Non-Polio Enterovirus A71 and D68 Infection of Human Neuromuscular Organoids Reveals Distinct Mechanisms of Neuromuscular Impairment

Enterovirus A71 (EV-A71) and enterovirus D68 (EV-D68) are recognised as causative agents of severe neurological complications, including acute flaccid myelitis (AFM). However, the molecular mechanisms underlying the neurovirulence and effects on neuromuscular integrity remain poorly understood. Here, we employed human induced pluripotent stem cell-derived neuromuscular organoids (NMOs) to investigate the cellular tropism and pathogenic effects of EV-A71 and EV-D68 in a human-relevant context. Both viruses infected neuronal populations within NMOs, with EV-A71 exhibiting higher levels of viral replication than EV-D68. Transcriptomic analysis revealed downregulation of neuronal and muscular gene networks following infection. EV-A71 preferentially suppressed neuronal pathways, while both viruses exerted comparable effects on muscle-associated gene expression. These transcriptional changes highlighted alterations in pathways governing neuronal and muscle function and communication, prompting examination of synaptic vesicle machinery components. At the protein level, both viruses were associated with sporadic cleavage of the neuronal SNARE protein synaptosomal-associated protein 25 (SNAP25). In addition, infection with either virus increased cleaved caspase-3 levels, consistent with activation of apoptotic signalling. Together, these findings indicate virus-specific downstream effects and establish NMOs as a robust platform for dissecting enterovirus–host interactions relevant to AFM.