosteroviridae\u003c/em\u003e was established with two genera\u0026mdash;\u003cem\u003eClosterovirus\u003c/em\u003e (monopartite genome, over 1000 nm long virions) and \u003cem\u003eCrinivirus\u003c/em\u003e (split genome, less than 1000 nm long virions with two modal lengths). Subsequent taxonomical revisions led to the formation of two new genera\u0026mdash;\u003cem\u003eAmpelovirus\u003c/em\u003e and \u003cem\u003eVelarivirus\u003c/em\u003e (Martelli, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The recent recognition of three new genera\u0026mdash;\u003cem\u003eBluvavirus\u003c/em\u003e, \u003cem\u003eMenthavirus\u003c/em\u003e and \u003cem\u003eOlivavirus\u003c/em\u003e\u0026mdash;by the ICTV (Sabanadzovic et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), makes the current number of recognized genera in the family to seven. Until 2024, the family \u003cem\u003eClosteroviridae\u003c/em\u003e comprised 58 recognized species across genera\u0026mdash;\u003cem\u003eAmpelovirus\u003c/em\u003e (13), \u003cem\u003eBluvavirus\u003c/em\u003e (1), \u003cem\u003eClosterovirus\u003c/em\u003e (17), \u003cem\u003eCrinivirus\u003c/em\u003e (14), \u003cem\u003eMenthavirus\u003c/em\u003e (1), \u003cem\u003eOlivavirus\u003c/em\u003e (3) and \u003cem\u003eVelarivirus\u003c/em\u003e (9) (Fuchs et al., 2020). In 2025, 31 additional species were recognized, while a species was excluded in the family (Aboughanem-Sabanadzovic et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eVirions of closterovirids are non-enveloped and appear as flexuous filaments (12 nm diameter x 650\u0026ndash;2200 nm) (Fuchs et al., 2020). Their genomes consist of a monopartite (except in criniviruses) or bi-/tripartite (in criniviruses) RNA molecule(s), with lengths ranging from 13 kb to 19.3 kb (combined length in case of criniviruses), making them the largest among the plant viruses (Candresse and Fuchs, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Fuchs et al., 2020). Sequence duplication and RNA recombination mediated non-viral coding sequence acquisition (heat shock protein 70-like protein (HSP70) and protease) account for the large genome size of closterovirids. Though the number and relative position of open reading frames (ORFs) differ across genera and species in the family \u003cem\u003eClosteroviridae\u003c/em\u003e, their genomes invariably encode a large replicase polyprotein with the conserved methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRP) motifs from ORFs 1a and 1b, typically expressed via a\u0026thinsp;+\u0026thinsp;1 ribosomal frameshift (RFS). Downstream of the replicase-encoding ORFs is the conserved five-gene module coding for a small hydrophobic protein (ca. 6 kDa), the HSP70, a ca. 60 kDa protein, a major (CP) and a minor coat protein (CPm) in the 5\u0026rsquo; to 3\u0026rsquo; direction. Exceptions to this are the members of the genus \u003cem\u003eClosterovirus\u003c/em\u003e that have CPm gene upstream of CP gene and subgroup II ampeloviruses that lack CPm gene. The closterovirid genome possibly has the 5\u0026rsquo; cap but its 3\u0026rsquo; end lacks the polyadenylated tail and the transfer RNA-like structure (Candresse and Fuchs, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Fuchs et al., 2020). Closterovirids are known to be semi-persistently transmitted by various sap-sucking hemipterans\u0026mdash;aphids vector closteroviruses and the sole known menthavirus, mealybugs and soft scales vector ampeloviruses and whiteflies vector criniviruses. Vectors are not known for bluvavirus, olivaviruses and velariviruses (Candresse and Fuchs, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Fuchs et al., 2020, Sabanadzovic et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent times, plant transcriptome data have increasingly been deposited in the public-domain sequence data repositories like National Centre for Biotechnology Information (NCBI). Besides the host transcipts, these datasets may also contain sequences of plant viruses if the sampled host was infected at the time of sampling. This facilitates the comprehensive exploration of non-host fraction of transcriptome data of a wide range of plant species for sequences of novel viruses- a process known as data-driven virus discovery (DDVD), which otherwise would require the costlier Next Generation Sequencing (NGS)-based virome studies (Lauber and Seitz, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Sidharthan and Baranwal, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). DDVD studies have considerably expanded the genetic diversity and host range of several plant virus groups like alphaflexiviruses (Sravani et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), amalgaviruses (Nibert et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Sidharthan et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), enamoviruses and poleroviruses (Sidharthan et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), kitaviruses (Reddy and Sidharthan, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), rhabdoviruses (Bejerman et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Bejerman and Debat, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), secoviruses (Sidharthan et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), tymoviruses (Bejerman and Debat, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and varicosaviruses (Bejerman et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our previous study, we identified the genome of a novel closterovirus in the transcriptome-assembled contigs of dwarf polish wheat (Sidharthan and Baranwal, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Considering the vast amount of plant transcriptome data deposited in the sequence read archives (SRA) database of NCBI over the transcriptome shotgun assembly (TSA) database (Lauber and Seitz, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we assumed that the existing plant transcriptome data in SRA database would contain sequences of novel closteroviruses and that their discovery would expand the phylogenetic diversity of the family \u003cem\u003eClosteroviridae\u003c/em\u003e. Thus, we mined the existing plant (meta)transcriptome data in the SRA database for novel closteroviral sequences and identified twenty-two putative novel taxa in the family \u003cem\u003eClosteroviridae\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Identification of plant (meta)transcriptome libraries with reads of putative novel closteroviruses\u003c/h2\u003e\u003cp\u003ePutative novel closterovirus-positive SRA libraries were identified in the Serratus RdRP search (Edgar et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) using the family \u003cem\u003eClosteroviridae\u003c/em\u003e as query with default parameters (alignment identity: \u0026ge;45%, score: \u0026ge;50). The resulting libraries derived from plants and sharing\u0026thinsp;\u0026lt;\u0026thinsp;90% identity with known closteroviruses were only retained for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Identification of putative novel closteroviruses from closterovirus-positive libraries and their genome recovery\u003c/h2\u003e\u003cp\u003ePlant SRA libraries tentatively identified to have reads of putative novel closteroviruses were imported into the Galaxy server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://usegalaxy.org/\u003c/span\u003e\u003cspan address=\"https://usegalaxy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Galaxy Community, 2022) and trimmed using the Trimmomatic tool v 0.39 (Bolger et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) to remove adapter sequences and