Genetics of host responses to Porcine Reproductive and Respiratory Syndrome virus (PRRSV)

This project uses novel and innovative methods to develop tools to help control the most important viral disease affecting pig industries in Europe and North America, viz. Porcine Reproductive and Respiratory Syndrome (PRRS). We will focus on infections with the PRRS virus (PRRSV) in the reproductive sow. Pigs differ genetically in their resistance to PRRS. Hence, our ultimate aims are to identify genetic markers that are associated with these between-animal differences and can therefore be used to breed for increased resistance. However, studying the impacts of PRRS in sows is expensive and difficult, so we will use novel methods to overcome this problem.

We have developed in-vitro methods to assess host responses to PRRSV infections: from a blood sample taken, we isolate monocytes from the white blood cells. By stimulation with CSF-1, these are then cultured into macrophages (MDM), the cell type that PRRSV infects. We then infect MDMs with PRRSV and measure their responses to infection, by image analysis and measuring genes that are expressed post infection. To relate these in-vitro phenotypes to infection outcomes in the living animal (in-vivo), we will use a herd in which gilts are vaccinated with a live vaccine, and therefore have a controlled PRRSV exposure. Blood samples will be taken before vaccination, to get MDMs, and at regular intervals post inoculation, and virus levels (viraemia) measured as an indicator of their resistance to infection. We will also perform the in vitro studies on naïve, unvaccinated, pigs, to study PRRVS responses in pigs naïve to the virus.

First, we will explore the genetic control of PRRS resistance, using viraemia as phenotypes. We will genotype each vaccinated pig with high density (750K) SNP arrays, these being DNA chips that detect genetic variation at about 750,000 locations across the genome. Unvaccinated pigs will be genotyped with a mix of 60K and 750K SNP chips. We will then do the in-vitro infection studies on MDMs from all animals. MDMs from each animal will be infected separately with each of three different PRRSV strains, giving detailed information per animal. Using the SNP chip genotypes we will identify genetic markers associated with all of the in-vitro measurements. We will also explore the relationships between the in-vitro and in-vivo measurements, to determine which in-vitro phenotypes are truly predictive of outcomes in live animals. Doing this on ca. 1150 pigs will give us good resolution to map loci affecting resistance and identify associated genetic markers.

Our analyses of the genetic markers and all phenotypes should identify loci with major impacts on PRRS resistance; the markers defining these loci can be used to select animals. However, we wish to know how these loci affect PRRS resistance and the consequences of breeding from pigs with these markers. To achieve this, we will choose the two most significant loci from our results, then identify animals of contrasting genotypes (10 high & 10 low for both). We will then infect MDMs from these animals with PRRSV and assess their responses over a 24 hour period, measuring viral load and expression of immune genes known to be involved in response to PRRSV infection. Further microarray analyses will assess global gene expression and comprehensively describe responses to infection. These results will provide insight into mechanisms of genetic differences in host resistance and pinpoint the consequences of selection using these markers.

Finally, we will bring together all our results, compare them with other available data, and determine SNP markers that may be used to breed pigs for increased resistance to PRRS. These results will be of immense value to the pig breeding industry, and our results and techniques will be of great interest to the wider scientific community, particularly those wishing to research animal disease genetics whilst minimising the impact on animals (i.e. respecting 3R principles).

This project addresses PRRS, the most important viral disease affecting the pig industry, focussing on reproductive sows. We will use in-vitro methods to quantify responses to infection, genetic variation in such responses and identify loci to breed pigs for increased resistance.

Our in-vitro methods utilise monocyte derived macrophages (MDM), created by stimulation of monocytes with CSF1. We will infect MDMs with PRRSV (three contrasting strains per pig) and measure responses to infection by image analysis. Measurements include % of PRRSV-positive cells, distribution and density of staining, shape, size, granularity, complexity, intensity of staining and viability of bystander cells. After genotyping each pig with a mix of 750K and 60K SNP arrays (imputing denser genotypes for 60K) we will perform a GWAS.

Our sample size will be ca. 1150 pigs, to give good experimental power. 500 sows will be vaccinated (MLV vaccine), and with the remainder PRRSV naïve. In vaccinated sows, blood samples will be taken at regular intervals post vaccination, with viraemia as the indicator of resistance. GWAS will be repeated for the in-vivo traits, and the in-vitro phenotypes will be calibrated against the in-vivo traits.

These analyses should identify loci affecting PRRS resistance. We will explore the function of these loci using animals of contrasting genotypes for the 2 most significant loci (10 high & 10 low for both). We will infect their MDMs with PRRSV and assess responses over a 24 hour period, using microarray-based transcriptomic profiling and viral load.

Our results will identify SNP markers to breed pigs for PRRS resistance, and pinpoint consequences of such selection. This will be of immense value to pig breeders, and our results and techniques will be of great interest to the wider scientific community, particularly those wishing to research animal disease genetics whilst respecting 3R principles.

The goal of this proposed project is to help tackle the endemic viral disease Porcine Reproductive and Respiratory Syndrome (PRRS). This is the most costly disease to pig industries in developed countries, and new control measures are urgently sought. Here we take a novel approach combining in vivo and in vitro approaches, to ultimately derive a marker panel which can be used to select pigs for increased resistance to infection and reduced disease impact (i.e. increased disease resistance). At the same time, we will enhance our knowledge of the mechanisms of resistance and the consequences of genetically increasing resistance. Critically, unlike most research efforts into this disease, we target the reproductive female, this being where the greatest costs of PRRS are incurred.

The major output from this project, if successful, will be an enhancement of the health, welfare and productivity of pigs, not only in the UK but also more widely in Europe and North America. This will add to food security and contribute towards evidence based policy-making and disease control at farm, industry and international levels. This leads to both economic and societal impacts, as PRRS is a costly and distressing problem to the pig industry, and the emergence of ever-more virulent strains in Eastern Europe and East Asia simply adds to these concerns.

This project will complement and enhance on-going international research efforts, especially in the US and Canada. Here, we fill gap in the current knowledge of host responses to PRRS, particularly for virus strains of relevance to the UK and Europe, providing information not only of value to the participating breeding company, Genus, but also to all breeding companies in this sector. Further, the ultimate results, viz. SNP markers associated with PRRS resistance, by their very nature are easily implemented into practical breeding programmes. Thus, the results should have widespread impact on pig breeders and pig producers across the board. Further, through the activities of the now-completed COST Action FA902 "Understanding and combating porcine reproductive and respiratory syndrome in Europe" [EuroPRRS] which was chaired by Co-I TAA, and also through the strategic partnership of Roslin with Zoetis, we have secured excellent links to the animal health sector with interests in PRRS. Our insights into the mechanisms of resistance will be of value to this sector as well.