J Dekkers, B Rowland, J Lunney, G Plastow, J Harding, S Bishop, B Kemp
Porcine Reproductive and Respiratory Syndrome (PRRS) is the most costly viral disease in pigs around the world. Estimated annual costs in the US alone are $664 million (Holtkamp et al. 2013). Although much emphasis has been placed on development of preventative measures to control the spread and impact of PRRS, including vaccination and regional eradication efforts, PRRS continues to be a major problem in the industry (Darwich et al. 2010, Huang and Meng, 2010, Chand et al. 2012).
Over the past decades, genetic selection has been very effective at developing and improving lines of pigs that produce high-quality lean pork in an efficient manner. This has been accomplished by systematic selection on estimates of the ‘breeding value’ of potential parent stock based on extensive phenotype recording for growth rate, backfat, meat quality, and reproduction. In principle, these same selection methods can be applied to identify and select pigs that are more resistant to diseases, provided resistance to disease of a pig is at least partially determined by the genetics of the pig (i.e. is heritable) and phenotypes related to disease resistance or susceptibility can be collected on breeding stock.
Although early research showed breed differences in the effects of PRRS virus (PRRSV) infection (Halbur et al. 1998; Petry et al. 2005, 2007; Vincent et al. 2006, Doeschl-Wilson et al. 2009), collection of phenotypic data on the effects of PRRSV infection in breeding stock is problematic because of the need to maintain high health status in breeding herds. However, if genes or genetic markers linked to genes that are associated with resistance or susceptibility of pigs to the PRRSV infection can be identified, this can provide opportunities to select pigs based on marker-assisted selection or genomic selection. Opportunities to investigate the role of host genetics on disease resistance have expanded greatly in the past decade through the rapid developments that have taken place in genomics and in genomics technology. These developments now allow animals to be genotyped for thousands of genetic markers (single nucleotide polymorphisms or SNPs) across the genome, allowing SNPs that are associated with traits of interest to be identified by genome-wide association studies (Goddard and Hayes, 2009).
The purpose of this presentation is to describe ongoing efforts to investigate opportunities to use the genetics of the pig as another tool in the fight against PRRS. Using novel deep genotype and deep phenotype approaches that combine state of the art genomics with state of the art virology, the ultimate goal is to find genomic markers and other biomarkers that can be employed in the development of breeding programs to lessen the impact of PRRSV on the commercial pig industry. Although genetic selection will not offer a single ‘magic bullet’ solution, especially given the complexity and variability of the PRRS virus, host genetics can be an additional and complementary approach to fight the impact of PRRSV on pork production.
Nursery pig model for host response to PRRSV
Initial studies under the PRRS Host Genomics Consortium
To capitalize on advances in genomics technology to study the role of host genetics in PRRSV infection and develop tools to select pigs for improved resistance or reduced susceptibility, the PRRS Host Genetics Consortium (PHGC) was initiated in 2008 (Lunney et al. 2011; Rowland et al. 2012). The PHGC involves experimental challenge of groups of 200 commercial nursery pigs at experimental facilities at Kansas State University with a specific strain of the PRRSV. Piglets are followed for 42 days following infection, with frequent blood sampling and weighing, along with collection of tissue for DNA for genotyping. All pigs are genotyped with the Porcine SNP60 BeadChip (Illumina), which includes >60,000 single nucleotide polymorphisms (SNPs) across the genome. Serum samples are evaluated to quantify PRRSV viremia using PCR.
