A Novel DNA Polymorphism is Associated with Susceptibility to Porcine circovirus 2
Lianna R. Walker, PhD student
University of Nebraska – Lincoln
Porcine Circovirus 2 (PCV2) is a small virus responsible for a set of symptoms collectively known as Porcine Circovirus Associated Diseases (PCVAD). Measures to properly manage and prevent PCVAD outbreaks cost swine industries more than $250 million a year in the US alone. PCV2 is found in swine populations across the globe, but only a small subset of animals in each population will show signs of disease. However, there is currently no way to predict which pigs will develop PCVAD and which pigs will remain unaffected so the entire herd must be vaccinated, posing an increased economic cost to producers. The current vaccines for PCV2 are fairly effective, but do not confer complete protection. Previous data obtained from field research has indicated differencesbetween breeds as well as individual animals in the incidence and severity of disease following PCV2 infection. This suggests that a pig’s own genetics may be partially responsible for susceptibility to PCV2.
In order to identify the genetic factors underlying susceptibility, over 1,000maternal crossbred pigs were experimentally infected with a PCV2b strain. Weekly bloodcollection and body weights were obtained from each animal over the course of the 28-day challenge. The amount of virus in the blood (viremia), antibody levels, and average daily gain (ADG) were measured weekly throughout the challenge. The total amount of virus across the entire challenge (viral load) was also calculated. Each animal was genotyped for ~60,000 DNA markers. Using these genotypes, a genome-wide association study (GWAS) was conducted for weekly viremia and antibody levels, viral load, and ADG. All together, the panel of 60,000 DNA markers accounted for 64% of the observed variation in overall viral load. Additionally, there were two genomic regions located onswine chromosome 7 and chromosome 12 that accounted for a larger amount of variation than all others, representing potential genomic regions that influence PCV2 viral load. The region located on chromosome 7 was located near a group of genes with known function in the host immune response called Swine Leukocyte Antigen Complex, whilethe other region located at the beginning of chromosome 12 has not been previously explored in swine.
Since the genomic region detected on chromosome 12 was not well characterized,gene prediction methods were combined with extensive DNA sequencing to identifypotential genes located in this region. Using these techniques, over 60 DNA variants (polymorphisms) across 5 genes located within this region were discovered. The obtaineddata pointed towards a DNA variant (SYNGR2 p.Arg63Cys) within a gene with unknown functions, called Synaptogyrin-2 (SYNGR2). SYNGR2 is predicted to regulate what molecules or substances enter into cells and is found in almost every type of cell in the body. This DNA polymorphism (SYNGR2 p.Arg63Cys) explained ~20% of the differencein PCV2 viral load between pigs and causes an alteration in the SYNGR2 protein that could impact the ability of this protein to function properly. The favorable genotype (Cys63/Cys63) was associated with lower viremia (Figure 1) and higher ADG (Figure 2) following PCV2 infection.
In order to validate the potential role of this gene in controlling PCV2 multiplication, a Porcine Kidney 15 cell line (PK15) was used as a cell culture validation model. First, the amount of SYNGR2 gene expression was experimentally reduced in the cells by more than 80% and then these cells were subsequently infected with PCV2. Areduction in SYNGR2 gene expression resulted in a significant decrease in the amount of virus produced following infection, indicating a direct role of the SYNGR2 gene in influencing PCV2 multiplication. Additionally, gene editing was performed to generate a PK15 mutant cell line (E1) with a non-functional SYNGR2 gene. These cells were theninfected with the same PCV2 isolate. Once again, a significant decrease in the amount of virus generated was observed in the E1 edited cells compared to normal PK15 cells, with little to no evidence of viral multiplication following infection.
All together, the findings of this research provide direct evidence for the role ofSYNGR2 in facilitating PCV2 multiplication and point towards a DNA polymorphism(SYNGR2 p.Arg63Cys) that may be responsible for the variation in PCV2 diseasesusceptibility. This knowledge enhances our understanding of how PCV2 may act to cause disease, but more importantly will aide in the development of genetic tests capable of predicting susceptibility prior to PCV2 exposure. The ability to distinguish susceptibleanimals at an early age and select for those with increased resistance has the potential to elevate animal welfare and reduce the high production costs associated with current PCV2 management regimes.
This study was led by Daniel Ciobanu from the Department of Animal Science of the University of Nebraska-Lincoln and involved Nebraska graduate students Lianna Walker, Taylor Engle and Emily Tosky, and also Thomas Burkey, and Hiep Vu from the Department of Animal Science, Stephen Kachman from the Department of Statistics, Dan Nonneman and Timothy Smith from the U.S. Meat Animal Research Center, Tudor Borza from Dalhousie University and Graham Plastow from the University of Alberta.The results of the study were recently published in PLOS Genetics.
Figure 1: Least square means of SYNGR2 p.Arg63Cys genotypes across measures of weekly viremia, measured as number of PCV2 genome copy number/ml of serum (log10), following PCV2 infection (n=268)
Figure 2: Least square means of SYNGR2 p.Arg63Cys genotypes across measures of weekly ADG (kg) following PCV2 infection (n=268)