Understanding foot-and-mouth disease virus (FMDV) replication

背景资料：英国的家养动物（牛、绵羊、山羊和猪）面临口蹄疫病毒的风险，口蹄疫病毒是一种高度传染性病毒，在世界许多地区流行。除了灾难性的急性感染外，该病毒还可能导致长期不明显的“持续性”感染，这些感染难以在现场诊断，并使疾病控制复杂化。口蹄疫病毒还可引起非洲野生反刍动物（如角马和布法罗）的持续感染，从而成为家畜感染的重要宿主。迫切需要制定更好的诊断和疾病控制战略。为了实现这一目标，我们需要更全面地了解病毒生命周期的分子细节。目的：拟议的研究将提供新的见解FMDV基因组的功能，允许快速复制/建立持续感染和确定物种特异性。它们将用于未来的减毒疫苗设计。新奇：口蹄疫病毒复制的许多方面还知之甚少。据我们所知，其他地方没有开展类似的工作。时间：最近，一些创新的研究表明，在病毒复制的病毒共感染的重要性。我们已经建立了一个系统来研究FMDV合并感染之间的串扰[Herod et al，2015 and 2016]。我们计划在这里利用这个系统，见下文。实验方法：我们将使用复制子（带有GFP报告基因）来研究哺乳动物细胞中的复制。由于没有结构蛋白，因此可以安全使用。该项目将涉及：1。利用诱变和复制引物技术，对口蹄疫病毒非结构蛋白和前体蛋白在复制过程中的作用进行了研究，重点是聚合酶（3D）和复制引物（VPg）.剖析复制的站点，即“复制工厂”，例如通过PALM/STORM和EM。

Background: Domesticated animals in the UK (cattle, sheep, goats and pigs) are at risk from FMDV, a highly contagious virus, endemic in many parts of the world. In addition to catastrophic acute infection, the virus can also cause long-term unapparent 'persistent' infections that are difficult to diagnose in the field and complicate disease control. FMDV can also cause persistent infections of wild ruminants (e.g. wildebeest and buffalo) in Africa, thus providing an important reservoir for infection of domestic livestock. There is an urgent need to develop improved strategies for diagnosis and disease control. In order to achieve this, we need a more comprehensive understanding of the molecular details of the viral lifecycle. Objectives: The proposed research will give novel insight into features of the FMDV genome that allow rapid replication/establishment of persistent infections and determine species specificity. They will be used in future attenuated vaccine design. Novelty: Many aspects of FMDV replication are poorly understood. To our knowledge, no similar work is being undertaken elsewhere. Timeliness: Recently, some innovative studies have suggested the importance of viral co-infection in viral replication. We have established a system to look at the cross talk between FMDV co-infections [Herod et al, 2015 and 2016]. We plan to exploit this system here, see below. Experimental approach: We will use a replicon (with GFP reporter) to study replication in mammalian cells. The absence of structural proteins makes this safe to use. The project will involve:1. Identifying new novel roles of FMDV non-structural proteins and precursors in replication, focusing on the polymerase (3D) and primer of replication (VPg) using mutagenesis and replication inhibitors.2. Dissecting the sites of replication i.e. 'replication factories' e.g. by PALM/STORM and EM.