Analysis of the first 8 PHGC trials, including over 1,500 pigs infected with PRRSV strain NVSL-97-7895, found that viral load, quantified as area under the curve of logarithm (base 10) viremia from 0 to 21 days post infection (dpi), had substantial heritability (h2=0.44) (Boddicker et al. 2012, 2013, 2014). Viremia after 21 days was characterized by ~1/3 of pigs showing viremia rebound, which likely reflects immune escape of the PRRSV. Genetics of the host was found to have little impact on whether or not a particular pig showed rebound, as the heritability of rebound was close to zero. Growth following infection was also shown to be moderately heritable (h2=0.29) (Boddicker et al. 2012, 2013, 2014). Growth rate and viral load were negatively correlated but correlations were not strong: the phenotypic correlation was - 0.29 and the estimate of the genetic correlation between growth rate and viral load was -0.46 (Boddicker et al. 2014). These results demonstrated that host response to experimental infection with the PRRSV has a sizeable genetic component.
To identify genetic markers and regions of the genome that are associated with viral load or growth rate following PRRSV infection, genotypes obtained by genotyping all pigs with the 60k SNP panel were evaluated in genome-wide association studies. Results identified a region on swine chromosome 4 (SSC4) of about 1⁄2 Mb that was associated with both viral load and growth rate following infection. This SSC4 region explained approximately 15% of the genetic variance for viral load and 11% of the genetic variance for growth rate, indicating the presence of a major gene associated with host response to PRRSV infection. The 1⁄2 Mb region was found to be in high linkage disequilibrium across all breeds and lines investigated, which means that it is a region with very little recombination. One SNP in the region, WUR10000125, was found to capture the full effects of the region on viral load and growth rate and this SNP (abbreviated WUR) was used in subsequent analyses. The WUR SNP was found to have significant effects in all trials, with individuals with genotype AB having 5% lower viral load and 14% higher weight gain than pigs with the AA genotype at this SNP. Although the frequency of BB pigs was low, the BB genotype had similar effects as the AB genotype, suggesting the B allele to be dominant over the A allele. The B allele was found to be present in all breeds and lines that contributed to the PHGC trials but at a low frequency (2 to 40%) (Boddicker et al. 2014).
Several other regions of the genome also showed associations with viral load and growth rate following infection (Boddicker et al. 2014). However, the effects of these regions were smaller than those identified for the SSC4 region. One region on SSC1 was found to be associated with mortality in one trial that showed signs of co-infection with other pathogens (Boddicker et al. 2014).
Experimental infection studies with a second PRRSV strain
A second generation of PHGC trials was conducted that involved experimental infection of commercial nursery pigs with another strain of the PRRSV. For these trials, pigs were infected with the more recent, KS-2006-72109 PRRSV strain, which is 89% identical to the NVSL 97 strain at the viral GP5 peptide sequence level. A total of five trials were completed with this virus. Results were very similar to those observed in the initial trials with the NVSL 97 strain, with similar estimates of heritabilities and correlations. The effect of the SSC4 region was also significant for this strain, indicating that the effects of this region may be present across multiple strains of the PRRSV. Estimates of the effect were, however, smaller than observed for the NVSL 97 strain, about 80% of the effect for viral load and about 36% of the effect on weight gain, which may reflect the differences in pathogenicity between these two virus strains (Hess et al. 2014).
Further analysis of the SSC4 region
The 1⁄2 Mb SSC4 region includes multiple candidate genes that have been shown to be associated with innate immune response, including members of the GBP family, CCBL2, GTF2B, and PKN2 (Boddicker et al. 2012). Because of the high linkage disequilibrium in the SSC4 region, fine mapping the causative mutation for the SSC4 effect by genetic mapping approaches is problematic. Instead, a functional genomics approach was used, involving the evaluation and analysis of the expression of genes in the region. For this purpose, blood samples at 5 time points from 8 pairs of littermates from one of the PHGC trials, with one piglet being AA and one piglet being AB for the WUR SNP, were subjected to RNA-seq analyses. Results identified one candidate gene in the SSC4 region that showed significantly higher expression in AB versus AA piglets across multiple time points during the trial (Eisley et al., 2013). In addition, allele-specific expression was identified for this gene in AB piglets, with the B allele being expressed more frequently than the A allele across multiple time points. Subsequent analyses of the sequence data identified a splice site variant in one of the introns of this gene, which results in premature stop codon in transcripts produced by the A allele, which are expected to result in production of an incomplete protein (Fritz-Waters et al., 2014). Further work to confirm this as the causative mutation for the SSC4 effect are underway.
