Engineering resistance to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

猪繁殖与呼吸综合征（PRRS）是一种严重危害猪的病毒性疾病。PRRS病毒（PRRSV）是一种快速进化的小包膜RNA病毒。虽然随着畜牧业和疫苗接种的变化，猪繁殖与呼吸综合征已经有所改善，但仍对猪的健康和福利产生重大影响。PRRS占美国养猪业传染病成本的约1/3，每年约6亿美元。PRRS是欧洲和北美养猪业损失最大的疾病，新的PRRSV变异株有可能更具破坏性。猪繁殖与呼吸综合征病毒（PRRSV）感染的是分化的巨噬细胞亚群，肺泡巨噬细胞是其主要靶细胞，体内、体外攻毒实验和田间研究越来越多地证明，宿主对PRRSV感染的反应和结果存在遗传变异。因此，存在遗传改良与猪在先天免疫水平上科普PRRS感染和疾病的能力相关的性状的范围。然而，抗病育种受到感染易感性的可用遗传变异的性质的限制。虽然存在宿主对PRRSV感染的反应的遗传变异的证据，但这些反应的遗传控制是多基因的，并且迄今为止没有证据表明主要基因赋予对PRRSV的完全抗性。遗传修饰养殖动物的方法的效率和复杂性的提高提供了产生对特定病原体具有遗传抗性的动物的替代方法。对病毒病原体（如PRRSV）的工程抗性的方法包括干扰病毒通过其获得进入的受体，例如通过消融受体或过表达可溶形式的受体，其可以结合病毒并阻断其进入。巨噬细胞特异性清道夫受体富含半胱氨酸（SRCR）CD 163已被证明是在这些过程中的关键作用。通过添加表达CD 163的转基因，可以将对PRRSV感染难治的细胞转化为PRRSV允许状态。用抗CD 163抗体处理易感的猪肺泡巨噬细胞以剂量依赖性方式减少PRRSV感染。有证据表明，CD 163蛋白的SRCR结构域5是参与PRRSV进入和释放的关键组分，本项目的目的是验证通过对CD 163基因的遗传修饰可以对细胞和猪进行工程改造以抵抗PRRSV感染的假设。我们提出了三种策略来工程化抗PRRSV：敲除CD 163基因;敲除CD 163基因的SRCR结构域5和过表达可溶形式的CD 163胞外结构域。

Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral disease of pigs that causes major economic losses. PRRS virus (PRRSV) is a rapidly evolving small enveloped RNA virus. Whilst improvements have been effected with changes in husbandry and vaccination, PRRS still has major impacts on pig health and welfare. PRRS accounts for ca.1/3 of the cost of infectious disease to the US pig industry, ~$600M p.a.. PRRS is the most costly disease to pig industries of Europe and North America and new PRRSV variants have the potential to be even more devastating. PRRSV infects subpopulations of differentiated macrophages, with alveolar macrophages being the major target cells.There is growing evidence from in-vitro and in-vivo challenge experiments and field studies that there is host genetic variation in responses to and outcomes of PRRSV infection. Thus, there is scope for genetically improving traits related to the capacity of pigs to cope with PRRS infection and disease at the innate immune level. However, breeding for disease resistance is constrained by the nature of the available genetic variation in susceptibility to infection. Whilst evidence for genetic variation in host responses to infection with PRRSV exists, the genetic control of these responses is polygenic and there is no evidence to date of major genes conferring complete resistance to PRRSV.The increased efficiency and sophistication of methods to genetically modify farmed animals offers alternative approaches to generate animals which are genetically resistant to specific pathogens. The approaches to engineering resistance to a viral pathogen, such as PRRSV, include interfering with the receptor(s) through which the virus gains entry, for example by ablating the receptor or over-expressing a soluble form of the receptor which could bind the virus and blocks its entry.Recent studies have revealed the molecular mechanisms through which PRRSV enters macrophages during infection. The macrophage specific scavenger receptor cysteine-rich (SRCR) CD163 has been shown to be a key role in these processes. Cells which are refractory to PRRSV infection can be converted to a PRRSV permissive state by the addition of transgenes expressing CD163. Treating susceptible porcine alveolar macrophages with anti-CD163 antibodies reduces PRRSV infection in a dose-dependent manner. There is evidence to indicate that the SRCR domain 5 of the CD163 protein is the key component involved in PRRSV entry and release.The aim of this project is to test the hypothesis that cells and pigs can be engineered to be resistant to infection with PRRSV by genetic modification of the CD163 gene. We propose three strategies to engineer resistance to PRRSV: knocking out the CD163 gene; knocking out the SRCR domain 5 of the CD163 gene and over-expressing a soluble form of the extracellular domains of CD163.