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

Quantitative qPCR was performed on a CFX Connect Real-Time PCR machine (BioRad, Hercules, CA, USA) using SSo Fast EvaGreen Supermix (Bio-Rad). 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 (Supplementary Materials Table S1 ). Cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. A melting curve (MC) analysis was included in each qPCR run confirm the identity of the PCR product. All assays were run in duplicate for each sample. The amount of virus RNA in each reaction was estimated by the CFX software through a calibration curve derived from a 10-fold dilution series of a cloned Abisko virus fragment of known concentration [11, 12] . These amounts were multiplied by the various dilution factors from processing the samples to estimate the number of virus genome copies per insect.