Both total (viral N protein-specific IgG based on ELISA, expressed as sample-to-positive (SP) ratio) and neutralizing antibody (Ab) response to the PRRSV were evaluated on blood samples collected at the end of each trial (42 dpi) for all pigs in multiple trials (Trible et al., 2013). Total Ab was shown to be heritable (h2=13%) but neutralizing Ab response was not. Genome-wide association studies identified the major histocompatibility complex (MHC) on SSC7 as having a major effect on total Ab, explaining 30% of the genetic variance (Hess et al., 2013). The WUR SNP of SSC4 was not found to be associated with antibody response, suggesting that its mode of action may not be through adaptive antibody- mediated immune responses.
Additional gene expression and immune response analyses
To further investigate the dynamics of host response to PRRRV infection and immunological and genetic pathways that are involved in this response, additional gene and protein expression analyses are underway. Initial work using microarrays showed a number of genes that were differentially expressed (Arceo et al., 2013). More detailed microarray and RNAseq analyses are continuing and focus on gene expression changes in blood over time and between pigs that show different responses in terms of viremia and weight gain (Choi et al., 2014). Serum cytokine and chemokines evaluations have highlighted the importance of early interferon-α levels. All PRRSV infected pigs have high 4 dpi serum interferon-α levels; however, pigs with higher viral loads continue to express interferon-α, whereas those with lower viral loads quickly return to pre-infection levels (Choi et al., 2013).
In practice, PRRS is often found to increase incidence of secondary infections, resulting in e.g. porcine respiratory disease complex (PRDC) and porcine circovirus associated disease (PCVAD). To investigate the genetic basis of host response to co-infection, several experimental trials in which nursery pigs are infected with both PRRSV and PCV are currently underway. To evaluate host response to PRRS vaccination, half of the pigs in these trials are first vaccinated with a modified live PRRSV.
Reproductive PRRS infection model
It is estimated that 45% of the total costs associated with PRRSV are attributed to reproductive disease (Holtkamp, 2013), yet proportionately little research has focused on reproductive PRRS compared to disease affecting pigs post- weaning. To investigate potential phenotypic and genotypic predictors of reproductive PRRS severity, a large scale experimental infection model was developed and conducted at the University of Saskatchewan (Harding et al., 2012). In this study, 114 pregnant gilts were experimentally infected with a type II PRRSV strain at 85 days of gestation, along with 19 sham-inoculated controls. Gilts were bled on day 0, 2, 6, and 19 post inoculation to evaluate viral load, changes in leukocyte subset counts, and cytokine responses. Gilts were euthanized at 21 days post inoculation (gestation day 106) to evaluate litter outcome and collect tissues from gilts and fetuses. Forty percent of fetuses were autolyzed or decomposed at termination. Genotyping using the porcine SNP60 BeadChip has been completed for all gilts, sires and over 850 viable and dead fetuses. Analysis of phenotypic and genotypic data is ongoing to elucidate mechanisms of fetal death and the phenotypic and genotypic traits associated with PRRS severity (Harding et al., 2013).
While experimental infection trials provide great opportunities to investigate the genetic basis of host response under controlled conditions, these finding must be validated in the field. In addition, field studies allow additional effects to be observed and investigated. To this end, multiple field trials have been initiated.
