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.