Characterisation of genomic signatures driving non-homologous recombination in enterovirus 71

肠道病毒71型(EV71)是一种小核糖核酸病毒，可引起大规模流行，症状包括手足口病(HFMD)、脑炎和脑膜炎，主要见于幼儿。EV71有一个7.4kb的单链正链RNA基因组，表达一个单一开放阅读框架的11个蛋白质产物和一个额外表达的小蛋白。在病毒复制周期中发生的点突变旁边，肠道病毒容易发生重组事件，这推动了病毒的进化。这些重组可分为同源重组、病毒后代的精确基因组组织和非同源重组，包括插入和缺失。缺失通常会导致病毒基因组(DVGs)的缺陷，因为病毒基因组的基本部分缺失或不起作用。DVGs的另一个亚型是缺陷病毒颗粒(Dis)，它的另一个特征是这些有缺陷的基因组通过隔离充分复制所需的重要资源或通过激活先天免疫反应来干扰共同感染的全长病毒基因组的复制。这个项目的目的是深入了解有助于产生DVG的基因组特征，以及其中一些特征是否可以预测用于DIS的识别。我们的目标是在不同的条件下产生EV71 DVGs，通过测序进行鉴定。从这些确定的DVG中，我们希望使用不同的标准(包括位置、删除长度、频率或数学适应模型)选择25个候选者，并测试DI活动的候选者。识别的DIS将使用不同的分子方法进行详细表征。然后，我们想要分析主要的DVG序列，并确定在测试的条件下有助于DVG形成的基因组学特征，并将它们与特征化的DIS进行比较。在此之后，我们的目标是用icSHAPE-MAP实验表征EV71在细胞中的二级RNA结构，然后将初级序列分析整合到生成的结构上。二级RNA结构有望使DVG分析具有新的质量。为此，我们将调查DVG基因组签名是否可以在结构背景下得到更好的解释。在分析的基础上，我们的目标是产生一个所有已识别的特征都被减少或耗尽的基因组(How-DVG)，以便病毒不再进行重组，无论是同源还是非同源。另一方面，我们希望最大化EV71基因组(hyper-DVG)中的DVG和DI信号，并探索它作为干扰野生型EV71复制的治疗疾病的有效启动平台，因此可以作为通过降低病毒滴度来减少疾病负担的概念验证。我们想要调查哪些DVG物种将占主导地位，以及他们如何有效地抑制野生型EV71滴度。将通过排序和重新分析来评估可能的DVG和DIS的确切产生。

Enterovirus 71 (EV71) is a picornavirus that causes large epidemics and results in symptoms from hand-foot-and-mouth disease (HFMD) to encephalitis and meningitis primarily in young children. EV71 has a small 7.4 kb positive-sense single-stranded RNA genome and expresses 11 protein products from a single open-reading frame as well as an additionally expressed small protein. Next to point mutations that occur during the viral replication cycle, enteroviruses are prone to recombination events, which drive viral evolution. These recombinations can be divided into homologous recombination, exact genomic organisation of the viral progeny, and non-homologous recombination, including insertions and deletions. Deletions often result in defective viral genomes (DVGs) as essential parts of the viral genome are missing or not functional. An additional subtype of DVGs is defective viral particles (DIs), which have the additional feature that these defective genomes interfere with the replication of co-infecting full-length virus genomes by either sequestering vital resources needed for sufficient replication or by activating innate immune responses. This project aims to gain an in-depth understanding of genomic features that help generate DVGs and whether some of them can be predictable for the identification of DIs. We aim to generate EV71 DVGs in different conditions, identified by sequencing. From these identified DVGs, we want to select 25 candidates using different criteria including location, deletion length, frequency or a mathematical fitness model and test the candidates for DI activity. The identified DIs will be characterised in detail using different molecular methods. We then want to analyse the primary DVG sequences and identify genomics signatures that contribute to the formation of DVGs in the conditions tested and compare them to characterised DIs. After this, we aim to experimentally characterise the secondary RNA structure of EV71 in cells using icSHAPE-MaP followed by the integration of the primary sequence analysis on the generated structure. The secondary RNA structure is expected to give the DVG analysis a new quality. For this, we will investigate whether the DVG genome signatures are better explained in a structural context. Based on the analysis, we aim to generate a genome where all identified features are reduced or depleted (hypo-DVG) so that the virus does not recombine, homologous or non-homologous, anymore. On the other hand, we want to maximise the DVG and DI signals in the EV71 genome (hyper-DVG) and explore it as an effective launching platform for therapeutic DIs that interfere with wild-type EV71 replication and therefore could serve as proof-of-concept for reducing disease burden by lowering virus titres. We want to investigate which DVG species will be dominant and how efficiently they suppress wild-type EV71 titres. The exact generation of possible DVGs and DIs will be assessed by sequencing and re-analysed.