Complete nucleotide sequence of a novel botourmiavirus from the rice blast fungus Magnaporthe oryzae isolate SH05

A novel mycovirus with the proposed name “Magnaporthe oryzae botourmiavirus 9” (MoBV9) was found in the rice blast fungus Magnaporthe oryzae isolate SH05. The virus has a positive single-stranded RNA genome of 2,812 nucleotides and contains a single open reading frame predicted to encode an RNA-dependent RNA polymerase that is closely related to those of some unclassified viruses of the family Botourmiaviridae, including Plasmopara viticola lesion associated ourmia-like virus 44, Plasmopara viticola lesion associated ourmia-like virus 47, and Cladosporium uredinicola ourmiavirus 1. Genome sequence comparisons and phylogenetic analysis supported the notion that MoBV9 is a new member of the family Botourmiaviridae.

Botourmiaviridae is a newly established virus family related to Narnaviridae, Mitoviridae, and Leviviridae, according to current taxonomic information from the International Committee on Taxonomy of Viruses (ICTV, Virus Taxonomy: 2019 Release). The family Botourmiaviridae includes four approved genera, Botoulivirus, Magoulivirus, Scleroulivirus and Ourmiavirus. Members of the genus Ourmiavirus are plant viruses with a bacilliform virion structure whose genome usually contains three (+) ssRNA segments, encoding an RNA-dependent RNA polymerase (RdRp), a coat protein (CP), and a movement protein (MP), respectively [6]. Since the MP and CP of ourmiaviruses show significant similarity to those of other plant viruses, including tombusviruses and sobemovirus, it has been proposed that ourmiavirus might have evolved by reassortment of genomic segments from viruses infecting fungi and plants [15]. Members of the genera Botoulivirus, Magoulivirus, and Scleroulivirus are mycoviruses. Similar to narnaviruses and mitoviruses, only one (+) ssRNA segment, encoding the RdRp, has been identified in all of these ourmia-like mycoviruses, and this segment has been demonstrated to be sufficient for replication, infection, and transmission [20]. Ourmia-like mycovirus-associated satellite-like RNAs have also been identified in Magnaporthe oryzae, but these are not essential for virus replication [14].
M. oryzae strain SH05 was isolated from a lesion of a rice neck-panicle collected in Fujian province, China, in 2014, stored on rice stem nodes at -20 °C, and cultured on potato dextrose agar at 28 °C. dsRNA was extracted by the CF-11 cellulose chromatography method [13], and the 2.8-kb viral genomic dsRNA of MoBV9 was extracted from an agarose gel after electrophoresis, purified using an Agarose Gel DNA Purification Kit 2.0 (Takara), and treated with DNase I and S1 nuclease to eliminate contaminating DNA and ssRNA. cDNA was synthesized using a tagged random primer (5′-CGA TCG ATC ATG ATG CAA TGCNNNNNN-3′) and amplified using a primer recognizing the tag sequence (5′-CGA TCG ATC ATG ATG CAA TGC-3′). The amplified cDNA products were cloned into the vector pMD19-T (Takara) and introduced by transformation into Escherichia coli strain Top10 for sequencing. To fill the gaps, the sequence data that were obtained were used to design dsRNA-specific primers, which were then used for RT-PCR. In order to determine the terminal sequences of the dsRNA, cDNA amplification of the 5′and 3′ends was performed using a ligase-mediated terminal amplification method as described previously [3,10]. In both orientations, every base was determined by sequencing at least three independent overlapping clones. The complete nucleotide sequence of the MoBV9 genome has been deposited in the GenBank database with accession number MT995746.1. The amino acid (aa) sequence of the putative RdRp of MoBV9 was aligned with other virus RdRp sequences using Clustal Omega (https:// www. ebi. ac. uk/ Tools/ msa/ clust alo/). RNA secondary structures of the termini of MoBV9 were predicted using the MFOLD web server (http:// www. unafo ld. org// RNA_ form. php) [22]. Conserved domains were identified using the NCBI Conserved Domain Database (CDD) (https:// www. ncbi. nlm. nih. gov/ cdd). On the basis of the aligned sequences, a phylogenetic tree was constructed by the neighbor-joining method using MEGA version 6.0 [8].

Sequence properties
dsRNA extraction and electrophoresis analysis showed that only one 2.8-kb dsRNA, representing the genome of MoBV9, could be detected in M. oryzae strain SH05 (Fig. 1A). The full-length nucleotide sequence of MoBV9 was determined and found to be 2,812 nucleotides (nt) long with a GC content of 54.1%. The 5′ untranslated region (UTR) is 179 nt long, and the 3′-UTR is 664 nt long, ending with a 9-nt poly (A) tail (Fig. 1B).
Using the standard genetic code, the MoBV9 genome was predicted to contain a single large open reading frame (ORF) on its positive strand, putatively encoding a 659-amino-acid (aa) protein with a molecular mass of 73.2 kDa (Fig. 1)   To analyze the phylogenetic position of MoBV9, a molecular phylogenetic tree was constructed using aa sequences of the RdRp regions of MoBV9 and 73 other selected viruses of the families Botourmiaviridae, Narnaviridae, Mitoviridae, and Leviviridae. As shown in Fig. 4, the neighbor-joining tree strongly suggested that MoBV9 is a new member of the family Botourmiaviridae (Fig. 4).

Declarations
Conflict of interest All authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.