Live attenuated nairovirus vaccines: targeted mutations in a recombinant virus

由昆虫、蜱虫或小型哺乳动物传播的病毒性疾病对人类和家畜的健康都是一个重大的危害。这些病毒中的许多引起所谓的病毒性出血热（VHF），其中感染导致皮肤下，内脏器官或身体孔口（如嘴，眼睛或耳朵）出血。病情严重的病人也可能出现休克、神经系统功能障碍、昏迷、谵妄和癫痫发作。目前还没有具体的治疗方法，只有两种已知的VHF有疫苗可用。对影响人类的VHF的研究受到限制，因为需要在保护研究人员和公众的特殊高封闭实验室中工作，并且没有任何动物模型（灵长类动物除外）显示出相同的症状。在这个项目中，我们将利用绵羊和山羊的自然发生的VHF（内罗毕绵羊病病毒（NSDV））。这种病毒对研究它的研究人员来说风险很低，自然宿主很容易获得。NSDV与克里米亚-刚果出血热病毒（CCHFV）非常密切相关，CCHFV是一种在非洲和亚洲大部分地区发现的人类病原体，死亡率约为30%。我们对NSDV的了解将有助于对CCHFV的研究，并增加我们对VHF的一般知识。该项目将研究NSDV干扰宿主防御的能力，特别是所谓的“先天”免疫系统，该系统在感染的最初几个小时内被激活。对抗病毒的先天免疫反应的最重要部分是受感染细胞产生“干扰素”。顾名思义，干扰素干扰病毒的生长，打开周围未感染细胞的许多内部防御系统，使病毒更难传播。许多病毒产生阻止受感染细胞产生干扰素的蛋白质，有些产生阻止干扰素作用的蛋白质，有些则两者兼有。NSDV和CCHFV所属的病毒组似乎以某种方式阻断这种干扰素反应，并且由于只有两种病毒蛋白质可能具有这种作用，因此找出哪一种应该相对简单。这两种蛋白质都是多功能的，所以我们将准确地识别出活性蛋白质的哪一部分是负责的，并改变这一部分，这样生成的蛋白质就可以继续做它在病毒生命周期中必须做的所有其他事情，而不再干扰干扰素的防御机制。我们预计，取消这种病毒对抗措施将大大降低病毒致病的能力，因为宿主的防御能力将不再受到阻碍。这种病毒，活的和传染性的，但不再引起疾病，可以作为疫苗，因为它会刺激生产特定的抗体和细胞准备攻击入侵NSDV。因此，我们将制造一种新病毒，其中正常蛋白质被有缺陷的突变体所取代，并在绵羊中测试天然病毒和突变病毒。

Virus diseases spread by insect, tick or small mammal vectors are a significant hazard to the health of both humans and livestock animals. Many of these viruses cause what is known as viral hemorrhagic fever (VHF) in which infection leads to bleeding under the skin, in internal organs, or from body orifices like the mouth, eyes, or ears. Severely ill patients may also show shock, nervous system malfunction, coma, delirium, and seizures. There are no specific treatments and only two of the known VHFs has a vaccine available. Study of the VHFs that affect people is limited by the need to work in special high containment laboratories that protect the researchers and the general public and by the absence of any animal model (other than primates) showing the same symptoms. In this project we will exploit a naturally occurring VHF of sheep and goats (Nairobi sheep disease virus (NSDV)). This virus is of low risk to the researchers studying it and the natural host is readily available. NSDV is very closely related to Crimea-Congo hemorrhagic fever virus (CCHFV), a human pathogen found in large parts of Africa and Asia which has a mortality rate of about 30%. What we learn about NSDV will help research on CCHFV as well as increasing our knowledge of VHFs in general. This project will investigate the ability of NSDV to interfere with the host defenses, in particular the the so-called 'innate' immune system, which is activated in the first hours of an infection. The most important part of the innate immune response against viruses is the production of 'interferons' by infected cells. As the name suggests, interferons interfere with virus growth, turning on lots of internal defence systems in the surrounding uninfected cells, and making it harder for the virus to spread. Many viruses produce proteins that stop infected cells from making interferons, some produce proteins that block the action of interferons, and some do both. The group of viruses to which NSDV and CCHFV belong appear to block this interferon response in some way and, as there are only two viral proteins that could be having this effect it should be relatively simple to find out which. Both proteins are multifunctional, so we will then identify exactly which part of the active protein is responsible and change just that part so that the resultant protein continues to do all the other things it has to do in the viral life cycle but no longer interferes with the interferon defense mechanism. We expect that taking away this viral countermeasure will greatly reduce the ability of the virus to cause disease, because the host's defenses will not be being blocked any more. Such a virus, live and infectious but no longer causing disease, could act as a vaccine, since it will stimulate the production of specific antibodies and cells ready to attack invading NSDV. We will therefore make a new virus in which the normal protein is replaced by the defective mutant, and test the native and mutant viruses in sheep.