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

肠病毒71型（EV71）是一种小核糖核酸病毒，可引起大规模流行，并导致从手足口病到脑炎和脑膜炎等症状，主要发生在幼儿中。EV71具有7.4 kb的小正义单链RNA基因组，表达来自单个开放阅读框的11个蛋白产物以及一个额外表达的小蛋白。除了在病毒复制周期中发生的点突变外，肠病毒还容易发生重组事件，从而推动病毒进化。这些重组可分为同源重组、病毒子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缺陷（dvg），因为病毒基因组的基本部分缺失或没有功能。DVGs的另一种亚型是缺陷病毒颗粒（DIs），其附加特征是这些缺陷基因组通过隔离充分复制所需的重要资源或激活先天免疫反应来干扰共感染全长病毒基因组的复制。该项目旨在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鉴定DIs。我们的目标是在不同条件下生成EV71 dvg，并通过测序进行鉴定。从这些已识别的dvg中，我们希望使用不同的标准（包括位置、缺失长度、频率或数学适应度模型）选择25个候选dvg，并测试候选dvg的DI活性。鉴定的DIs将使用不同的分子方法进行详细的表征。然后，我们想要分析初级DVG序列，并确定在测试条件下有助于DVG形成的基因组学特征，并将其与特征DIs进行比较。之后，我们的目标是使用icSHAPE-MaP实验表征细胞中EV71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结构有望为DVG分析提供一个新的质量。为此，我们将研究DVG基因组特征是否在结构背景下得到更好的解释。根据分析，我们的目标是生成一个基因组，其中所有已识别的特征都减少或耗尽（低dvg），以便病毒不再重组，同源或非同源。另一方面，我们希望最大化EV71基因组中的DVG和DI信号（hyper-DVG），并探索其作为干扰野生型EV71复制的治疗性DIs的有效启动平台，因此可以通过降低病毒滴度来减轻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，以及它们抑制野生型EV71滴度的效率如何。可能的dvg和DIs的确切产生将通过测序和重新分析来评估。

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.