sequences with average quality score\u0026thinsp;\u0026lt;\u0026thinsp;25. Trimmed libraries were \u003cem\u003ede novo\u003c/em\u003e assembled using MEGAHIT v 1.2.9 (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and subjected to BLASTx analysis (evalue cutoff: 1e-5) against a database made of protein sequences of known closteroviruses (retrieved from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/labs/virus/vssi/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/labs/virus/vssi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using the NCBI BLAST\u0026thinsp;+\u0026thinsp;tool v 2.16.0 (Cock et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The largest matching hit to an individual closteroviral genus in each library was subjected to ORF prediction using the NCBI\u0026rsquo;s ORF finder tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to determine the presence of the conserved closteroviral ORFs, and the encoded RdRP, HSP70 and CP protein sequences were analysed through BLASTp against NCBI\u0026rsquo;s non-redundant protein sequences (nr) database. If the sequences of analysed proteins shared\u0026thinsp;\u0026lt;\u0026thinsp;75% identity to the existing sequences in GenBank and the intact contig length corresponded to the genome length of related closterovirus, it was regarded as coding-complete genome of a putative novel closterovirus. If the coding-complete genome of a putative novel closterovirus could not be recovered, the corresponding trimmed library was \u003cem\u003ede novo\u003c/em\u003e assembled using rnaSPAdes v 4.2.0 (Bushmanova et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and/or Trinity v 2.15.1 (Grabherr et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) assemblers and the resulting contigs were analysed to identify the coding-complete genome. In two instances, the overlapping contigs assembled by MEGAHIT and rnaSPAdes were manually assembled to obtain the coding-complete genome. Further, if the contigs were relatively shorter and did not possess all the conserved closteroviral ORFs, they were regarded as partial genome sequences of the identified putative novel closteroviruses. In instances where more than one putative novel closterovirus-positive SRA library was available for a host species, the library with the maximum number of closteroviral reads in each independent bioproject was analysed. Genome coverage estimates were obtained using the QualiMap v 2.3 (Okonechnikov et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) tool after mapping the trimmed reads onto the recovered closteroviral genome sequences using the HISAT2 v 2.2.1 (Kim et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) tool.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Genome annotation of putative novel closteroviruses\u003c/h2\u003e\u003cp\u003eORFs determined in the recovered genomes were annotated based on BLASTp analysis of encoded proteins against nr database and motifs predicted using the MOTIF Search tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/tools/motif/\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/tools/motif/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Molecular weight of encoded proteins was determined using the Expasy tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/compute_pi/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/compute_pi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) while the transmembrane helices (TMHs) were predicted using the Phobius tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phobius.sbc.su.se/\u003c/span\u003e\u003cspan address=\"https://phobius.sbc.su.se/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Phylogenetic and sequence identity analyses\u003c/h2\u003e\u003cp\u003eProtein sequences of RdRP, HSP70 and CP genes of identified and recognized closteroviruses were aligned using the MUSCLE tool implemented in MEGA7 v 7.0.26 (Kumar et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Best-fit models were identified for the individual protein alignment using the \u0026lsquo;Find Best DNA/Protein Models\u0026rsquo; tool available in MEGA7. Using the determined best-fit model, maximum-likelihood (ML) tree was constructed for each protein in MEGA7 with 100 bootstrap replicates. Pairwise sequence identity values were obtained using the Sequence Demarcation Tool v 1.2 (Muhire et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e3.1. Identification of putative novel members of the family \u003cem\u003eClosteroviridae\u003c/em\u003e in plant (meta)transcriptomes\u003c/h2\u003e\n \u003cp\u003eIn total, sequences of 22 putative novel closterovirids were identified in 21 plant genera belonging to 19 plant families. Of these, 15 are dicot families, 3 are monocot families and 1 is a gymnosperm family. In each of the family Asteraceae, Betulaceae and Juglandaceae, two putative novel closterovirids were identified. Coding-complete genomes of 18 identified novel closterovirids were obtained while for the remaining 4 viruses, only partial genome sequences were recovered. Sequences of three viruses were identified in two independent (meta)transcriptome libraries of the same plant species/genus. Mean depth of identified viral genome sequences ranged from 8.1x to 2,325.8x across libraries (Table 1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e3.2. Identification of putative novel ampeloviruses in plant (meta)transcriptomes\u003c/h2\u003e\n \u003cp\u003eGenomes of six putative novel ampeloviruses—black pepper ampelovirus 1 (BPAV1), Leucadendron ampelovirus 1 (LeuAV1), Matucana ampelovirus 1 (MatAV1), oak ampelovirus 1 (OaAV1), pecan ampelovirus 1 (PecAV1), turmeric ampelovirus 1 (TurAV1)—were identified in transcriptomes of six plant species (5 dicots and 1 monocot), of which five are coding-complete (except LeuAV1) and one is partial (LeuAV1) (Table\u0026nbsp;1). In case of OaAV1, coding-complete genome of two isolates sharing 88% sequence identity were obtained from two independent bioprojects of oak. Genome organization of BPAV1 and TurAV1 were similar and contained seven ORFs. ORFs 1a and 1b, possibly via a + 1 RFS, encode a replicase polyprotein with viral MTR (PF01660), HEL (PF01443) and RdRP (PF00978) motifs. ORF2 encodes a putative transmembrane protein (5–6 kDa) with no predicted motif (BPAV1)/ MP p6 (PF06716) motif. ORFs 3, 4 and 5 encodes HSP70 (59–60 kDa), heat shock protein 90 homolog (HSP90h) (61 kDa) and CP (28–36 kDa), respectively with HSP70 (PF00012), HSP90h (PF03225) and closterovirus CP (PF01785) motif while ORF6 encodes a 24 kDa hypothetical protein with no predicted motif. The encoded proteins of both the viruses, except the transmembrane protein of BPAV1, shared a maximum of 67% identity (at maximum query coverage) with the corresponding sequences of known ampeloviruses in BLAST analysis (Table S1). Genomes of LeuAV1, MatAV1, OaAV1 and PecAV1 invariably encode replicase polyprotein (from ORFs 1a and 1b via possible + 1 RFS, except OaAV1 where ORF1 directly encodes replicase), a small transmembrane MP-like protein (5–6 kDa), HSP70 (59–61 kDa), HSP90h (54–58 kDa, excluding the partial sequence of LeuAV1), CP (32–39 kDa) and CPm (55–61 kDa) in the order except the reverse orientation of CP and CPm ORFs in MatAV. Replicase encoded by MatAV1 contains an additional 2OG-Fe(II) oxygenase superfamily motif (PF13532) in between MET and HEL motifs. BLASTp analysis of these encoded proteins revealed a maximum of 72% identity with the corresponding sequences of known ampeloviruses. Additionally, each of these viral genomes contain ORFs encoding hypothetical proteins: two ORFs in LeuAV1 (downstream of the CPm ORF) and MatAV1 (one between the CPm and CP ORFs, and the other downstream of the CP ORF); four ORFs in OaAV (downstream of CPm ORF); and six ORFs in PecAV1 (one between ORF1b and MP-like ORF, and the remaining ORFs located downstream of the CPm ORF). This includes those encoding a 31 kDa thaumatin-like protein (PF00314) and a 25 kDa 3'→5' exonuclease-like protein (PF01612) of PecAV1, and a papain family cysteine protease (PF00112) of OaAV1 isolates (Table S1). Pairwise sequence identity matrix revealed 38.9–69.2% amino acid sequence identity of RdRP, HSP70 and CP encoded by the identified novel ampeloviruses with that of known members (Figs. S1–S3, Table S2). Phylogenetic analysis based on RdRP, HSP70 and CP sequences grouped LeuAV1, MatAV1, OaAV1 and PecAV1 with subgroup I ampeloviruses, while BPAV1 and TurAV1 grouped with subgroup II ampeloviruses (Figs.