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Background and Aims Enterovirus 71 (EV71), a member of the Picornaviridae family, poses a significant threat as a cause of hand-foot-and-mouth disease (HFMD), primarily impacting small children. The potential for severe neurological complications, leading to mortality rates of up to 19 %. With the escalating outbreaks in the Asia-Pacific region, the exploration of novel therapeutic approaches is indispensable. While current vaccines target specific serotypes, their efficacy lacks evidence of cross-protection. This underscores the importance of investigating targeted antiviral therapies. However, progress is hindered by gaps in understanding EV71’s replication cycle. This study aims to examine the unmapped facets of enterovirus RNA replication coupling with genome packaging and viral assembly. Methods We present the establishment of a fluorescent reporter that enables specific EV71 replication site labelling without altering the viral genome, hence preserving the natural course of genome replication. This system will serve as a crucial tool for guiding subsequent research phases, including correlative light and electron microscopy for precise lamella preparation and cryo-electron tomography data collection. Results In our preliminary results, we utilised AlphaFold-guided experimental design to inform the development of our fluorescent reporter system. The reporter was successfully cloned, and we are currently testing its functionality on the fluorescence microscope. Conclusions These initial steps mark significant progress towards our goal of visualising EV71 genome replication in situ and gaining insights into its replication mechanisms.