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

肠道病毒 71 中驱动非同源重组的基因组特征的表征

• 批准号: 528194537
• 负责人: Dr. Björn Meyer
• 金额: --
• 依托单位: Institut für Medizinische Mikrobiologie und Krankenhaushygiene
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/528194537?language=en
• 关键词: Characterisation; genomic; signatures; driving; non

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.

肠道病毒71型（EV 71）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（HFMD）到脑炎和脑膜炎的症状，主要发生在幼儿中。EV 71具有7.4kb的小的正义单链RNA基因组，并且表达来自单个开放阅读框的11种蛋白产物以及另外表达的小蛋白。除了在病毒复制周期中发生的点突变之外，肠道病毒还容易发生重组事件，从而驱动病毒进化。这些重组可以分为同源重组、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。缺失通常导致缺陷病毒基因组（DVG），因为病毒基因组的重要部分缺失或不起作用。DVG的另一种亚型是缺陷型病毒颗粒（DI），其具有额外的特征，即这些缺陷型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应答来干扰共感染全长病毒基因组的复制。本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识别DI。我们的目标是在不同条件下产生EV 71 DVG，并通过测序进行鉴定。从这些鉴定的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个候选物，并测试候选物的DI活性。将使用不同的分子方法详细表征所鉴别的DI。然后，我们要分析的主要DVG序列，并确定基因组学的签名，有助于DVG的形成在测试的条件下，并将它们与表征的DI。在此之后，我们的目标是实验性地使用icSHAPE-MaP的EV 71在细胞中的二级RNA结构，然后通过整合的主要序列分析生成的结构。二级RNA结构有望为DVG分析提供新的质量。为此，我们将研究DVG基因组签名是否在结构背景下得到更好的解释。基于该分析，我们的目标是生成一个基因组，其中所有鉴定的特征都被减少或消除（hypo-DVG），以便病毒不再重组，无论是同源的还是非同源的。另一方面，我们希望最大化EV 71基因组中的DVG和DI信号（hyper-DVG），并探索其作为干扰野生型EV 71复制的治疗性DI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。我们希望研究哪些DVG物种将占主导地位，以及它们如何有效地抑制野生型EV 71滴度。将通过测序和重新分析评估可能的DVG和DI的确切生成。