Near-Complete Sequence of a Highly Divergent Reovirus Genome Recovered from Callinectes sapidus

This report describes the sequence of a reovirus genome, discovered in Callinectes sapidus in Brazil. The genome sequence of CsRV2 consists of 12 segments that encode 13 putative proteins. The predicted RNA-dependent RNA polymerase is highly similar to that of Eriocheir sinensis reovirus 905, suggesting that CsRV2 also belongs to the genus Cardoreovirus.

T he Atlantic blue crab, Callinectes sapidus, is an estuarine keystone species that functions as both predator and prey in food webs and supports a multimillion dollar fishery along the western Atlantic coast from the U.S. mid-Atlantic to southern Brazil (1,2).
Reoviruses are nonenveloped icosahedral viruses with genomes comprised of 9 to 12 segments of linear double-stranded RNA (dsRNA). They have been found in diverse host species, including crabs (3). In studies on viruses of C. sapidus captured near Tramandaí, Brazil, we discovered a novel reovirus dsRNA that showed an electrophoretic genome organization distinct from that of Callinectes sapidus reovirus 1 (CsRV1) (4-9) but similar to that of Eriocheir sinensis reovirus 905 (EsRV905) (10), with a pattern of 3/4/5 and an estimated size of ;21.4 kbp based on gel migration (Fig. 1). We refer to the putative reovirus represented by this dsRNA as CsRV2.
Total RNA was extracted from the muscle of an infected C. sapidus specimen using a phenol-guanidinium method and visualized on agarose gels (5). dsRNA was purified by CF11 cellulose chromatography as previously described (4) and used for cDNA synthesis with barcoded octamers (59-GGCGGAGCTCTGCAGATATC-NNNNNNNN-3') (11). The resulting cDNA was amplified by PCR using barcode primers (59-GGCG GAGCTCTGCAGATATC-39). PCR products of 250 to 500 bp were selected and obtained by agarose gel purification, and DNA library preparation was performed using the NEBNext Ultra DNA library prep kit following the manufacturer's recommendations (New England BioLabs [NEB], Ipswich, MA). The library was sequenced in a 2 Â 250-bp paired-end configuration on the MiSeq platform with a MiSeq reagent kit v3 (Illumina, San Diego, CA). CLC Genomics Workbench 9.5.2 (Qiagen) was used for quality trimming, removal of barcode sequences, and de novo assembly of the sequencing reads. The 12 viral contigs integrated 51,168 reads (67.1% of the total reads) and defined 21,109 nucleotides (nt) at 560-fold average coverage with a mean G1C content of 41.4%. The sizes of most contigs are consistent with the apparent dsRNA bands on the electrophoresis gel (Table 1 and Fig. 1), with the exception of S2 and S11, which are smaller than the dsRNA bands. The 12 contigs lack conserved 59 or 39 termini.
The 12 assembled segments of CsRV2 were annotated by BLASTX and BLASTN comparisons with GenBank using default parameters. A single open reading frame (ORF) was identified in each segment, with the exception that segment 5 (S5) contains two partially overlapping ORFs. S1 shows significant similarity to the putative EsRV905 RdRp gene, with 72% nucleotide identity and 79% amino acid identity (GenBank accession no. AY542965 and Q698V5, respectively), suggesting that CsRV2 belongs to the same genus as EsRV905, Cardoreovirus. Aside from the RdRp gene, no other sequences of EsRV905 are available in public databases. The GenBank entry with the second highest similarity (29%) to S1 of CsRV2 is the RdRp of the Liao ning virus, in the genus Seadornavirus (YP_460026) (12)(13)(14).
Data availability. The complete genome sequence of CsRV2 has been deposited in GenBank under accession no. MW208677 to MW208688. Raw sequencing data are registered in the NCBI SRA database under accession no. SRR13068891.