Complete genome sequence of a novel mycovirus from Pleurotus citrinopileatus

The complete genome sequence of a novel single-stranded [+ ssRNA] positive-sense (+) RNA mycovirus, designated as "Pleurotus citrinopileatus ourmiavirus 1" (PcOV1), isolated from Pleurotus citrinopileatus strain CCMJ2141, was determined. The complete genome of PcOV1 is composed of 2,535 nucleotides. It contains a single open reading frame (ORF), which encodes a protein of 657 amino acids (aa) containing conserved domains of an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on RdRp sequences revealed that PcOV1 is a new member of the genus Ourmiavirus in the family Botourmiaviridae. This is the first virus from P. citrinopileatus to be characterized.


Provenance and sequencing of strains
Twenty-four P. citrinopileatus strains were collected from the herbarium of the Mycological Institute of Jilin Agricultural University, China. The strains were maintained on potato dextrose agar (PDA) slants at 4°C. Mycelia for total RNA extraction were inoculated onto PDA plates covered with cellophane sheets and incubated in the dark at 25°C for 10 days. Total RNA was extracted from ca. 0.2 g of mycelia using an RNAsimple Total RNA Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. The extracted total RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Madison, WI, USA) and separated by electrophoresis in a 1% gel with 1x TAE buffer for 20 min at 125 V. The RNA from each strain was pooled into a single tube, and rRNA was depleted using a Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA). The rRNA-depleted total RNA (1200 ng/µL) was used as a template to synthesize cDNA. A cDNA library was constructed using an Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The quality of the library was estimated using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The library was sequenced on an Illumina NovaSeq 6000 system (Illumina, USA) with 150bp paired-end reads (PE150) at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).
A total of 78,860,122 raw reads were generated, and 77,919,412 (97.57%) clean reads were obtained after quality filtering. The clean reads were assembled de novo into 38,745 contigs using Trinity [31] and subjected to BLASTx analysis using the NCBI GenBank non-redundant protein database (https://www.ncbi.nlm.nih.gov). One contig (con-tig_17942: 1902 nt length) showed the highest sequence similarity (40.23% identity with 66% query coverage) to Heterobasidion ourmia-like virus 1 (UHK02571.1). The total RNA from each fungal strain was subjected to reverse transcription using Moloney murine leukemia virus (M-MLV) reverse transcriptase (TaKaRa, Dalian, China) and cDNA synthesis using random hexanucleotide primers (dN6 primers: 5′-CGATCGATCATGATGCAATGCNNNNNN-3′). Specific primers (Supplementary Table S1) were designed based on contig_17942 to identify the putative viral sequence to obtain the target strain. Strain CCMJ2141 was confirmed to harbor the putative viral sequence.
To determine the complete viral genome sequence, cDNA amplification of the 5' and 3' ends was performed using the rapid amplification of cDNA ends (RACE) method with multiple sets of overlapping primers for nested PCR designed based on contig 17942 (Supplementary Table S1) [32] using RACE and a high-efficiency DNA gel recovery kit (DingGuo, Beijing, China). The DNA was cloned into the pMD18-T vector (TaKaRa, Dalian, China) and used to transform Escherichia coli strain DH5α. Recombinant plasmids were extracted, amplified with M13 universal primers from individual colonies, and sequenced in both directions by the Sanger method at Sangon Biotech (Shanghai) Co., Ltd. At least three independent clonal inserts of each fragment were sequenced to reduce experimental error. The viral fragment sequences were assembled into contiguous sequences based on common overlapping regions, using DNAMAN 9.0 software (version 8.0; Lynnon Biosoft, Quebec, Canada).
The ClustalX program was used to make a multiple sequence alignment of RdRp aa sequences [33]. Phylogenetic analysis was conducted using the maximum-likelihood (ML) method using PhyML3.0 software [34,35]. MEGA X software [36] was used to visualize and edit the phylogenetic tree. To confirm the presence of the assembled viral contig, the primer pair Ple1900F/Ple1900S was designed to cover the genomic region. RT-PCR was performed, and PCR products were analyzed using gel electrophoresis and sequenced at Sangon Biotech (Shanghai) Co., Ltd.

Sequence properties
Meta-transcriptome and RACE-PCR analysis results showed that P. citrinopileatus strain CCMJ2141 was infected with a novel mycovirus, which we have named "Pleurotus citrinopileatus ourmiavirus 1" (PcOV1). The complete genome sequence of PcOV1 is 2535 nucleotides (nt) long. The GC content is 51%, with a base composition of 24.1% A, 20.9% C, 30.1% G, and 24.9% U. The 5' and 3' untranslated regions (UTRs) of the mycovirus are 233 and 35 nt in length, respectively. The PcOV1 genome contains a single ORF that is 1974 nt in length, starting at nt 223 and terminating at nt 2500. The ORF potentially encodes a protein of 657 aa with a predicted molecular mass of 7.54 kDa and an isoelectric point of 4.67 that contains conserved motifs that are characteristic of an RNA-dependent RNA polymerase (RdRp) (Fig. 1). The full-length nucleotide sequence of PcOV1 was deposited in the GenBank database and assigned the accession number MW772941.
A BLASTp search showed that the amino acid sequence of the PcOV1 RdRp protein is 42.42% identical to that of Armillaria mellea ourmia-like virus 2 (66% query coverage) and 41.04% identical to that of Heterobasidion ourmia-like virus 1 (57% query coverage). A multiple sequence alignment and MOTIF search of the RdRp sequences of PcOV1 and representative viruses of the family Botourmiaviridae (Supplementary Table S2) revealed six highly conserved motifs ( Supplementary Fig. S1). A maximum-likelihood phylogenetic tree constructed based on the RdRP amino acid sequences of PcOV1 and representative members of five genera of the family Botourmiaviridae, as well as some viruses of the family Narnaviridae [35,36] (Fig. 2), showed that PcOV1 clustered together with members of the genus Ourmiavirus and was separate from the members of other genera of the family Botourmiaviridae. These findings suggest that the + ssRNA mycovirus (PcOV1) isolated from P. citrinopileatus is a new member of the genus Ourmiavirus in the family Botourmiaviridae.