\u0026nbsp;1–3).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e3.3. Identification of a putative novel bluvaviruses in plant (meta)transcriptomes\u003c/h2\u003e\n \u003cp\u003eGenome (18.3 kb) of a novel bluvavirus—Rhododendron bluvavirus 1 (RhBlV1)—was identified in the transcriptome of \u003cem\u003eRhododendron arboretum\u003c/em\u003e and contained twelve ORFs. ORFs 1a and 1b, possibly via + 1 RFS, encode replicase with viral protease (PF05533), MTR, HEL and RdRP motifs. ORFs downstream to ORF1b encode a 11 kDa hypothetical protein, 7 kDa putative transmembrane protein, HSP70 (64 kDa), HSP90h (61 kDa), CP (32 kDa) and five hypothetical proteins (23 kDa, 24 kDa, 7 kDa, 8kDa and 9 kDa) in the order. BLASTp analysis revealed the maximum identity of 57% of any encoded RhBlV1 protein with the corresponding protein of blueberry virus A (BVA) (Table S1). Sequence identity matrix revealed 32.4–56.6% amino acid sequence identity of RdRP, HSP70 and CP encoded by RhBlV1 with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic analysis grouped RhBlV1 with BVA in RdRP, HSP70 and CP-based trees (Figs.\u0026nbsp;1–3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.4. Identification of putative novel closteroviruses in plant (meta)transcriptomes\u003c/h2\u003e\n \u003cp\u003eCoding-complete genomes (15.4–18.3 kb), encoding nine to eleven ORFs, of seven novel closteroviruses—Alternanthera closterovirus (AltCV1), Artemisia closterovirus 1 (ArtCV1), Cerastium closterovirus 1 (CerCV1), Chrysanthemum closterovirus 1 (ChrCV1), cowslip closterovirus 1 (CwCV1), Euphorbia closterovirus 1 (EuCV1) and thyme closterovirus 1 (ThCV1)—were identified in transcriptomes of seven dicot plants (Table\u0026nbsp;1). All the identified novel closteroviral genomes encode replicase (with viral preotease, MTR, HEL and RdRP motifs) from ORFs 1a and 1b possibly via + 1 RFS, a small transmembrane protein with MP p6 motif (5–7 kDa), HSP70 (64–67 kDa), HSP90h (61–62 kDa), CPm (24–26 kDa), CP (22–25 kDa) and a protein with RNA silencing suppressor motif(s) (PF11757, PF11479; 20–26 kDa) in the order. Besides, the genomes contain a common ORF encoding a hypothetical protein (17–21 kDa) in between CP and silencing suppressor-encoding ORFs. ArtCV1, ChrCV1, CwCV1 and EuCV1 genomes contain an additional hypothetical protein (31–33 kDa)-encoding ORF in between ORF1b and ORF encoding MP-like protein, while ChrCV1 genome also possesses an ORF, encoding a CP-like protein (48 kDa), immediately downstream of ORF1b. BLASTp analysis revealed that the maximum sequence identity of any protein encoded by the identified novel closteroviruses to the corresponding protein of known closteroviruses was 80% (Table S1). While RdRP encoded by identified novel closteroviruses shared 67.7–84.5% amino acid sequence identities with that of known and identified closterovirids, the encoded HSP70 and CP shared 31.5–71.8% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic trees constructed based on RdRP, HSP70 and CP sequences grouped the identified seven novel closteroviruses with known closteroviruses (Figs.\u0026nbsp;1–3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.5. Identification of putative novel olivaviruses in plant (meta)transcriptomes\u003c/h2\u003e\n \u003cp\u003eA coding-complete (18.2 kb) and a 5’ end partial genome (13.1 kb), each containing fourteen ORFs, were identified in transcriptomes of mango and pecan, respectively, representing two novel olivaviruses: mango olivavirus 1 (MgOV1) and pecan olivavirus 1 (PecOV1). ORFs encoding replicase (from ORFs 1a and 1b), a putative transmembrane protein (5–7 kDa), HSP70 (66–67 kDa), HSP90h (57–60 kDa), CP (25–36 kDa), thaumatin-like protein (PF00314; 26–28 kDa) and a 3’→5’ exonuclease-like protein (PF01612; 25–26 kDa) were determined in both the genomes, though their order differed. The remaining ORFs of variable lengths encoded hypothetical proteins (8–38 kDa) with no predicted motif. BLASTp analysis of proteins encoded by novel olivaviruses revealed a maximum of 50% identity to the corresponding sequence of known olivaviruses (Table S1). Sequence identity matrix revealed 31.4–55.9% amino acid sequence identity of RdRP, HSP70 and CP encoded by MgOV1 and PecOV1 with that of known closterovirids (Figs. S1–S3, Table S2). MgOV1 and PecOV1 grouped with olivaviruses in RdRP, HSP70 and CP-based phylogenetic trees (Figs.\u0026nbsp;1–3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.6. Identification of putative novel velariviruses in plant (meta)transcriptomes\u003c/h2\u003e\n \u003cp\u003eCoding-complete genomes (16.6–17.0 kDa) of two velariviruses—Camellia velarivirus 1 (CamVV1) and hazelnut velarivirus 1 (HzVV1)—were identified in transcriptomes of \u003cem\u003eCamellia chekiangoleosa\u003c/em\u003e and hazelnut, respectively. CamVV1 genome contains nine ORFs, while thirteen ORFs were determined in HzVV1 genome, both encoding replicase (from ORFs 1a and 1b), a small putative transmembrane protein (4–5 kDa), HSP70 (62–63 kDa), HSP90h (61 kDa), CP (37–38 kDa) and CPm (26–73 kDa) in common. The remaining ORFs encoded hypothetical proteins of size 26–73 kDa with no predicted motif. BLASTp analysis revealed the 61% maximum sequence identity of any protein encoded by the novel velariviruses to the known velariviruses (Table S1). Sequence identity matrix revealed 35.5–62.1% amino acid sequence identity of RdRP, HSP70 and CP encoded by CamVV1 and HzVV1 with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic analysis based on RdRP, HSP70 and CP grouped CamVV1 and HzVV1 with known velariviruses (Figs.\u0026nbsp;1–3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.7. Identification of other putative novel viruses potentially representing novel closteroviral taxa in plant (meta)transcriptomes\u003c/h2\u003e\n \u003cp\u003eCoding-complete clostero-like viral genome was identified in transcriptomes of \u003cem\u003eBetula pendula\u003c/em\u003e and \u003cem\u003eMusa textilis\u003c/em\u003e, while partial clostero-like viral genome was identified in \u003cem\u003eAgave tequilana\u003c/em\u003e, \u003cem\u003eMusa\u003c/em\u003e hybrid cultivar and \u003cem\u003ePicea abies\u003c/em\u003e. Genome (22.1 kb) of virus identified in \u003cem\u003eBetula\u003c/em\u003e, named tentatively as birch clostero-like virus 1 (BiClV1), contained fourteen ORFs encoding replicase (from ORFs 1a and 1b with possible + 1 RFS), a small putative transmembrane protein (6 kDa), HSP70 (67 kDa), HSP90h (59 kDa), CP (35 kDa) and eight hypothetical proteins (9–46 kDa). Additionally, the replicase of BiClV1 contained the 2OG-Fe(II) oxygenase superfamily motif at the N-terminal region before the endopeptidase motif. A reverse transcriptase(RTase)-like motif (PF13456) was determined in the hypothetical protein (22 