Recombinant B3 clade enterovirus D68 strains are efficiently rescued in 293T cells and infect human spinal cord organoids.

Enterovirus D68 (EV-D68) is associated with respiratory disease in children. Between 2014 and 2018, biennial EV-D68 outbreaks coincided with peaks of a polio-like neurologic condition called acute flaccid myelitis (AFM). We hypothesized that specific mutations within the currently circulating B3 clade of EV-D68 impacted neurovirulence. However, recovery of these strains from infectious clones by published methods proved unsuccessful. Therefore, we tested different cell lines, reagents, and conditions to enhance efficiency of recombinant (r)EV-D68 rescue. In this study, we present a tractable rescue system to define virulence determinants in B3 clade strains. Using this approach, we successfully rescued historic and contemporary rEV-D68 strains to high titer after limited cell culture passage. All strains in our study replicated efficiently in the parental RD cell line and in human respiratory epithelial cell lines, with BEAS-2B cells exhibiting greater permissivity than A549 cells. While B2 and B3 clade strains could infect the SH-SY5Y neuroblastoma cell line, the neurovirulent B2 clade strain rUSA/IL/2014-18952 was more dependent on neuron differentiation than B3 clade strains and replication was not sustained under multi-cycle growth conditions. Conversely, replication of rUSA/IL/2014-18952 was more efficient in human spinal cord organoids, which model the cellular heterogeneity of the spinal cord, while replication of B3 clade strains was more modest. Immunofluorescence staining confirmed infection, with viral antigen colocalized with neurons. These findings suggest shifting dynamics of EV-D68 neurovirulence and provide a critical platform for further assessment of viral determinants of neurovirulence in B3 clade strains. 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 neuropathogenesis is critical to understanding recent shifts in AFM prevalence; however, these investigations are limited to a small subset of infectious clones distantly related to currently circulating B3 clade strains. In this study, we leverage improved rescue strategies to characterize recombinant (r)EV-D68 strains from the dominant B3 clade. While all rEV-D68 replicate efficiently in the parental cell line and in human respiratory epithelial cells, B3 clade strains achieved greater titers in cultured neurons than a neurovirulent B2 clade strain, with less dependence on neuronal differentiation state. All B3 clade strains established infection in human spinal cord organoids, but replication varied between strains. Therefore, our study presents a tractable rescue system to begin to dissect viral determinants of shifting neurotropism within contemporary EV-D68 clades.