Nucleic acid extraction and real-time PCR
Prior to real-time PCR analysis, viral nucleic acids from all samples (inoculum, blind passage, virus suspension) were extracted using the NucleoMag VET kit (Macherey-Nagel, Düren, Germany) on the automated KingFisher 96 flex platform (Thermo Fisher Scientific, Schwerte, Germany) according to the manufacturer’s recommendations. Subsequently, nucleic acids were analyzed using a published real-time PCR (King et al., 2003) in combination with an internal control based on an EGFP detection system (Hoffmann et al., 2006) on a Biorad CFX real-time cycler (Bio-Rad Laboratories, Hercules, USA). For each real-time PCR, a quantification cycle (Cq) value was determined according to the PCR cycle number at which the fluorescence of the reaction crosses a value that is statistically higher than the background which is determined by the Biorad CFX software. Using a dilution series of an in-house ASFV DNA standard, the genome copies in the respective samples were determined. For generation of the ASFV standard, DNA from an ASFV “Armenia08” PBMC culture supernatant was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Subsequently, the DNA concentration was determined by spectrophotometry using a Nanodrop 2000c (Thermo Fisher Scientific, Schwerte, Germany) and the exact number of DNA molecules was calculated using an online tool (http://www.molbiol.edu.ru/eng/scripts/01 07.html).