kDa) encoded by the last but one ORF from the 5’ end, while the proteins encoded by the last two ORFs shared sequence similarities (28–32% at 75–92% query coverage) with plant proteins. BLASTp analysis also revealed the maximum 43% sequence identity of any BiClV1 protein with that of known closterovirids (Table S1). Sequence identity matrix revealed 28.1–44.6% amino acid sequence identity of RdRP, HSP70 and CP encoded by BiClV1 with that of known closterovirids (Figs. S1–S3, Table S2). Genomes of the virus identified in \u003cem\u003eMusa\u003c/em\u003e hosts, named tentatively as abaca clostero-like virus 1 (AbaClV1), shared 81% nucleotide sequence identity with each other and contained ten ORFs. These ORFs encode replicase (possibly via + 1 RFS from ORFs 1a and 1b), a small putative transmembrane protein (5 kDa), HSP70 (62 kDa), HSP90h (60 kDa), CPm (48 kDa), CP (32 kDa), a 24 kDa hypothetical protein, a thaumatin-like protein (31 kDa) and a 20 kDa hypothetical protein. RdRP sequence of AbaClV1 shared a maximum of 50% identity with that of known criniviruses in BLASTp analysis, which was the highest sequence identity of any AbaClV1 protein with known members (Table S1). RdRP, HSP70 and CP encoded by AbaClV1 isolates shared 30.7–52.7% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Five partial contigs (2.1–10.3 kb) representing genomes of a novel clostero-like virus, named as Agave clostero-like virus 1 (AgClV1), were identified in transcriptome data of two independent bioprojects of Agave. AgClV1 contained seven ORFs, in total, across the partial contigs, encoding replicase (possibly via + 1 RFS of ORFs 1a and 1b), a small putative transmembrane protein (4 kDa), HSP70 (63 kDa), HSP90h (61 kDa), CP (28 kDa) and a hypothetical protein. The maximum sequence identity shared by any intact protein encoded by AgClV1 contigs with the corresponding sequence of known closterovirids was 53% in BLAST analysis (Table S1). RdRP, HSP70 and CP encoded by AgClV1 isolates shared 31.3–55.9% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Four partial contigs (2.8–12.9 kb) that represented the genome of a clostero-like virus, named as spruce clostero-like virus 1 (SpClV1), was identified in two related bioprojects of \u003cem\u003eP. abies\u003c/em\u003e. Across the partial contigs, SpClV1 contained ten ORFs encoding replicase (from ORFs 1a and 1b through possible + 1 RFS), a putative transmembrane protein (7 kDa), HSP70, HSP90h, CP (28 kDa) and four hypothetical proteins (11–20 kDa). SpClV1 proteins shared a maximum of 49% identity with the corresponding sequence of known closterovirids in BLAST analysis (Table S1). RdRP, HSP70 and CP encoded by SpClV1 isolates shared 26.8–54.6% amino acid sequence identities with that of known closterovirids (Figs. S1–S3, Table S2). Phylogenetic analysis based on RdRP, HSP70 and CP placed AbaClV1, AgClV1, BiClV1 and SpClV1 in distinct sub-clades within closterovirids. Among these, AbaClV1 and AgClV1 were related to each other and to criniviruses and velariviruses (Figs.\u0026nbsp;1–3).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe family \u003cem\u003eClosteroviridae\u003c/em\u003e includes members that cause economically important diseases in cultivated plants (German-Retana et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Rybicki and Foster, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As of 2025, 87 species were recognized by the ICTV across seven genera in the family (Fuchs et al., 2020, Aboughanem-Sabanadzovic et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the present study, we identified sequences of 22 putative novel closterovirids in 21 plant genera, by mining the yet-unexplored plant (meta)transcriptome data in SRA database for novel closteroviral sequences. This discovery represents 0.25-fold expansion of the family \u003cem\u003eClosteroviridae\u003c/em\u003e. Importantly, the first identification of a putative novel clostero-like virus\u0026mdash;SpClV1\u0026mdash;in a gymnosperm host not only expands the host range of closteroviruses to gymnosperms but also reiterates the vast diversity of gymnosperm virosphere (Bejerman et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Mifsud et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Phylogenetic placement of SpClV1 in a distinct subclade within closterovirids supports the notion of divergent evolution of viruses of gymnosperms from those of angiosperms (Bejerman et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Sidharthan et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Sidharthan et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). TurAV1, a subgroup II ampelovirus, was identified in a host belonging to Zingiberaceae, whose members also host two other subgroup II ampelo-/ampelo-like viruses\u0026mdash;Alpinia vein clearing virus (Larrea-Sarmiento et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and ginger chlorotic fleck-associated virus 2 (Bhat et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is noteworthy that atleast six novel closterovirids were identified in tree hosts in the present study, including the two novel members of the genus \u003cem\u003eOlivavirus\u003c/em\u003e that predominantly contains members reported from tree hosts (Read et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The economically important hosts in which novel closterovirids were reported in the present study include birch, black pepper, hazelnut, mango, oak, pecan, spruce and turmeric.\u003c/p\u003e\u003cp\u003eOn the basis of sequence-based species demarcation criteria of closterovirids (i.e. \u0026lt;75% amino acid sequence identity based on RdRP, HSP70 and CP to known members) and phylogenetic relatedness to known members, BPAV1, LeuAV1, MatAV1, OaAV1, PecAV1 and TurAV1 are regarded as novel ampeloviruses, RhBlV1 as a novel bluvavirus, ChrCV1 and EuCV1 as novel closteroviruses, MgOV1 and PecOV1 as novel olivaviruses, and CamVV1 and HzVV1 as novel velariviruses. Though the RdRP of AltCV1, ArtCV1, CerCV1, CwCV1, and ThCV1 shared\u0026thinsp;\u0026gt;\u0026thinsp;75% identity with RdRP of known or identified closterovirids, their HSP70 and CP sequences shared\u0026thinsp;\u0026lt;\u0026thinsp;75% identities with that of known or identified closterovirids. It is to note that some recognized closteroviruses\u0026mdash;carrot closterovirus 1, carrot closterovirus 2, carrot yellow leaf virus, mint virus 1, Rehmannia virus 1 and tobacco virus 1\u0026mdash;also share\u0026thinsp;\u0026gt;\u0026thinsp;75% RdRP sequence identity with that of other closteroviruses (Adams et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Kwon et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Sch\u0026ouml;negger et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, AltCV1, ArtCV1, CwCV1, and ThCV1 can be regarded as novel closteroviruses, atleast based on their HSP70 and CP sequence divergence.