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

该项目使用新颖和创新的方法来开发工具，以帮助控制影响欧洲和北美养猪业的最重要的病毒病，即。猪繁殖与呼吸综合征(PRRS)。我们将重点关注繁殖母猪感染PRRS病毒(PRRSV)。猪对PRRS的抵抗力在遗传上有所不同。因此，我们的最终目标是确定与这些动物之间的差异相关的遗传标记，从而可以用于繁殖以增加抵抗力。然而，研究PRRSV对母猪的影响是昂贵和困难的，所以我们将使用新的方法来克服这个问题。我们开发了体外方法来评估宿主对PRRSV感染的反应：从采集的血液样本中，我们从白细胞中分离单核细胞。在CSF-1的刺激下，这些细胞被培养成巨噬细胞(MDM)，这是PRRSV感染的细胞类型。然后，我们用PRRSV感染MDM，并通过图像分析和测量感染后表达的基因来测量它们对感染的反应。为了将这些体外表型与活体动物(体内)的感染结果联系起来，我们将使用一个牛群，其中小母猪接种了活疫苗，因此有控制的PRRSV暴露。将在接种疫苗前采集血液样本，以获取MDM，并在接种后定期采集血样，并测量病毒水平(病毒血症)，作为其对感染的抵抗力的指标。我们还将对未接种疫苗的幼稚猪进行体外研究，以研究幼稚猪对病毒的反应。首先，我们将以病毒血症为表型，探索PRRS耐药性的遗传控制。我们将用高密度(750K)SNP阵列对每头接种疫苗的猪进行基因分型，这些SNP阵列是DNA芯片，可以检测基因组中约750,000个位置的遗传变异。未接种疫苗的猪将用60K和750K SNP芯片混合进行基因分型。然后，我们将对所有动物的MDM进行体外感染研究。每只动物的MDM将分别感染三种不同的PRRSV毒株，并提供每只动物的详细信息。使用SNP芯片基因型别，我们将识别与所有体外测量相关的遗传标记。我们还将探索体外和体内测量之间的关系，以确定哪些体外表型可以真正预测活体动物的结果。在大约1150头猪身上做这项工作将给我们很好的分辨率来定位影响抗性的基因座并识别相关的遗传标记。我们对遗传标记和所有表型的分析应该确定对PRRS抗性有主要影响的基因座；定义这些基因座的标记可以用于选择动物。然而，我们希望知道这些基因座是如何影响PRRS抗性的，以及利用这些标记进行猪繁殖的后果。为了实现这一点，我们将从我们的结果中选择两个最重要的基因座，然后识别出具有不同基因型别的动物(两种类型各有10个高和10个低)。然后，我们将用PRRSV感染这些动物的MDM，并评估它们在24小时内的反应，测量病毒载量和已知参与PRRSV感染的免疫基因的表达。进一步的微阵列分析将评估全球基因表达并全面描述对感染的反应。这些结果将深入了解宿主抗性遗传差异的机制，并准确地确定使用这些标记进行选择的后果。最后，我们将综合所有结果，将它们与其他现有数据进行比较，并确定可能用于提高猪对PRRS抗性的SNP标记。这些结果将对养猪业具有巨大的价值，我们的结果和技术将引起更广泛的科学界的极大兴趣，特别是那些希望在研究动物疾病遗传学的同时将对动物的影响降至最低的人(即尊重3R原则)。

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).