Interferon-stimulated genes as resilience factors for PRRSV infection

猪繁殖与呼吸综合征（PRRS）是猪的一种流行性病毒性疾病，造成重大经济损失。PRRS病毒（PRRSV）是一种快速进化的小包膜RNA病毒，是PRRS的病原体。虽然随着畜牧业和疫苗接种的变化，情况有所改善，但PRRS仍然对猪的健康和福利产生重大影响。PRRS约占美国养猪业传染病成本的三分之一，每年达6.5亿美元。PRRS是对欧洲、中国和北美养猪业造成最大损失的疾病，最近在中国和东南亚暴发的高致病性PRRSV变种表明，新的PRRSV变种具有更具破坏性的潜力。地方性PRRSV的存在还会抑制免疫力并增加对其他感染的易感性，从而导致复杂的疾病和增加抗菌素的使用。PRRSV具有狭窄的宿主细胞趋向性，仅限于单核细胞/巨噬细胞谱系的细胞。像所有病毒一样，PRRSV依靠宿主细胞机制进行自我复制并产生新的病毒颗粒。越来越多的体外和体内挑战实验和实地研究证据表明，宿主对PRRSV感染的反应和结果存在遗传变异。特别是参与细胞免疫反应的基因，干扰素（IFN）反应，已被发现是遗传弹性的候选者。然而，动物挑战实验甚至实地研究的一个普遍问题是，由于动物太少，它们通常没有足够的力量来解剖遗传效应，这使得它们错过了弹性变异。干扰素是抵御病毒感染的关键第一道防线，通过触发一系列抗病毒蛋白的表达和激活而起作用。它们通过触发细胞因子和趋化因子的释放来形成适应性免疫反应。干扰素反应从根本上参与了宿主对病毒感染的反应。更好地了解干扰素反应以及病毒如何抵消其影响，对我们如何治疗病毒感染和开发疫苗具有重要意义。I型干扰素是对抗病毒感染的先天免疫的主要细胞因子，根据动物物种的不同包括不同的亚型。IFN- γ是II型IFN的唯一代表。虽然I型IFN在大多数细胞类型中产生，以响应不同的病毒，但II型IFN由有限类型的细胞产生，包括自然杀伤细胞、活化的T淋巴细胞、巨噬细胞和神经元。虽然IFN的抗病毒性质早已被记录在案，但IFN如何抑制不同病毒的具体机制却知之甚少。最近，由IFN诱导的500多个人类基因已被单独克隆，允许过表达和研究它们对病毒的影响。这已被证明是一种特别有效的方法，对IFN在人类细胞中的作用有了重大发现。在BBSRC拨款提案“鉴定限制甲型流感病毒跨物种传播的干扰素刺激基因”（BB/S00114X/1）中，我们为猪生成了一个排列的I型ISG文库。在本提案中，我们将把这一独特的工具与尖端的多能干细胞技术和新生成的猪II型ISG文库相结合，以鉴定在限制PRRSV感染中发挥作用的基因。这些基因将被进一步研究，以评估它们是否适合在猪中进行遗传选择或基因组编辑，或者是否可能被抗病毒药物靶向以对抗PRRSV感染。该提案不仅将确定有效的PRRS抗性基因或抗病毒策略，而且还将产生宝贵的技术资源和方案，可用于其他猪病原体，特别是感染免疫细胞的猪病原体，如非洲猪瘟病毒、鼠伤寒沙门氏菌或弓形虫。

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic viral disease of pigs that causes major economic losses. The causative agent of PRRS, 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 about a third of the cost of infectious disease to the US pig industry amounting to $650M per year. PRRS is the most costly disease to pig industries of Europe, China and North America and new PRRSV variants have the potential to be even more devastating as demonstrated by recent outbreaks of highly pathogenic variants of the virus in China and Southeast Asia. The presence of endemic PRRSV also supresses immunity and increases susceptibility to other infections, leading to complex disease and increased use of antimicrobials. PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. Like all viruses PRRSV relies on the host cell machinery to replicate itself and generate new viral particles.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. In particular genes involved in the cellular immune response, the interferon (IFN) response, have been found to be candidates for genetic resilience. However, a general issue with animal challenge experiments and even field studies is that they usually have insufficient power to dissect genetic effects as there are too few animals, which makes them miss resilience variants.IFNs are a crucial first line of defence against viral infection, acting by triggering the expression and activation of a suite of antiviral proteins. They shape the adaptive immune response by triggering release of cytokines and chemokines. The IFN response is fundamentally involved in the host response to virus infections. Developing a better understanding of the IFN response and how viruses counteract its effects has important implications for how we treat viral infections and the development of vaccines. Type I IFNs are the main cytokines for innate immunity against viral infections and include various subtypes depending on the animal species. IFN-gamma is the sole representative of type II IFN. While type I IFNs are produced in most cell types in response to different viruses, type II IFN is produced by limited types of cells including natural killer cells, activated T lymphocytes, macrophages, and neurons.While the antiviral nature of IFN has long been documented, the specific mechanisms of how IFN inhibits different viruses is poorly understood. Recently, more than 500 human genes induced by IFN have been individually cloned, allowing overexpression and investigation of their effects on viruses. This has proven to be a particularly powerful approach, resulting in significant discoveries on the action of IFN in human cells. In the BBSRC grant propoasal "Identification of interferon stimulated genes that restrict cross-species transmission of influenza A virus" (BB/S00114X/1) we generated an arrayed type I ISG library for the pig. In this proposal we will combine this unique tool with cutting-edge pluripotent stem cell technology and a newly generated pig type II ISG library to identify genes playing a role in restricting PRRSV infection. These genes will be further investigated to assess whether they are suitable candidates for genetic selection or genome editing in pigs or may be targeted by antiviral drugs to combat PRRSV infection. This proposal will not only identify valid PRRS resilience genes or antiviral strategy but also generate valuable technical resources and protocols that can be used for other porcine pathogens, in particular ones infecting immune cells, such as African swine fever virus, salmonella typhimurium, or toxoplasma gondii.