Molecular characterization of a novel ourmia‑like virus from the phytopathogenic fungus Botryosphaeria dothidea

Here, we describe a novel ourmia-like virus, Botryosphaeria dothidea ourmia-like virus 2 (BdOLV2), derived from the phytopathogenic fungus Botryosphaeria dothidea strain ZM180192-1 infecting maize in Henan province of China. The complete genome sequence of BdOLV2 consists of a positive-sense single-stranded RNA (+ ssRNA) segment with a length of 2,532 nucleotides (nt). The sequence contains a large open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) consisting of 605 amino acids (aa) with a molecular mass of 68.59 kDa. This RdRp protein contains eight typical conserved motifs associated with ourmia-like viruses. BLASTp analysis revealed that the RdRp protein of BdOLV2 had the highest similarity (62.10%, 58.15%, and 55.75% identity, respectively) to a virus previously identified as "Botourmiaviridae sp.", Macrophomina phaseolina ourmia-like virus 2, and Macrophomina phaseolina ourmia-like virus 2-A. Phylogenetic analysis based on the RdRp aa sequence indicated that BdOLV2 is a new member of the genus Magoulivirus in the family Botourmiaviridae.

In this study, we determined the complete genome sequence of a novel ourmia-like virus from B. dothidea strain ZM180192-1, which we have tentatively named "Botryosphaeria dothidea ourmia-like virus 2" (BdOLV2). Phylogenetic analysis showed that BdOLV2 is closely related to members of the genus Magoulivirus in the family Botourmiaviridae.

Provenance of the virus material
Strain ZM180192-1 was first obtained from maize leaf in Henan Province of China in 2018 and identified as B. dothidea based on its internal transcribed spacer (ITS), elongation factor-1α (EF-1α), and β-tubulin sequences. The strain was cultured on potato glucose agar at 25° C for 3 days in the dark to observe the colony morphology.
Two hundred thirty strains of the family Botryosphaeriaceae were isolated from diseased samples of different plants in Henan Province, China. The mycelium of 230 samples from Botryosphaeriaceae were combined in a pool and used for high-throughput sequencing (Novogene, Beijing, China). Clean reads were obtained by removing adapter sequences, low-quality bases, and host RNA-seq data. The putative viral RNA-seq data were then spliced using the Inchworm, Chrysalis, and Butterfly programs in Trinity software. The spliced sequences were compared with sequences in the GenBank, NR, and CDD databases using DIAMOND software to identify virus-related sequences [25]. Among the high-quality viral contigs obtained, we focused on the viral c36267_g1_i1 sequence of BdOLV2, which shared the highest sequence similarity to a virus identified as "Botourmiaviridae sp." (partial genome; identity, 62.15%; query coverage, 80%; GenBank accession number, URG17163.1) (Supplementary File S1). Total RNA was extracted using TRIzol Reagent (Adlai, Beijing, China), and cDNA was synthesized using a SuperScript III First-Strand Synthesis System Kit (Vazyme, Nanjing, China). Specific primers were designed based on the c36267_g1_i1 sequence, and the presence of the virus in the host (ZM180192-1) was confirmed by RT-PCR. An electrophoresis image of the RT-PCR product is shown in Supplementary File S5. Another sequence, c37748_g1_i1, was found by BLASTx alignment to share the highest sequence similarity with Plasmopara viticola lesion associated narnavirus 4 (complete coding region; identity, 71.48%; query coverage, 99%; GenBank accession number, QIR30283.1), and this virus was also detected in strain ZM180192-1 (Supplementary File S6). The primer sets used in this study are shown in Supplementary Table S2. dsRNA was extracted from the mycelium by a previously described column separation method, with minor modifications [27]. The dsRNA was then digested with DNase I and S1 nuclease (Takara, Dalian, China) to eliminate DNA and ssRNA, and the treated dsRNA was electrophoresed in a 1.0% (w/v) agarose gel stained with Goldview and viewed on a UV transilluminator (Fig. 1A). Two dsRNA fragments with estimated sizes between 1.0 and 3.0 kbp were isolated, one of which was the genome of BdOLV2 (associated with c36267_g1_i1) and the other of which belonged to a member of the genus Narnavirus (associated with c37748_g1_i1). The terminal sequences of BdOLV2 were obtained using nested primers designed based on the central sequences of BdOLV2 and ligase-mediated rapid amplification of cDNA ends (RLM-RACE) [28]. The PCR products were purified and ligated into the pMD18-T vector (Takara, Dalian, China), and this construct was introduced by transformation into chemically competent cells of Escherichia coli DH5α (Tsingke, Beijing, China). At least three independent positive recombinant plasmids of each product were sequenced in both directions to ensure the accuracy of the sequence (Sangon, Shanghai, China).
The final viral genome sequences were assembled using DNAMAN software (version 5.2.2). ORF Finder (http:// www.unafold.org/mfold/applications/rna-folding-form. php) and Conserved Domain Database (CDD) Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) were used to predict potential ORFs and identify conserved domains, respectively, in the BdOLV2 genome. The NCBI database was used for sequence comparisons, and a multiple alignment with these reference sequences was made using CLUSTALX 2.0. The alignment was annotated and colored using GeneDoc software. A phylogenetic tree was constructed using MEGA 6.0 software by the maximumlikelihood (ML) method with default parameters (Poisson model) and a bootstrap test consisting of 1000 replicates.

