Study area and sample collection
The study was conducted in north-eastern Poland where ASF was introduced in February 2014, <1 km from the border with Belarus, and has subsequently spread through the region in wild boar (Podgórski et al., 2020) and domestic pigs (Taylor et al., 2021). By mid 2016, ASF cases in wild boar were distributed continuously in the infected region covering about 4500 km2 and this is where samples used in this study originate (Fig. 1). The area is mostly field-woodland mosaic characterized by extensive agriculture and low human population density (60 people/km2) and relatively high forest cover (31%). The landscape is flat (highest elevation 298 ms a.s.l.) with no significant natural or anthropogenic barriers to wild boar movement. Wild boar are distributed continually throughout the area with densities at the start of the ASF epidemic ranging between 0.5 and 5 ind./km2 at the forest district level (Regional Directorate of State Forests, Białystok, Poland).
Following the introduction of ASF, an intensive surveillance program was implemented in the affected area. The program conducted laboratory tests of all hunted wild boar (active surveillance) and all wild boar found dead (passive surveillance). We used surveillance data routinely collected by the National Reference Laboratory for ASF at the National Veterinary Research Institute in Puławy, Poland. Wild boar samples were classified as ASF-positive (herefater ”case”) if presence of viral DNA was confirmed in real-time PCR or antibodies were detected using ELISA test and confirmed with a immunoperoxidase test (IPT). Detailed description of the surveillance design and laboratory procedures can be found in (Woźniakowski et al., 2016). A total of 5487 wild boars were sampled in the study area from February 2014 to July 2016 and 168 of them tested positive for ASF. It was not technically possible to genotype all of the sampled individuals. Instead, we selected a subsample of 1078 individuals (including all ASF-positive) evenly distributed across the study area for further analyses. However, many samples, particularly originating from the carcasses, were of poor quality and yielded too few microsatellite loci (those yielding less than 13 out of 16 loci were excluded). Finally, we were able to satisfactorily genotype 323 samples (including 80 ASF cases) which comprised a final dataset.