Pseudorabies virus (PRV) – the causative agent of Aujeszky’s disease – was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk–based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome–wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA–SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA–SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production.

Sample Collection
Between 2012 and 2020, blood, hair, and tissue samples were collected from adult feral swine (n = 2,490) throughout the invaded range within the contiguous United States as an extension of damage management efforts led by the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) Wildlife Services (WS).

Serological Assay
Serum samples were tested for PRV antibodies at the Kentucky Federal Brucellosis Laboratory (Frankfort, Kentucky, USA) using the "short protocol" for Pseudorabies–gB Enzyme–Linked Immunosorbent Assay (PRV–gB ELISA; IDEXX Laboratories Inc., Westbrook, Maine, USA). Samples were considered positive for PRV if the S/N value ≤ 0.60 and negative if the S/N value > 0.70.

Genotype Data
DNA extraction and genotyping were conducted at GeneSeek (Neogen Corporation, Lincoln, Nebraska, USA). DNA extraction was performed using the MagMAX™ DNA Multi-Sample Ultra Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and genotyping was completed using the GeneSeek Genomic Profiler (GGP) for Porcine 80k array (68,516 loci; Illumina BeadChip microarrays [San Diego, California, USA] licensed exclusive to GeneSeek, Neogen Corporation, [Lincoln, Nebraska, USA]). Bi–allelic SNP were mapped to the Sscrofa11.1 reference genome assembly (Warr et al., 2020), and unmapped and non–autosomal markers were removed, leaving 62,128 loci for further consideration. Standard quality control filters for genotype data were implemented using SNP & Variation Suite (SVS; version 8.9.0; Golden Helix, Bozeman, Montana, USA), specifically removing samples with call rates < 0.90 and then pruning loci with call rates < 0.90 or minor allele frequency < 0.05. Following quality control measures, 56,024 SNP were retained.

True Seroprevalence for PRV
True seroprevalence for PRV, accounting for imperfect detection was predicted for each year in each watershed (hydrological unit code [HUC] eight). Predictions were made using a hierarchical Bayesian model with 28,030 feral swine sampled across 37 states in the contiguous United States between 2010 and 2020. Similar to the paired genetic and serological samples that serve as the focus of this study, these samples were more broadly collected (temporally and spatially) by USDA APHIS Wildlife Services as a component of disease surveillance and population control efforts.

Percent European Wild Boar Ancestry
The methods described in Smyser et al. (2020) were used to estimate the percent European wild boar ancestry of individual feral swine included in our analysis. Briefly, ADMIXTURE (Alexander et al. 2009) was used in a supervised framework to query an individual genotype against a comprehensive reference set for Sus scrofa, comprised of 105 domestic breeds, 23 wild boar populations, and 4 sister taxa. This method proportionately associates the origin of individual feral swine genotypes among the 17 ancestry groups that comprise the Sus scrofa wild–domestic species complex reference set.

References

Alexander, D.H., Novembre, J., and Lange, K. (2009). Fast model-based estimation of ancestry in unrelated individuals. Genome Res 19(9), 1655-1664. doi: 10.1101/gr.094052.109.

Smyser, T.J., Tabak, M.A., Slootmaker, C., Robeson, M.S., Miller, R.S., Bosse, M., et al. (2020). Mixed ancestry from wild and domestic lineages contributes to the rapid expansion of invasive feral swine. Molecular ecology 29(6), 1103-1119. doi: https://doi.org/10.1111/mec.15392.

Warr, A., Affara, N., Aken, B., Beiki, H., Bickhart, D.M., Billis, K., et al. (2020). An improved pig reference genome sequence to enable pig genetics and genomics research. GigaScience 9(6), giaa051. doi: https://doi.org/10.1093/gigascience/giaa051.