Experiment 2: Recovery from sterile sand, beach sand, swamp mud, and forest soil on macrophages
The collected soils were tested along with two controls: blood-only and blood mixed with 6 grams of sterile sea sand. All soils and controls were spiked with 1.2 mL of ASFV-positive blood with a titer of 6.00 log10 HAD50/mL and three replicates per condition. The forest soils were extremely dry, so 12 ml of media were added to all samples for virus isolation. We continued experiment 2 and subsequent experiments at room temperature (25°C), since we had not seen big differences between soil stored at 4°C or 25°C in experiment 1. We limited our testing to 14 days in experiment 2 as it seemed unlikely that live virus would be detected beyond one week. In addition to the protocol described in experiment 1, we also used a dedicated kit for DNA extraction from soil (DNeasy PowerSoil; Qiagen, Hilden, Germany) from 0.25 grams of all matrices and time points.