Enterovirus D68 B3 clade strains are efficiently recovered from cDNA infectious clones in 293T cells and infect human spinal cord organoids.

Enterovirus D68 (EV-D68) is associated with respiratory disease in children. Between 2014 and 2018, EV-D68 infection was linked to peaks of a polio-like neurologic condition called acute flaccid myelitis (AFM), but currently circulating strains are not. We hypothesized that specific mutations within B3 clade strains impact neurovirulence. Few studies have investigated B3 clade strains, and none have recovered these viruses from cDNA infectious clones to understand how novel mutations in B3 clade strains impact neurovirulence. In this study, we examine the neurovirulence of recovered (r) B3 clade strains in relevant human cell culture and 3D organoid models. We demonstrate that rB3 clade strains replicate efficiently in the permissive RD cell line and in human respiratory epithelial cell lines, with BEAS-2B cells exhibiting greater permissivity than A549 cells. rB3 clade strains infect neural cells, including SH-SY5Y cells, a neuroblastoma cell line, and human spinal cord organoids, which model the cellular heterogeneity of the spinal cord. Immunofluorescence staining confirmed infection, with viral antigen colocalized with neurons. These findings are consistent with previous studies from our laboratory and others on the neurovirulence of clinical isolates of B3 clade strains. Overall, we provide a critical platform for rigorous interrogation of viral determinants of neurovirulence within the B3 clade.IMPORTANCEEnterovirus D68 (EV-D68) can cause acute flaccid myelitis (AFM), a debilitating neurological condition of the spinal cord in children. Identifying viral determinants of EV-D68 neurovirulence is critical to understanding recent shifts in AFM prevalence; however, these investigations have been limited to a small subset of infectious clones distantly related to currently circulating B3 clade strains. In this study, we examine characteristics of recovered (r)EV-D68 strains from the dominant B3 clade. While all rEV-D68 replicate efficiently in a permissive cell line and in human respiratory epithelial cells, titers varied between strains in cultured neuroblastoma cells. Similarly, all B3 clade strains established infection in human spinal cord organoids, but replication varied between strains. Our study provides an essential platform for investigation into viral mutations within relevant B3 clade strains driving shifts in AFM prevalence.