Material and Methods
Experimental design and amendments to the protocols over time
After learning that the soil underneath a carcass can be positive for ASFV genome, we started a pilot experiment based on existing protocols for the analysis of soil samples. During these experiments, it was essential to continuously improve our methods to increase the robustness and sensitivity of the detection of infectious ASFV. In detail, we accomplished this by increasing the virus inoculation volumes and then by increasing the sensitivity and robustness of virus isolation detection methods. Besides, for arid soil types it was necessary to adjust the volume of medium used to recover the virus from the soil.
During the study, it turned out that primary swine macrophages were too sensitive to toxic effects from the soil matrices. Therefore, we switched to a permanent wild boar lung (WSL) cell line. In contrast to the non-dividing macrophages, these cells can divide quickly and seem to be more resistant to matrix effects. Using this optimized cell culture technique, we had the benefit of using ASFV “Kenya1033” genotype IX in which the gene encoding the viral CD2 homologue was replaced with that of a red fluorescent reporter protein (Hubner et al., 2018). With the WSL cells and the marker virus, the detection of viral replication was accelerated to 5-7 days after titration.