Sequence properties
Examination of the colony morphology of B. dothidea strain ZM180192-1 on PDA showed that it has fluffy aerial mycelia, initially white, which grow to fill the plate within 3-4 days, becoming denser over time (Fig. 1B).
The full-length genome sequence of BdOLV2 from strain ZM180192-1 was obtained by RT-PCR and RNAligase-mediated rapid amplification of cDNA ends (RLM-RACE) (Supplementary File S3) and submitted to the GenBank database (accession number OP784426). The complete genome of BdOLV2 is 2532 nt in length, consisting of 18.44% A, 23.93% U, 31.48% G, and 26.15% C. The 5' and 3' termini of the genome contain untranslated regions (UTRs) of 31 nt and 683 nt, respectively, and potential stem-loop structures were predicted by RNAfold (Fig. 1D). The BdOLV2 genome contains an open reading frame (ORF) encoding a putative 605-aa RdRp protein with a molecular mass of 68.59 kDa. A Conserved Domain Database (CDD) Search revealed the presence of the conserved RdRp catalytic core domain of + ssRNA viruses at aa position 149-355 (ps-ssRNAv RdRp-like superfamily; accession number CL40470; E value, 2.97e-72) (Fig. 1C). A multiple alignment of the RdRp sequence of BdOLV2 with the corresponding sequences of other ourmia-like viruses of the family Botourmiaviridae revealed the presence of eight conserved motifs (I-VIII) associated with ourmia-like viruses, including a highly conserved GDD triplet within motif VI ( Fig. 2A) [21,29]. The names and accession numbers of these viruses are as follows: Acremonium sclerotige- BLASTp analysis revealed that the RdRp protein of BdOLV2 shares the highest sequence similarity with three viruses: "Botourmiaviridae sp." (partial genome; identity, 62.10%; query coverage, 77%; GenBank accession no., URG17163.1), Macrophomina phaseolina ourmia-like virus 2 (complete coding sequence; identity, 58.15%; query coverage, 74%; GenBank accession no., QOE55587.1), and Macrophomina phaseolina ourmia-like virus 2-A (complete coding sequence; identity, 55.75%; query coverage, 99%; GenBank accession no., QOE55588.1) (Supplementary Table S4). The level of RdRp sequence similarity of BdOLV2 to these viruses is far below the International Committee on Taxonomy of Viruses (ICTV) (https://ictv. global/report/chapter/botourmiaviridae/botourmiaviridae/ magoulivirus) cutoff of < 90% sequence identity for demarcating species in the genus Magoulivirus. To further assess lanes D and S1, dsRNA treated with DNase I and S1 nuclease, respectively; lane D + S1, dsRNA was treated with both DNase I and S1 nuclease. One of the fragments is the genome of BdOLV2 (associated with c36267_g1_i1), and the other belongs to a mycovirus of the genus

Conflict of interest The authors have no conflict of interest.
Ethical approval This article does not contain any studies with human the taxonomic status of BdOLV2, a phylogenetic tree was constructed based on the RdRp aa sequences of BdOLV2 and other selected mycoviruses in the family Botourmiaviridae. The results showed that BdOLV2 clustered closely (100% bootstrap support) with Macrophomina phaseolina ourmia-like virus 2-A and Macrophomina phaseolina ourmia-like virus 2 of the genus Magoulivirus (Fig. 2B).
In conclusion, we have identified a novel ourmia-like virus, BdOLV2, from B. dothidea strain ZM180192-1 that belongs to the genus Magoulivirus of the family Botourmiaviridae.