\u003c/p\u003e\u003cp\u003eTo our knowledge, the unusually longer BiClV1 genome (22.1 kb) is, thus far, the largest undivided RNA genome known among the closterovirids and plant viruses in general. Generally, replication of RNA viruses with larger genomes require larger replicase (Agranovsky, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This is the case with BiClV1, which encodes a larger replicase\u0026mdash;likely the largest among closterovirids. To note, BiClV1 genome encodes two proteins sharing sequence similarities with plant proteins, one of which contains an RTase-like domain. This domain is typically associated with a retrotransposon in plants (Marchler-Bauer et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, the role of this domain in a closterovirid needs to be studied further. Although 2OG-Fe(II) oxygenase superfamily motif was determined in between MET and HEL motifs in the replicase of a known ampelovirus (Ito and Nakaune, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and MatAV, the location of this motif in the replicase was different in BiClV1. Based on the genome organization and phylogenetic divergence, BiClV1 can be classified as a new member of a putative new genus, with tentative name \u003cem\u003eBetulavirus\u003c/em\u003e, within the family \u003cem\u003eClosteroviridae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAbaClV1, whose proteins shared sequence similarities with criniviruses, possessed a monopartite RNA genome, unlike the criniviruses with split genomes (Fuchs et al., 2020). Phylogenetically, AbaClV1 was related to criniviruses and velariviruses. Largely, AbaClV1 genome resembled velariviruses than criniviruses. However, it differed from velariviruses in the reverse orientation of CPm and CP ORFs and possession of an additional ORF encoding a thaumatin-like protein, similar to the ones reported in olivaviruses (Ruiz-Garc\u0026iacute;a et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Interestingly, AbaClV1 was identified in (meta)transcriptomes of two Musacaeous hosts, representing two independent bioprojects that includes a virus-indexing project on \u003cem\u003eMusa\u003c/em\u003e germplasm (Rong et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Rong et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported the identification of two contigs sharing sequence similarities (63% and 66%) with genome segments of lettuce chlorosis virus, a crinivirus, in a library derived from a Musa hybrid cultivar. In the same library, we identified the 5\u0026rsquo; partial monopartite genome of AbaClV1 (14.1 kb), which corresponded to the 85% length of the AbaClV1 coding-complete genome identified in an abaca transcriptome library. Based on the genome organization and phylogenetic divergence based on RdRP and HSP70 regions, AbaClV1 can be classified as a new member of a putative new closterovirid genus, for which the tentative name \u003cem\u003eMusavirus\u003c/em\u003e is proposed.\u003c/p\u003e\u003cp\u003eRdRP, HSP70 and CP encoded by AgClV1 and SpClV1 shared\u0026thinsp;\u0026lt;\u0026thinsp;56% sequence identities with known closterovirids. In phylogenetic trees, both the viruses formed distinct clades within closterovirids. Thus, these viruses may also represent potential novel taxa within the family \u003cem\u003eClosteroviridae\u003c/em\u003e. However, their coding-complete genomes could not be recovered in the present study.\u003c/p\u003e\u003cp\u003eGenome organization of the identified olivaviruses slightly differed from that of known olivaviruses as they possess three additional ORFs encoding hypothetical proteins in between ORF1b and the ORF encoding putative transmembrane protein. This includes the thaumatin-like protein-encoding ORF of PecOV1, like in olive yellowing leaf-associated virus, while a similar ORF was present in between HSP90h and CP ORFs in MgOV1, like in Actidinia virus 1 and persimmon virus B (Ruiz-Garc\u0026iacute;a et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, an ORF encoding a protein with 3\u0026rsquo;\u0026rarr;5\u0026rsquo; exonuclease-like motif was determined in both the identified olivaviral genomes, though its position differed in both the viruses. Similar protein was encoded by citrus associated ampelovirus 1 (CaAV-1) (Liu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Interestingly, during the time of manuscript preparation, a genome sequence named \u0026lsquo;Mangifera virus 1 isolate MG (PV101221)\u0026rsquo; deposited from India that shared 88% nucleotide sequence identity (at 94% query coverage) with MgOV1 genome identified in this study in a mango transcriptome dataset deposited from Israel, was available in the GenBank. Genome organization of Mangifera virus 1 and MgOV1 were similar, except for the three unannotated ORFs in Mangifera virus 1 and an additional ORF determined in MgOV1. Thus, the sequence named Mangifera virus 1 represents the near coding-complete genome of MgOV1, and this further validates the host of MgOV1 as mango.\u003c/p\u003e\u003cp\u003eTypically, the closterovirids possess ORFs 1a and 1b that via a\u0026thinsp;+\u0026thinsp;1 RFS encode replicase (Fuchs et al., 2020). Surprisingly, genomes of OaAV1, obtained from two independent bioprojects, contained only one ORF1 encoding a replicase with viral MET, HEL and RdRP motifs. This atypical feature was also observed in the recently reported citrus associated ampelovirus 2, a subgroup I ampelovirus (Liu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) that was related to OaAV1. Taken together, these findings hint at the possible existence of an alternative strategy to +\u0026thinsp;1 RFS for the expression of RdRP gene, atleast in subgroup I ampeloviruses. Nevertheless, the absence of potential\u0026thinsp;+\u0026thinsp;1 RFS in these ampeloviruses needs to be experimentally verified in further studies (Liu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Interestingly, OaAV1 isolates encode a papain family protease from a downstream ORF, a feature that is atypical of ampeloviruses. Similar to CaAV-1, PecAV1 encodes a thaumatin-like protein and a protein with 3\u0026rsquo;\u0026rarr;5\u0026rsquo; exonuclease-like motif (Liu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Likewise, thaumatin and 3\u0026rsquo;\u0026rarr;5\u0026rsquo; exonuclease motifs were determined in protein p33 and p25, respectively, of the Pistachio ampelovirus A (PAVA) (MF198462). Interestingly, in HSP70 and CP-based phylogenetic trees, CaAV-1, PAVA and PecAV1 formed a distinct group within subgroup I ampeloviruses. Thus, although typical of olivaviruses (Sabanadzovic et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), thaumatin-like protein is not exclusive to olivaviruses as it is encoded by a few ampeloviruses and the clostero-like virus AbaClV1. The evolutionarily distinct, animal nidovirids and plant closterovirids\u0026mdash;both possessing the largest undivided RNA genomes in their respective domains\u0026mdash;share strikingly similar genomic features, including the expression of 5\u0026rsquo;-proximal ORFs via ribosomal frameshifting to encode papain-like protease(s), viral MET, HEL and RdRP. Additionally, some nidovirids, as part of the ORF1b polyprotein, encode a 3\u0026rsquo;\u0026rarr;5\u0026rsquo; exonuclease\u0026mdash;related to the DEDD family\u0026mdash;that performs proofreading activity during RNA replication. However, a similar protein was not previously reported in closterovirids (Agranovsky, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Identification of a DEDDy 3\u0026rsquo;\u0026rarr;5\u0026rsquo; exonuclease-like protein in three ampeloviruses and two olivaviruses in the present and an earlier study (Liu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) tempts us to speculate their possible role in enhancing the fidelity of RNA replication in these viruses (Agranovsky, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, further experimental evidences are needed to confirm this speculation. It is noteworthy that the 3\u0026rsquo;\u0026rarr;5\u0026rsquo; exonuclease-like protein is encoded by a separate downstream ORF in closterovirids, unlike in nidovirids, which encode it as a part of the replication-associated polyprotein (Agranovsky, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe present study has certain limitations. As with other data-mining studies, the host assignment of the identified viruses was based solely on the metadata provided in the SRA database, due to the lack of access to the original samples for validation. Consequently, the end sequences of the coding-complete viral genomes could not be precisely determined through wet-lab based assays. Therefore, these findings should be considered preliminary until further validation (Bejerman et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nevertheless, the identification of a few novel closteroviruses in different independent libraries derived from the same or closely related hosts strengthens the evidence for their host association.