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

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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.