Near-complete sequence of a novel reovirus genome identified from Callinectes sapidus

The Atlantic blue crab, Callinectes sapidus, is an estuarine keystone species that functions as both predator and prey in food webs and supports a multi-million dollar fishery along the western Atlantic coast from the US mid-Atlantic to southern Brazil. Throughout their range, blue crabs are host to viral, bacterial, fungal, protozoan, and metazoan pathogens. Reoviruses are non-enveloped 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 brachyuran crustaceans. This work describes the sequence of a novel reovirus genome, discovered in Callinectes sapidus. The double-stranded RNA genome of CsRV2 consists of 12 segments that encode 13 putative proteins. High nucleotide sequence identity with Eriocheir sinensis Reovirus 905 revealed this virus belongs to Cardoreovirus within the Reoviridae family.

Methods
Viral RNA was detected from total RNA isolated from the muscle of infected C. sapidus walking leg. DsRNA purified by CF11 cellulose chromatography, was used for cDNA synthesis with barcoded octamers (5’-GGCGGAGCTCTGCAGATATC-NNNNNNN-3’) and the resulting cDNA was amplified by PCR using the barcode primers (5’-GGCGGAGCTCTGCAGATATC-3’). PCR products of 250-500 bp were obtained by gel purification and DNA library preparation was performed using NEBNext Ultra DNA Library Prep kit following the manufacturer’s recommendations (Illumina, San Diego, CA, USA). The library was sequenced in a 2 × 250 paired-end configuration on the Illumina MiSeq platforms. After quality trimming and removal of barcode sequences, the reads were assembled into contigs using CLC Genomics Workbench 9.5.2 (Qiagen).  In total, 12 viral genomic contigs were constructed, integrating data from a total of 51,168 reads (67.1% of the total reads), generating a total of 21,109 nucleotides at 560-fold average coverage with a mean G+C content of 41.4%.