Autophagy represents a new host-pathogen interface for identification of infectious bronchitis virus proteins that determine virulence

传染性支气管炎是由禽冠状病毒传染性支气管炎病毒(IBV)引起的一种重要的鸡地方病。禽肉是一种重要的食物来源，在一年中，全球大约饲养了40x109只鸡。对禽肉日益增长的需求导致了集约化养殖方法的引入，但生产率往往受到传染病的限制，这些疾病通过高密度的鸡群迅速传播。对于这项建议来说，重要的是，英国政府在2005年发表的一份报告((http://www.defra.gov.uk/science/Project_Data/Document图书馆/ZZ0102/ZZ01021215_FRP.doc)显示，鸡的传染病导致英国家禽业经济损失的头号原因是由传染性支气管炎病毒引起的。该病毒不仅会引起呼吸道疾病，还会对母鸡的肾脏和产蛋器官造成损害，影响鸡蛋的产量和质量。尽管有活疫苗和灭活疫苗可用，但IBV仍然是一个主要问题。该病毒发病率高，在世界范围内普遍存在，在英国流行，并表现出广泛的抗原变异和短暂的免疫力。这些因素导致高感染率和在感染或接种疫苗后缺乏交叉保护。大多数疫苗是通过喷雾或饮用水给家禽接种的。这两种方法都相当随意。疫苗研发人员的“圣杯”是拥有在雏鸟孵化前由机器人给雏鸟接种的疫苗。不幸的是，没有现有的IB疫苗可以在卵子中接种，因为病毒会阻止雏鸡孵化。控制IBV的一种方法是有系统地生产针对强毒株提供保护的减毒活疫苗。IAH康普顿的冠状病毒小组已经开发出修改或移除与IBV毒力相关的基因所需的反向遗传学。IAH冠状病毒小组与DEFRA和主要的商业疫苗开发商InterveInternational一起，正在操纵IBV的基因，以在毒力减弱和诱导免疫能力之间获得最佳平衡。为了将这项技术应用于活疫苗的合理设计，现在有必要识别导致IBV毒力的基因，并了解它们是如何发挥作用的。对这项提议来说，重要的是，最近在哺乳动物冠状病毒方面的工作，以及康普顿在禽类IBV冠状病毒上的工作表明，毒力可能由细胞控制病毒复制的方式决定。我们生产的IBV不会产生一系列称为3a、3b、5a和5b的小蛋白。这些病毒在细胞培养中正常生长，并在卵子中接种后允许雏鸟孵化。这表明我们可以通过移除非必需基因来减弱IBV，但这是一个经验过程，因为我们不知道3(Ab)和5(Ab)蛋白在毒力背景下如何发挥作用。目前向BBSRC申请赠款的目的是为了建立我们正在进行的经验观察背后的科学。支持这一提议的实验表明，IBV的毒力可能与控制复制的蛋白质有关，并且这些蛋白质与细胞膜相关，这些细胞膜有可能在病毒离开细胞之前摧毁病毒。我们现在想要了解复制酶蛋白如何避免破坏，并通过这种方式决定毒力。这将使我们能够微调我们的突变体，使病毒存活足够长的时间来感染雏鸟，并诱导免疫反应，但太弱，不能伤害雏鸟，阻止它们孵化。

Infectious bronchitis is an important endemic disease of chickens and caused by the avian coronavirus infectious bronchitis virus (IBV). Poultry meat is an important food source and during the course of a year approximately 40 x 109 chickens are reared globally. The increasing demand for poultry meat has lead to the introduction intensive farming methods but productivity is often limited by infectious diseases which spread rapidly through high density chicken populations. Importantly for this proposal, a report sponsored by the UK government published in 2005 (http://www.defra.gov.uk/science/Project_Data/Document Library/ZZ0102/ZZ0102 1215_FRP.doc) revealed that the number one cause of economic loss in the UK poultry industry resulting from infectious diseases of chickens was caused by IBV. The virus is not only responsible for respiratory disease, but also causes damage to the kidneys and to egg producing organs of hens, affecting both the production and quality of eggs. Despite the availability of live and inactivated vaccines, IBV continues to be a major problem. The virus causes high morbidity, is ubiquitous world wide, and endemic in the UK, and shows extensive antigenic variation and short lived immunity. These factors lead to high rates of infection and poor cross-protection following infection or vaccination. Most vaccines are given to poultry by spray or in drinking water. Both approaches are rather hit-and-miss. The 'holy grail' of vaccine developers is to have vaccines that can be given by robotic machine to chicks before they hatch. Unfortunately, no existing IB vaccine can be given in ovo because the viruses stop the chicks hatching. One means of controlling IBV is to have a systematic way of generating live attenuated vaccines that provide protection against virulent strains. The reverse genetics necessary for the modification or removal of genes associated with virulence from IBV has been developed by the coronavirus group at the IAH Compton. Together with DEFRA and Intervet International, a major commercial vaccine developer, the IAH coronavirus group are manipulating the genes of IBV to get an optimum balance between attenuation of virulence and capacity to induce immunity. To apply this technology to the rational design of live vaccines it is now necessary to identify genes that contribute to IBV virulence, and understand how they function. Importantly for this proposal, recent work on mammalian coronaviruses, and work on the avian IBV coronavirus at Compton, suggests that virulence may be determined by the way in which cells control virus replication. We have produced IBVs that do not make a series of small proteins called 3a, 3b, 5a and 5b. These viruses grow normally in cell culture and allow chicks to hatch after inoculation in ovo. This shows that we can attenuate IBV by removing non-essential genes, but this is an empirical process because we do not know how the 3(ab) and 5(ab) proteins function in the context of virulence. The purpose of the present grant application to BBSRC is to establish the science behind the empirical observations that we are making. Experiments underpinning this proposal have shown that virulence of IBV may be associated with proteins that control replication, and that these proteins associate with membranes in cells that have the potential to destroy the virus before it can leave the cell. We now want to understand how the replicase proteins avoid destruction, and in this way determine virulence. This will enable us to fine tune our mutants to make viruses that survive long enough to infect chicks 'in ovo', and induce an immune response, but are too weak to harm the chicks and prevent them from hatching.