Identification of host and viral determinants of Enterovirus 71 disease severity

Enterovirus 71 (EV-A71) is an important human pathogen that causes widespread epidemics of hand, foot, and mouth disease, sometimes leading to severe neurological complications and death, particularly in children and immunocompromised patients. In 2013, we identified a mutation (VP1 L97R) in the VP1 viral capsid protein, which arose during a disseminated EV-A71 infection in an immunosuppressed patient and conferred affinity for heparan sulfate (HS), a viral attachment receptor. The mutation was absent in the patient respiratory tract, emerged in the digestive tract and became dominant in the blood and cerebrospinal fluid. We identified a second spontaneous mutation (VP1 E167G) resulting from cell adaptation, which further increased the affinity for HS. In immunocompromised mice, the VP1 L97R mutation increased dissemination and neurotropism, while it increased sensitivity to neutralizing antibodies in immunocompetent mice. Surprisingly, the strong HS-binder, bearing the two capsid mutations VP1 97R and 167G, was attenuated in both immunocompetent and immunosuppressed mice models, contradicting clinical observations highlighting an association between complicated neurological diseases and infections with HS-dependent strains. The general aim of this grant proposal is to decipher the mechanisms by which EV-A71 can invade the brain, and more specifically, how HS are involved in EV-A71 pathogenesis. To this end, we propose to apply a multi-level panel of approaches, including (i) in vitro studies to understand how mutations that enable the binding to HS make the virus more susceptible to host neutralizing antibodies, via electron microscopy and various biochemical methods; (ii) ex vivo characterization of HS expression in human biopsies and murine tissues and evaluation of the susceptibility of the different tissues to EV-A71 infection, using precision cut tissue slices (PCTS); (iii) in vivo analyses of the impact of HS present in the endothelial glycocalyx on viral dissemination and neurotropism in a model of infection of mice treated with heparinase; and (iv) identification of host factors differently modulating viral replication of HS-dependent and HS-independent EV-A71 variants, through the implementation of the first genome-wide CRISPR/Cas9 knockout screening in human gut organoids. With this project, we hope to extend the general knowledge on the involvement of HS in EV-A71 pathogenesis and to promote similar studies on the pathogenesis of other viruses using HS as receptor, such as SARS-CoV-2. As further benefits, our study will demonstrate the relevance of organoids and PCTS for the study of viral pathogenesis and the identification of antiviral targets.