Development of enterovirus trans-encapsidation assays as tools to understand viral entry.

Enteroviruses (EVs) are globally important human and animal pathogens which cause a diverse spectrum of disease, ranging from febrile illness to paralysis. Despite decades of research, parts of the EV lifecycle remain poorly understood. Replicons, in which reporter genes replace the structural protein coding region, have proved useful for the study of EV biology. However, it is not possible to study the molecular mechanism(s) of entry, capsid uncoating and genome release without the production of virus particles. To utilise the benefits provided by replicons for the study of viral cell entry, it would be necessary to supply the structural proteins in trans. Here, we present an EV trans-encapsidation (TE) system in which reporter replicons are transfected into cells modified to express the viral structural proteins. The nascent replicons are packaged in trans to form virus particles containing fluorescent or luminescent replicon genomes. This enables the real-time assessment of EV entry and replication through quantification of fluorescence using live-cell imaging. We demonstrate that these TE particles are biologically accurate proxies to EVA71 virions and show utility for the study of EV entry, uncoating and replication. Additionally, we demonstrate the use of TE particles as platforms for drug discovery and immunological screening, applicable to the development of antiviral therapeutics and assessment of immunisation outcomes.