\u003c/p\u003e\u003cp\u003eIn conclusion, coding-complete or partial genomes of twenty-two putative novel closterovirids were identified across a broad range of plant genera. Some of the identified viruses may represent novel genera within the family \u003cem\u003eClosteroviridae\u003c/em\u003e, including a virus with the largest known RNA genome among closterovirids. Further studies in the respective virus-associated hosts are needed to validate the host associations and understand the distribution and biology of these novel viruses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5. Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks the original submitters of Next Generation Sequencing data to the NCBI SRA database whose data were analysed in this study and the NCBI SRA for making the data publicly available. The author acknowledges the support of the Group Coordinator Research and the Director, ICFRE-IFB, Hyderabad.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Author contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV. Kavi Sidharthan: Conceptualization, Methodology, Formal analysis and investigation, Writing-original draft preparation, Writing - review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Data availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequences obtained in this study were submitted to the NCBI TPA database. In addition, they are included in the Supplementary file 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Declaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares that there is no competing interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no funding for this study\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAboughanem-Sabanadzovic N, Bar-Joseph M, Candresse T, Maree HJ, Melzer M, Menzel W, Minafra A, Mollov D, Tzanetakis IE, Rubino L, Sabanadzovic S (2025) Create 31 new species in the genera \u003cem\u003eAmpelovirus\u003c/em\u003e, \u003cem\u003eClosterovirus\u003c/em\u003e, \u003cem\u003eCrinivirus\u003c/em\u003e, \u003cem\u003eOlivavirus\u003c/em\u003e and \u003cem\u003eVelarivirus\u003c/em\u003e and abolish one species in the genus \u003cem\u003eClosterovirus\u003c/em\u003e. https://ictv.global/system/files/proposals/pending/2025/Plant%20virus%20%28P%29%20proposals/2025.019P.N.v2.Closteroviridae_31nsp_abolish_1sp.docx\u003c/li\u003e\n\u003cli\u003eAdams IP, Skelton A, Macarthur R, Hodges T, Hinds H, Flint L, Nath PD, Boonham N, Fox A (2014) Carrot yellow leaf virus is associated with carrot internal necrosis. 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Frontiers in Plant Science 13:804968\u003c/li\u003e\n\u003cli\u003eZheng H, Yu MY, Han Y, Tai B, Ni SF, Ji RF, Pu CJ, Chen K, Li FQ, Xiao H, Shen Y (2022) Comparative transcriptomics and metabolites analysis of two closely related Euphorbia species reveal environmental adaptation mechanism and active ingredients difference. Frontiers in Plant Science 13:905275\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Details of putative novel closteroviruses identified in plant (meta)transcriptomes available in public domain\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"1075\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVirus name/Tentative species name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcronym\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource library/ Bioproject\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenome length\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(nt)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean depth (x)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample cultivar or isolate or ecotype or variety/tissue\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlant Species name/ Family\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eData Reference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus: \u003cem\u003eAmpelovirus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eBlack pepper ampelovirus 1/ \u003cem\u003eAmpelovirus piperis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eBPAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR3404569/ PRJNA318916\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e12,080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e141.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003ePanniyur 1/ Leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003ePiper nigrum\u003c/em\u003e L./ Piperaceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMahadevan et al., 2024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eLeucadendron ampelovirus 1/ \u003cem\u003eAmpelovirus leucadendri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eLeuAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eERR6130833/ PRJEB45774\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e5,967*/ 3,728*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e11.53/8.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBrunioides/ NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eLeucadendron brunioides\u003c/em\u003e Meisn./ Proteaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eScharmann et al., 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eMatucana ampelovirus 1/\u003cem\u003eAmpelovirus matucanae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eMatAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR13805638/ PRJNA705387\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e16,247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e34.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eHBG13/ Pistil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eMatucana madisoniorum\u003c/em\u003e (Hutchison) G.D.Rowley/ Cactaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eRamanauskas and Igić, 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 197px;\"\u003e\n \u003cp\u003eOak ampelovirus 1/ \u003cem\u003eAmpelovirus quercus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003eOaAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR3636733/ PRJNA322128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e17,795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e2,325.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Stem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eQuercus robur\u003c/em\u003e L./ Fagaceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eBroberg et al., 2018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eERR1354110/ PRJEB13357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e16,005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e31.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eHearn et al., 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003ePecan ampelovirus 1/ \u003cem\u003eAmpelovirus caryae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ePecAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR17335006/ PRJNA792564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e16,130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e26.