Reproduction PRRS Outbreak Herd
Serum samples were collected on 641 pregnant sows in a sow herd that broke with PRRS. All sows were genotyped using the Porcine SNP60 BeadChip (Serao et al. 2013). The date of the outbreak was identified based on rolling averages of farrowing traits. The date of blood sampling was approximately 7 weeks after the outbreak. Blood samples were evaluated for PRRS viral N protein Ab by ELISA, expressed as SP ratio. Antibody response was found to be highly heritable (h2=45%) and had strong genetic correlations with multiple reproductive traits after the outbreak, ranging from -0.72 for number of mummified piglets to +0.73 for number born alive. This suggests that antibody response following PRRSV infection (or vaccination) could be used as an indicator trait to select for reduced susceptibility to reproductive PRRS. A genome-wide association study identified two regions on SSC7 that explained large proportions of the genetic variance in SP ratio, including one region in the MHC, which explained 25% of the genetic variance. This region was similar to the region identified in the experimental challenge studies of nursery pigs.
Gilt acclimation study
To study the genetic basis of host response to PRRS and other diseases on the sow herd in a field setting, groups of 10 to 47 naïve commercial F1 replacement gilts were introduced in “health-challenged” herds. Gilts were directly introduced into the cooperating herds, using standard passive acclimation protocols, i.e. no direct challenge for any disease. Data on a total of 923 F1 gilts from 13 genetic sources that were introduced into 18 herds were used in a preliminary analysis. A total of 15 farms vaccinated gilts against PRRSV upon arrival, using a modified live virus vaccine. Individual weights and blood samples were collected on the day of introduction and after the acclimation period (~40 days). Blood samples were also collected at first parity weaning. Blood samples were analyzed for PRRS by ELISA and expressed as SP ratio. Most gilts were negative on day 0. All gilts were genotyped using the Porcine SNP60 BeadChip.
All traits had low heritability, except PRRS antibody SP ratio after acclimation, which was heritable (h2 = 0.44). Genome-wide association studies identified multiple regions on SSC7 associated with antibody SP ratio, including the MHC region. Effects of the SSC4 WUR SNP on antibody SP ratio and growth were not significant. Collection of additional health and reproduction data through third parity is currently underway.
Field studies in grow-finish pigs
Multiple field studies are currently underway to evaluate host response to disease in growing pigs in the field. Using the gilt acclimation concept, these studies involve introducing 200 clean nursery pigs into ‘health-challenged’ finishing barns. Pigs are repeatedly weighed and bled and followed all the way to market.
Host response of nursery pigs to PRRSV infection was found to have a sizeable genetic component under controlled experimental challenge studies. In particular, a major gene for host response to PRRS was identified on SSC4, with a strong candidate gene that is involved in innate immune response. The WUR SNP in this region could be used to select for pigs that are less affected by PRRSV infection in terms of growth rate. However, these effects need to be validated under more complex disease scenarios and in the field. Several such studies are currently under way. Studies on the genetic basis of host response to reproductive PRRS are also underway. While this comprehensive suite of studies is unlikely to identify pigs that are completely resistant to PRRSV, they are starting to unravel the genetic basis of host response to PRRS, leading to the ability to select pigs that are less susceptible to PRRSV infection and to the effects of PRRSV infection on performance. In addition, insight into host response to PRRSV infection can lead to new avenues for development of more effective vaccines and therapeutics.
The projects described in this paper are supported by National Pork Board grants, USDA NIFA (awards 2008-55620- 19132, 2010-65205-20433 and 2013-68004-20362), the Canadian Swine Health Board, Genome Canada, Genome Alberta and the Alberta Livestock and Meat Agency, Genome Prairie and the Saskatchewan Agriculture and Development Fund, PigGen Canada, Canadian Swine Breeders Association, Canadian Centre for swine Improvement, the NRSP-8 Swine Genome and Bioinformatics Coordination projects, and the PRRS Host Genetics Consortium consisting of USDA ARS, Kansas State University, Iowa State University, Michigan State University, Washington State University, Purdue University, University of Nebraska-Lincoln, University of Saskatchewan, University of Alberta, PIC/Genus, Newsham Choice Genetics, Fast Genetics, Genetiporc, Inc., Genesus, Inc., TOPIGS, PigGen Canada, Inc., IDEXX Laboratories, and Tetracore, Inc. SB also acknowledges the funding contribution of the BBSRC.