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

The Abiskovirus genomic (positive) and replicative (negative) RNA strands were quantified separately using four pairs of strand-specific assays; two located in open reading frame 1 (ORF-1) and two located in ORF-2/3. Strand-specific cDNA was generated in 10 µL reactions containing 1 µL RNA plus 2 µM tagged cDNA primer (Supplementary Materials Table S1 ) heated to 70 • C for 5 min and cooled on ice; 20u MuMLV reverse transcriptase (Thermo Fisher K1612, Waltham, MA, USA), 10u RiboLock RNAse inhibitor (Thermo Fischer EO03819), 1 mM dNTP plus the MuMLV 5x reaction buffer. For each sample, a no-template, a no-primer, and a no-reverse transcriptase reaction were included in its suite of cDNA reactions, as essential cDNA negative controls [11, 13] . Also included was a full suite of cDNA reactions for a virus-negative adult E. autumnata sample (25A), as a biological negative control. The cDNA reactions were incubated 60 min at 37 • C followed by 10 min at 70 • C. Excess cDNA primer was removed by adding 1u Exonuclease-I (Thermo Fischer 70073Z2500UN), incubating 30 min at 37 • C followed by 15 min at 70 • C, followed by diluting the cDNA 10-fold in sterile water [11, 13] . The cDNAs were quantified by qPCR with the CFX Connect Real-Time PCR machine (BioRad) and the SSo Fast EvaGreen Supermix, using the tag to the cDNA primer and a virus-specific primer (Supplementary Materials Table S1 ) to ensure exclusive amplification of cDNA generated only with the tagged primer. The reaction volume was 20 µL containing 1 µL of the diluted cDNA template and 0.3 µM each of the forward and reverse primers. Cycling conditions were 95 • C for 10 min, followed by 35 cycles at 95 • C for 15 s, 58 • C for 30 s and 72 • C for 5 s, followed by a Melting Curve analysis to verify the identity of the PCR products. The no-primer and no-RT cDNA reactions were amplified with an equimolar mix of tag primer and a pool of all virus-specific primers, so as to enable the amplification of all possible illegitimate products. All reactions were run in triplicate, on separate plates. The Melting Curve data was used to identify positive amplifications, after which the mean and standard deviation of the corresponding quantification cycle (Cq) values were calculated and converted to estimated copies of cDNA per reaction, ±standard deviation, through external calibration curves established from 10-fold dilution series of the purified and quantified PCR products, covering four orders of magnitude dynamic range. The no-template cDNA controls and no-template qPCR controls were negative for all assays.