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eOconee/ Kernel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarya illinoinensis\u003c/em\u003e (Wangenh.) K. Koch/ Juglandaceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eZhang et al., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eTurmeric ampelovirus 1/ \u003cem\u003eAmpelovirus curcumae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eTurAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR13594120/ PRJNA698442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e12,997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e157.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eIISR Prathibha/ Rhizome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eCurcuma longa\u003c/em\u003e L./ Zingiberaceae (monocot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSheeja et al., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus: \u003cem\u003eBetulavirus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eBirch clostero-like virus 1/ \u003cem\u003eBetulavirus betulae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eBiClV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eERR3173759/ PRJEB29260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e22,120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e209.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003ev5834/ Stem (developing phloem)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eBetula pendula\u003c/em\u003e Roth/ Betulaceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eAlonso‐Serra et al., 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus: \u003cem\u003eBluvavirus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eRhododendron bluvavirus 1/ \u003cem\u003eBluvavirus rhododendri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eRhBlV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR4449165/ PRJNA345272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e18,250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e44.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eHimachal Pradesh/ Flower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhododendron arboreum\u003c/em\u003e Sm./ Ericaceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eChoudhary et al., 2018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus: \u003cem\u003eClosterovirus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eAlternanthera closterovirus 1/ \u003cem\u003eClosterovirus alternantherae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eAltCV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eERR2040215/ PRJEB21674\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e15,852\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e273.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlternanthera brasiliana\u003c/em\u003e (L.) Kuntze/ Amaranthaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e1000 Plant Transcriptomes Initiative, Unpublished\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eArtemisia closterovirus 1/ \u003cem\u003eClosterovirus artemisiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eArtCV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR15595118/ PRJNA752933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e16,576\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e760.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eYQ7/ Root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eArtemisia annua\u003c/em\u003e L./ Asteraceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMa et al., 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eCerastium closterovirus 1/ \u003cem\u003eClosterovirus cerastii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eCerCV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR1979688/ PRJNA280277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e15,402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e53.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eCerastium arvense\u003c/em\u003e L./ Caryophyllaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eBrockington et al., 2015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eChrysanthemum closterovirus 1/ \u003cem\u003eClosterovirus chrysanthemi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eChrCV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR15321559/ PRJNA751454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e18,179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e313.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eRibonette/ Leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eChrysanthemum\u003c/em\u003e \u0026times; \u003cem\u003emorifolium\u003c/em\u003e (Ramat.) Hemsl./ Asteraceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eChirkov et al., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eCowslip closterovirus 1/ \u003cem\u003eClosterovirus primulae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eCwCV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eERR5762867/ PRJEB44353\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e17,184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e2,160.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Flower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003ePrimula veris\u003c/em\u003e L./ Primulaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ePotente et al., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eEuphorbia closterovirus 1/ \u003cem\u003eClosterovirus euphorbiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eEuCV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR13511996/ PRJNA693983\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e16,432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1,032.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eEuphorbia ebracteolata\u003c/em\u003e Hayata/ Euphorbiaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eZheng et al., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eThyme closterovirus 1/ \u003cem\u003eClosterovirus thyme\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eThCV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR6262814/ PRJNA417241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e15,449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e26.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eTv8/ Leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eThymus vulgaris\u003c/em\u003e L./ Lamiaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMollion et al., 2018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus: \u003cem\u003eMusavirus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 197px;\"\u003e\n \u003cp\u003eAbaca clostero-like virus 1/ \u003cem\u003eMusavirus musae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003eAbaClV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eERR6912813/ PRJEB47952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e16,509\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e273.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eTangongon/ Pseudostem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eMusa textilis\u003c/em\u003e N\u0026eacute;e/ Musaceae (monocot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eEreful et al., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR16881896/ PRJNA777477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e14,134*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e90.