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).