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

宿主对猪繁殖与呼吸综合征病毒（PRRSV）反应的遗传学

• 批准号: BB/M012891/1
• 负责人: Alan Archibald
• 金额: $ 86.13万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM012891%2F1
• 关键词: Genetics; host; responses; Porcine; Reproductive

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).

该项目使用新颖和创新的方法开发工具，以帮助控制影响欧洲和北美养猪业的最重要的病毒性疾病，即猪繁殖与呼吸综合征（PRRS）。我们将重点关注繁殖母猪中的PRRS病毒（PRRSV）感染。猪在对PRRS的抗性方面存在遗传差异。因此，我们的最终目标是确定与这些动物间差异相关的遗传标记，从而可用于育种以提高抗性。然而，研究猪繁殖与呼吸综合征对母猪的影响既昂贵又困难，因此我们将使用新的方法来克服这一问题。我们已经开发了体外方法来评估宿主对猪繁殖与呼吸综合征感染的反应：从采集的血液样本中，我们从白色血细胞中分离单核细胞。通过用CSF-1刺激，这些细胞然后被培养成巨噬细胞（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动物（体内）的感染结果相关联，我们将使用一个母猪接种活疫苗的畜群，因此具有受控的PRRSV暴露。将在接种前和接种后定期采集血液样本，以获得MDM，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猪进行体外研究，以研究未感染该病毒的猪的PRRVS反应。 首先，我们将探索PRRS抗性的遗传控制，使用病毒血症作为表型。我们将用高密度（750 K）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。将使用60 K和750 K SNP芯片的混合物对未接种疫苗的猪进行基因分型。然后，我们将对所有动物的MDM进行体外感染研究。将用三种不同的PRRSV毒株分别感染每只动物的MDM，并提供每只动物的详细信息。使用SNP芯片基因型，我们将鉴定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真正预测活体动物的结果。在CA上做这个。1150头猪将为我们绘制影响抗性的基因座和鉴定相关遗传标记提供良好的分辨率。我们对遗传标记和所有表型的分析应确定对PRRS抗性具有主要影响的基因座;定义这些基因座的标记可用于选择动物。然而，我们希望了解这些基因座如何影响PRRS抗性以及用这些标记进行猪育种的后果。为了实现这一点，我们将从我们的结果中选择两个最显著的基因座，然后识别具有相反基因型的动物（10个高基因型和10个低基因型）。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响应PRRSV感染的免疫基因的表达。进一步的微阵列分析将评估整体基因表达，并全面描述对感染的反应。这些结果将提供深入了解宿主抗性遗传差异的机制，并查明使用这些标记选择的后果。最后，我们将汇集我们所有的结果，将它们与其他可用数据进行比较，并确定SNP标记，可用于育种猪提高对PRRS的抗性。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，特别是那些希望研究动物疾病遗传学，同时最大限度地减少对动物的影响（即尊重3R原则）的人。

项目成果

Quasispecies evolution of the prototypical genotype 1 porcine reproductive and respiratory syndrome virus early during in vivo infection is rapid and tissue specific.

• DOI: 10.1007/s00705-017-3342-0
• 发表时间: 2017-08
• 期刊: Archives of virology
• 影响因子: 2.7
• 作者: Lu ZH;Wang X;Wilson AD;Dorey-Robinson DLW;Archibald AL;Ait-Ali T;Frossard JP
• 通讯作者: Frossard JP

Analysis of the genetic diversity and mRNA expression level in porcine reproductive and respiratory syndrome virus vaccinated pigs that developed short or long viremias after challenge.

• DOI: 10.1186/s13567-018-0514-1
• 发表时间: 2018-02-15
• 期刊: Veterinary research
• 影响因子: 4.4
• 作者: Cortey M;Arocena G;Ait-Ali T;Vidal A;Li Y;Martín-Valls G;Wilson AD;Archibald AL;Mateu E;Darwich L
• 通讯作者: Darwich L

Combining laboratory and mathematical models to infer mechanisms underlying kinetic changes in macrophage susceptibility to an RNA virus.

• DOI: 10.1186/s12918-016-0345-5
• 发表时间: 2016-10-22
• 期刊: BMC systems biology
• 作者: Doeschl-Wilson A;Wilson A;Nielsen J;Nauwynck H;Archibald A;Ait-Ali T
• 通讯作者: Ait-Ali T