Real-time PCR analysis of virus suspensions, contaminated SDPP and
blind passages
The virus suspension used to contaminate SDPP contained high genome copy
numbers (5.66 log10/ml, Figure 1). As a control, the
original virus suspension was stored up to 35 days under the same
conditions as the SDPP samples. Irrespective of the storage temperature
(4°C or room temperature, respectively), ASFV genome copy numbers were
constant over the entire observation period in the virus suspension
(copy numbers between 5.66 and 5.39 log10/ml). However,
copy numbers do not allow any conclusions on the amount of infectious
virus.
In comparison, the contaminated SDPP, used as inoculum for the blind
passage, contained lower genome copy numbers than the initial virus
suspension due to dispersion on SDPP. Such a dispersion effect was also
seen after contamination of field crops (Fischer et al., 2020). Overall
ASFV genome was detectable in all samples, indicating a successful
contamination of SDPP (Figure 1). Furthermore, genome copy numbers of
SDPP did not display any distinct decline over time, irrespective of the
storage temperature. In the un-treated plasma negative control (NC) no
ASFV genome was detected (Table S1).
PCR analyses after blind passages of contaminated SDPP stored at 4°C
revealed that these specimens were highly genome positive during the
entire observation period (copy numbers between 7.32 and 6.52
log10/ml, Figure 2A). These values exceed the viral load
of the initial virus suspension, indicating a considerable amplification
of ASFV on macrophages during the blind passages. In contrast, only a
single blind passage replicate of SDPP stored at room temperature for
one week was PCR positive (4.88 log10 genome copies/ml,
Figure 2A and Table S2).