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eMusa hybrid cultivar/ Musaceae (monocot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eRong et al., 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus: \u003cem\u003eOlivavirus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eMango olivavirus 1/ \u003cem\u003eOlivavirus mangiferae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eMgOV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR19975612/ PRJNA855362\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e18,177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e21.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eShelly/ Fruit peel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eMangifera indica\u003c/em\u003e L./ Anacardiaceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ePatel et al., 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003ePecan olivavirus 1/ \u003cem\u003eOlivavirus caryae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ePecOV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR13411591/ PRJNA680537\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e13,110*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e45.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eOaxaca/ Catkin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarya illinoinensis\u003c/em\u003e (Wangenh.) K. Koch/ Juglandaceae (dicot)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eLovell et al., 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus: \u003cem\u003eVelarivirus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eCamellia velarivirus 1/ \u003cem\u003eVelarivirus camelliae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eCamVV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR10121554/ PRJNA565081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e16,623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1,327.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eRISF_HS/ Endocarp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eCamellia chekiangoleosa\u003c/em\u003e Hu/ Theaceae (dicot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eYan et al., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 197px;\"\u003e\n \u003cp\u003eHazelnut velarivirus 1/ \u003cem\u003eVelarivirus coryli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eHzVV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR10541224/ PRJNA591492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e17,021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e103.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eDawei/ Ovule\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eCorylus heterophylla\u003c/em\u003e Fisch. ex Trautv./\u0026nbsp;Betulaceae\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eLiu et al., 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 1075px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnclassified members\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 197px;\"\u003e\n \u003cp\u003eAgave clostero-like virus 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 85px;\"\u003e\n \u003cp\u003eAgClV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR789742/ PRJNA193469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e10,328*/2,143*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e26.07/48.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWeber azul/ NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eAgave tequilana\u003c/em\u003e F.A.C.Weber/ Asparagaceae\u003cem\u003e\u0026nbsp;\u003c/em\u003e(monocot)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGross et al., 2013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR789754/ PRJNA193469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e5,173*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e23.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eERR2040717/ PRJEB21674\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e5,072*/2,067*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e28.40/13.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Whole plant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e1000 Plant Transcriptomes Initiative, Unpublished\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 197px;\"\u003e\n \u003cp\u003eSpruce clostero-like virus 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003eSpClV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR11565953/ PRJNA622086\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e12,863*/4,674*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e18.62/92.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 142px;\"\u003e\n \u003cp\u003eNA/ Root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003ePicea abies\u003c/em\u003e (L.) H.Karst./ Pinaceae (gymnosperm)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eDOE Joint Genome Institute, 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSRR9030810/ PRJNA537855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e6,259*/2,836*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e17.70/15.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*indicates partial genome segment length and NA indicates information not available.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"ICFRE-Institute of Forest Biodiversity","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Closteroviridae, data-driven virus discovery, diversity, host range, novel","lastPublishedDoi":"10.21203/rs.3.rs-7560636/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7560636/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe family \u003cem\u003eClosteroviridae\u003c/em\u003e comprises filamentous, RNA genome-containing viruses that infect plants. In the present study, public domain SRA libraries derived from plants were mined for novel closteroviral sequences, resulting in the identification of twenty-two putative novel closterovirids across twenty-one plant genera. The identified viruses were represented by eighteen coding-complete and four partial genomes. Based on genome organization, pairwise sequence identity and phylogenetic analysis, the viruses were classified in the following genera: \u003cem\u003eAmpelovirus\u003c/em\u003e (6), \u003cem\u003eBluvavirus\u003c/em\u003e (1), \u003cem\u003eClosterovirus\u003c/em\u003e (7), \u003cem\u003eOlivavirus\u003c/em\u003e (2) and \u003cem\u003eVelarivirus\u003c/em\u003e (2), while four other viruses may represent four novel genera within the family. Other significant findings of the study include: (i) the identification of a 3\u0026rsquo;\u0026rarr;5\u0026rsquo; exonuclease-like protein in ampeloviruses and olivaviruses, (ii) the identification of an ampelovirus that encodes a polyprotein containing an RNA-dependent RNA polymerase motif without employing a\u0026thinsp;+\u0026thinsp;1 ribosomal frameshift, (iii) the identification of a virus with the largest known genome among closterovirids, and (iv) the identification of a monopartite crini-like virus in \u003cem\u003eMusa\u003c/em\u003e hosts potentially representing a novel genus. Besides, expanding the known closterovirid diversity by 0.25-fold, this study provides a base for future research aimed at understanding the biology and distribution of the identified novel viruses.\u003c/p\u003e","manuscriptTitle":"Plant (meta)transcriptome data mining identified twenty-two putative novel taxa in the family Closteroviridae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 05:13:13","doi":"10.21203/rs.3.rs-7560636/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a28d4edc-9afc-4a26-a24e-c0788a6d3ac4","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-09T05:13:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 05:13:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7560636","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7560636","identity":"rs-7560636","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}