Single-cell characterization of enterovirus infection of spinal cord organoids

The mechanisms by which enteroviruses, particularly enterovirus D-68 (EV-D68) and enterovirus A-71 (EV-A71), contribute to acute flaccid myelitis (AFM), a severe neurological condition characterized by sudden muscle weakness and paralysis, remain poorly understood. To investigate the cellular tropism and infection dynamics of these enteroviruses, we utilized human spinal cord organoids (hSCOs) derived from induced pluripotent stem cells (iPSCs). We performed single-cell RNA sequencing (scRNA-seq) to profile the cellular composition of hSCOs and identify infected cell types post-infection. We found that hSCOs exhibit a diverse cellular landscape, including neurons, astrocytes, oligodendrocyte progenitor cells (OPCs), and multipotent glial progenitor cells (mGPCs), with astrocytes predominant in earlier developmental stages and neurons more abundant in later stages. Upon infection with two EV-D68 strains (US/IL/14-18952 and US/CO/18-23089) and one EVA71 strain (Tainan/4643/1998), we observed distinct viral tropism. EV-D68-18952 showed a significant increase in infected neurons, while EV-D68-23089 exhibited higher infection rates in cycling astrocytes and OPCs. In contrast, EVA71 demonstrated a broader tropism, with a statistically significant increase in infected mGPCs, suggesting enhanced infection efficiency across multiple cell types. These findings provide novel insights into the cell-type specificity of EV-D68 and EVA71 in the spinal cord, offering a deeper understanding of potential mechanisms underlying AFM pathogenesis. Understanding the dynamics of infection at single-cell resolution will inform future therapeutic strategies aimed at mitigating the neurological impact of enteroviral infections. Overall design: Human spinal cord organoids (hSCOs) were infected in pools of 12 organoids each and inoculated with virus at 105 PFU/pool. After 1 hour of incubation at room temperature, hSCOs were washed 3X with PBS and moved to new wells before incubation with fresh medium. No further media changes were performed for the rest of the experiment. The EV-D68 infected pool was incubated at 33oC and EV-A71 at 37oC for 48 hours post infection (hpi). After 48hpi, the pools were dissociated to a single-cell suspension with Accumax (Invitrogen). They were incubated for 15 minutes in the water bath at 37?, gently mixed, and then proceeded with the proposed 10X Genomics protocol (Cell Preparation Guide - CG00053 Rev C). Once the cell's concentration is achieved the cells are moved to analyze using scRNA-seq.