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（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，全球每年大约饲养40 × 109只鸡。对禽肉日益增长的需求导致引进集约化养殖方法，但生产力往往受到传染病的限制，这些传染病在高密度的鸡群中迅速传播。重要的是，英国政府于2005年发表的一份报告（http://www.defra.gov.uk/science/Project_Data/Document Library/ZZ0102/ZZ0102 1215_FRP.doc）显示，IBV是英国家禽业因鸡传染性疾病造成经济损失的头号原因。这种病毒不仅会导致呼吸道疾病，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，但IBV仍然是一个主要问题。该病毒引起高发病率，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。这些因素导致感染率高，感染或接种疫苗后交叉保护差。大多数疫苗是通过喷雾或饮用水给家禽接种的。这两种方法都是碰运气的。疫苗开发人员的“圣杯”是研制出可以在小鸡孵化前由机器人给它们注射疫苗的疫苗。不幸的是，由于病毒会阻止雏鸡的孵化，现有的乙肝疫苗无法在蛋鸡中接种。控制IBV的一种手段是采用一种系统的方法来生产减毒活疫苗，以提供对毒性毒株的保护。美国国际大学康普顿分校的冠状病毒研究小组已经开发出了修改或去除IBV毒力相关基因所需的反向遗传学。与DEFRA和主要商业疫苗开发商Intervet International一起，IAH冠状病毒研究小组正在操纵IBV的基因，以在减弱毒力和诱导免疫能力之间取得最佳平衡。为了将这项技术应用于活疫苗的合理设计，现在有必要确定导致IBV毒力的基因，并了解它们如何起作用。对于这一建议来说，重要的是，最近对哺乳动物冠状病毒的研究以及康普顿大学对禽类IBV冠状病毒的研究表明，毒力可能是由细胞控制病毒复制的方式决定的。我们已经生产出不制造3a， 3b， 5a和5b等一系列小蛋白质的ibv。这些病毒在细胞培养液中正常生长，并在卵中接种后使小鸡孵化。这表明我们可以通过去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）蛋白在毒力背景下是如何起作用的。目前向BBSRC申请拨款的目的是建立我们正在进行的实证观察背后的科学。支持这一建议的实验表明，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中的膜有关，这些膜有可能在病毒离开细胞之前将其摧毁。我们现在想了解复制酶蛋白是如何避免破坏的，并通过这种方式确定毒性。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“卵中”的小鸡，并诱导免疫反应，但太弱而无法伤害小鸡并阻止它们孵化。

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.