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.