Sampling of Cordyceps chanhua
The strain RCEF6000 isolated from cicada in Anhui Province of China was identified as Cordyceps chanhua based on its morphological features and molecular data (sequence of the ITS region and the translation elongation factor 1-α gene). The strain was incubated on SDAY medium (1% w/v peptone, 4% w/v dextrose, 0.2% w/v yeast, 1.5% w/v agar) at 25 °C for 5 days (Shi et al. 2019).
dsRNA enrichment, metagenomic sequencing, RT-PCR, and RACE cloning
The dsRNA from harvested mycelia was extracted, purified and electrophoresed using a method described previously. C. chanhua strain RCEF6000 was found to harbor five distinct dsRNA bands of approximately 3.5, 2.6, 2.4, 1.8, and 1.6 kb in length, which we named dsRNA 1-5, respectively (Fig. 1A). All dsRNAs were sequenced on an Illumina HiSeq 2500 platform at BGI (Shenzhen, China), producing approximately 9,500 contigs, which (>200 nt) were used to search for similar sequences in the GenBank database using BLASTx. The blast and RT-PCR results confirmed that the strain was infected by two different mycoviruses including a partitivirus with (dsRNA 4, dsRNA 5) and a alternavirus (dsRNA 1, dsRNA 2 and dsRNA 3). Further analyses indicated that contig 8618 (3,465 bp) corresponding to dsRNA 1 had the highest sequence identity (66.99%) to Aspergillus foetidus dsRNA mycovirus, while contig 3757 (2,607 bp) corresponding to dsRNA 2 and contig 3831 (2,359 bp) corresponding to dsRNA 3 had 48.61% and 56.91% amino acid sequence identity to Aspergillus foetidus dsRNA mycovirus, respectively. Thus, dsRNA 1, dsRNA 2 and dsRNA 3 were segments of a potential novel mycovirus, designated as Cordyceps chanhua alternavirus 1 (CcAV1). The terminal sequences of CcAV1 were determined previous protocols described by Coutts and Livieratos (Coutts and Livieratos 2003), and amplified PCR products were then cloned into pMD18-T (TAKARA) and sequenced separately three times.
Bioinformatic and phylogenetic analyses of the sequence data
The complete genome sequence of CcAV1 was deposited in the GenBank database (accession numbers OK481552, OK481553 and OK481554). The putative ORFs of three dsRNAs were predicted using ORFfinder (https:// www.ncbi. nlm. nih. gov/ orffinder/) (Fig. 1B), and the amino acid sequence of the putative RdRp of CcAV1 was aligned with those of other dsRNA viruses using the Multiple Alignment using Fast Fourier Transform (MAFFT) program (Katoh et al. 2018). A phylogenetic tree was constructed by the maximum-likelihood (ML) method with the LG+G+I+F model and 1000 bootstrap replicates, using MEGA X (Kumar et al. 2019). The resulting phylogenetic tree was exported to Figtree 1.4.4 (http://tree.bio.ed.ac.uk/softw are/figtree/).