Characterization of a Novel RNA Virus Discovered in the Autumnal Moth Epirrita autumnata in Sweden

de Miranda, Joachim R.; Hedman, Harald; Onorati, Piero; Stephan, Jörg; Karlberg, Olof; Bylund, Helena; Terenius, Olle

One microgram each of total RNA of two highly diseased E. autumnata larvae was sequenced by the SNP and SEQ platform of the SciLifeLab facility in Uppsala, Sweden using the default Illumina HiSeq technology (San Diego, CA, USA). The Abisko virus sequence was assembled from around 0.5 million end-paired ca. 100 nt sequences using the Trinity transcriptome assembly software [8], after filtering out low quality reads and removing the adapter sequences. The consensus sequence, coverage data, and variability profile were created using mpileup from Samtools v.0.1.8 (www.htslib.org, [9] ) and an in-house script. The assembled Illumina sequence was confirmed through chain-termination sequencing [10] of uncloned Reverse Transcription-PCR fragments produced by primers EaNV-F8005 and EaNV-B9465 (Supplementary Materials Table S1 ), spanning the major junctions of the genome (Figure 1 ). The 3 terminus was obtained by 3 RACE (Rapid Amplification of cDNA Ends), through priming the natural poly-A tail of the virus with anchored oligo-dT (V[T] 16 ) for cDNA synthesis, followed by PCR with the EaNV-3RACE-F2 virus-specific forward primer (Supplementary Materials Table S1 ), using the FirstChoice RLM-RACE Kit (Applied Biosystems, Foster City, CA, USA). The identity of the reverse transcription quantitative polymerase chain reaction (RT-qPCR) fragments Viruses 2017, 9, 214 3 of 12 from the prevalence survey was also confirmed through sequencing. These PCR products were sequenced by Macrogen (Seoul, South Korea).