Sample preparation
Spray dried porcine plasma (SDPP) from a commercial producer (APC, Villarrobledo, Spain) was used in this study. Seventy grams of SDPP (humidity max. 9.0%) were contaminated with 10.5 ml of an ASFV suspension with a titer of 106HAD50/ml (ASFV strain “Armenia08”). The contamination procedure was performed in a zipper bag and divided into two steps (5 ml + 5.5 ml virus suspension), separated by a 15 min drying period, using an intranasal mucosal atomization device (MAD Nasal; Wolfe Tory Medical, Salt Lake City, USA) to nebulize the virus suspension. After additional 15 min drying and shaking at room temperature, contaminated SDPP was dispensed as 2 g aliquots in 50 ml tubes (Sarstedt, Nümbrecht, Germany) and stored at 4°C or at room temperature. SDPP samples were taken as biological triplicates on days 0, 7, 14, 21, 28, 35 and stored at -80°C until further analysis. As negative control (NC), triplicate samples of uncontaminated SDPP were stored immediately at -80°C together with an aliquot of the original virus suspension (VS T0). Furthermore, 900 µl of the virus suspension were also incubated at 4°C or at room temperature for 7 days or 35 days and subsequently stored at -80°C.
Prior to analysis, the -80°C stored SDPP samples were resuspended thoroughly with 10 ml sterile distilled water with an 1% Antibiotic-Antimycotic mix (Gibco Antibiotic-Antimycotic 100x; Thermo Fisher Scientific, Schwerte, Germany), by shaking and vortexing. Two ml of the reconstituted plasma were stored at -80°C for real-time PCR analysis to prove successful contamination. The remaining plasma was used as inoculum for a blind passage on peripheral blood mononuclear cell (PBMC) derived macrophages to determine whether any residual infectious ASFV could be detected.