Enterovirus D68 VP1 and VP3 determine neurotropism in human spinal cord organoids

Enterovirus D68 (EV-D68) is a non-polio enterovirus that can cause a polio-like paralysis condition, acute flaccid myelitis (AFM). EV-D68 associated AFM cases waned in the US after 2018 and the reasons for this are unknown. It has recently been demonstrated that EV-D68 containing point mutations in viral structural proteins VP1 and VP3 resulted in decreased paralysis in different neonatal mouse models. However, phenotypes of these mutations in a human multicellular central nervous system (CNS) model are unknown. We hypothesized that mutations in VP1 and VP3 will similarly direct neurotropism in human spinal cord organoids (hSCO). To investigate this, we recreated viruses with mutations in VP3 (I88V) or VP1 (L1I/N2D/T98A/E283K or L1P/V148A/K282R) and infected hSCOs. We found that VP3 I88V and VP1 L1I/N2D/T98A/E283K resulted in decreased titer and viral protein staining, consistent with attenuated neurovirulence in previously published murine models. When these mutations were combined, their effects on neurotropism were not additive. Sequence analysis of recently circulating EV-D68 strains reveals that VP3 I88 and VP1 E283 have remained the dominant amino acid residues since 2014, whereas VP1 sites 1, 2, and 98 have higher population diversity, indicating that these residues may be contributing to newly reduced neurovirulence after 2018.