Discovery of a host genetic variant for PCV2 resistance with origins in European domestic swine
Allele more predominant in European than Asian breeds, with the highest frequency in Duroc.
By Lianna R. Walker
Contrary to its small size and elusive nature, Porcine Circovirus type 2 (PCV2) has had a lasting and continuous impact on the swine industry. Retrospective analysis of archival samples indicated PCV2 emerged in the 1960’s, however, it was not identified as the causative agent of a set of detrimental symptoms and syndromes known as Porcine Circovirus Associated Diseases (PCVAD) until decades later.
Even though only a fraction of infected pigs will display clinical disease symptoms, a global genotype shift from PCV2a to PCV2b in the early 2000’s marked a substantial increase in both the incidence and severity of PCVAD leading to the implementation of herd-wide vaccination.
Nonetheless, continued viral surveillance suggested another genotype shift underway in many parts of the world, with strains belonging to the PCV2d subtype gaining prevalence over the previously predominant PCV2b strains. Although the ultimate impact of this shift on disease progression is not yet clear, it illustrates the constant threat imposed by viral pathogens to industry sustainability.
Advancements in genomic technology and selection programs has fueled a desire to utilize host genetic variation as means of improving disease resistance and tolerance. However, given the ability of viruses to counter evolve, it is not only important to identify causal host variants, but to also understand their function and context within the host-virus evolutionary arms race.
Previously, a large-scale genome-wide association analysis uncovered a DNA polymorphism within the host Synaptogyrin-2 (SYNGR2) gene statistically associated with the total amount of circulating virus (viral load) and immune response following in vivo PCV2b experimental challenge. This polymorphism (SYNGR2 p.Arg63Cys) confers an amino acid substitution from Arginine (Arg) to Cysteine (Cys) within the first intraluminal loop and important functional domain of the SYNGR2 protein involved in vesicle biogenesis.
A clear role of SYNGR2 in PCV2b replication was demonstrated by in vitro infection of an edited porcine kidney 15 (PK15) cell line predicted to have a non-functional SYNGR2 protein. However, direct experimental evidence for the function of the SYNGR2 p.Arg63Cys polymorphism or consistency of its effect across PCV2 subtypes had yet to be provided.
CRISPR-Cas9 gene editing was utilized to induce a single nucleotide substitution within the wildtype PK15 cell line (wtSYNGR2+p.63Arg) and generate clones homozygous for the favorable SYNGR2 p.63Cys allele associated with lower viral load. In vitro infection with genetically distinct isolates belonging to the PCV2b and PCV2d subtypes, revealed a consistent reduction in viral replication in the edited clones (emSYNGR2+p.63Cys and 2emSYNGR2+p.63Cys) compared to the unedited wildtype cell line (wtSYNGR2+p.63Arg).
These findings not only validate the causality of the SYNGR2 p.Arg63Cys polymorphism in host susceptibility to two distinct PCV2 strains, but also suggest that PCV2 evolution has yet to overcome the protection provided by the SYNGR2 p.63Cys allele.
However, these findings provide insight from only the viral perspective of the evolutionary arms race. To investigate the evolutionary origin and history of this allele within the swine lineage, a comparative analysis of SYNGR2 sequences from over 750 wild and domestic pigs was conducted. The SYNGR2 p.Arg63Cys variant was the only polymorphism in this dataset predicted to alter the encoded protein sequence.
The favorable SYNGR2 p.63Cys allele was found exclusively within domestic pigs and more predominant in European (43.1%) than Asian (8.1%) breeds, with the highest frequency in Duroc (68%). Haplotype analysis of SYNGR2 identified a single haplotype (Hap1) with the SYNGR2 p.63Cys allele that was identical to another haplotype (Hap2) prevalent in both domestic and wild boar except for the SYNGR2 p.63Arg allele.
Interestingly, Hap2 was found to be nearly fixed within European wild boar (98%) and completely absent from Asian wild boar, indicating a likely emergence of the SYNGR2 p.63Cys allele post-domestication in ancestral European swine. Further support for this hypothesis is provided by the Iberian breed.
The high frequency of Hap2 (88%) within this breed coincides with the noted continuous introgression with European wild boar. However, Iberian is also one of the few European breeds with no known written or molecular evidence of Asian introgression. Therefore, the presence of Hap1 within this breed heavily suggests a likely European origin of the favorable SYNGR2 p.63Cys allele.
Despite the seemingly recent emergence of this allele post-domestication, the high frequency of the SYNGR2 p.63Cys allele within Duroc and notable frequencies across European breeds suggests potential positive selection within domestic swine.
The Duroc breed is known for lean growth and has historically exhibited strong selective pressure for this trait throughout the 20th century. Even mild PCV2 infections can have substantial effects on growth, therefore, it is possible that the SYNGR2 p.63Cys allele may have been unknowingly selected for in this breed due to the relationship between PCV2 viral load and overall fitness.
Positive selection within a population leads to a rapid increase in frequency of the favored allele, but also of other alleles present on the same haplotype resulting in an extended region of homozygosity called a selective sweep. Recombination events will eventually increase the genetic diversity surrounding the allele subjected to selection, however, this process takes time.
Therefore, the genetic diversity present across the SYNGR2 gene was assessed between domestic pigs with alternate homozygous genotypes for SYNGR2 p.Arg63Cys. A dramatic reduction in heterozygosity was observed amongst Cys/Cys compared to Arg/Arg individuals, supporting the hypothesis of recent positive selection favoring the SYNGR2 p.63Cys allele in domestic pigs.
All together, these findings validate the causality of a natural variant previously associated with PCV2 susceptibility to which the virus has yet to overcome. Furthermore, evolutionary analysis within the swine lineage suggests the favorable allele likely arose post-domestication and was unknowingly selected for during the formation of modern European breeds.
While the exact function of the SYNGR2 gene and the SYNGR2 p.Arg63Cys polymorphism in PCV2 infection is not yet clear, this information has the potential to improve disease resilience across swine as a predictor of PCVAD or through targeted selection. However, the current viral landscape is not static, but ever changing.
Therefore, continuous interrogation of viral evolution in response to increased host resilience, not just the identification of adaptive host variants, will be imperative in maintaining animal welfare long-term.
This study was led by scientists from the Department of Animal Science (Lianna R. Walker, Hiep L. Vu, Daniel C. Ciobanu) and School of Biological Sciences (Kristi L. Montooth) of the University of Nebraska-Lincoln. The project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2019-05380 from the USDA National Institute of Food and Agriculture. The results of the study were recently published in PLOS Genetics.
Walker is a post-doctoral research associate in molecular genetics in the University of Nebraska-Lincoln Animal Science Department.
