Live attenuated nairovirus vaccines: targeted mutations in a recombinant virus

通过昆虫、扁虱或小型哺乳动物媒介传播的病毒疾病对人类和牲畜的健康都是一种重大威胁。这些病毒中的许多会引起众所周知的病毒性出血热(VHF)，感染会导致皮肤下、内脏或口腔、眼睛或耳朵等身体孔道出血。重病患者还可能表现为休克、神经系统功能障碍、昏迷、精神错乱和癫痫发作。目前还没有特定的治疗方法，在已知的VHFs中只有两种有疫苗可用。由于需要在保护研究人员和普通公众的特殊高密封性实验室工作，以及没有任何动物模型(灵长类动物除外)显示出同样的症状，对影响人类的VHFs的研究受到限制。在这个项目中，我们将开发一种自然发生的绵羊和山羊的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，而且自然宿主很容易获得。NSDV与克里米亚-刚果出血热病毒(CCHFV)关系密切，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CCHFV的研究，并增加我们对VHFs的总体知识。该项目将调查NSDV干扰宿主防御的能力，特别是在感染的最初几个小时被激活的所谓的“先天”免疫系统。针对病毒的先天免疫反应中最重要的部分是受感染的细胞产生‘干扰素’。顾名思义，干扰素会干扰病毒的生长，启动周围未感染细胞中的大量内部防御系统，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，一些病毒产生阻止干扰素活动的蛋白质，还有一些两者兼而有之。NSDV和CCHFV所属的病毒组似乎以某种方式阻止了这种干扰素反应，由于只有两种病毒蛋白可能具有这种效果，因此找出哪种应该相对简单。这两种蛋白质都是多功能的，所以我们将准确地确定活性蛋白质的哪一部分负责，并只改变这一部分，以便生成的蛋白质继续做它在病毒生命周期中必须做的所有其他事情，但不再干扰干扰素防御机制。我们预计，去除这种病毒对策将极大地降低病毒致病的能力，因为宿主的防御将不再被阻断。这种活的、有传染性但不再致病的病毒可以作为疫苗，因为它将刺激特定抗体和细胞的产生，准备攻击入侵的NSDV。因此，我们将制造一种新的病毒，其中正常蛋白质被有缺陷的突变体取代，并在绵羊身上测试本土和突变病毒。

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.