Infection of Enterovirus D68 clinical isolates in a neonatal mouse model: viral kinetics, tissue tropism and histopathology

Enterovirus D68 (EV-D68), a primary causative agent of respiratory illness, is increasingly associated with neurological complications, including acute flaccid myelitis (AFM). While clinical AFM cases are frequently linked to clade B, the neurovirulent potential across other clades remains under-characterized. In this study, we characterized five EV-D68 clinical isolates from clades A and B using in vitro assays and a neonatal mouse model. We identified a subclade A2 isolate, 12MYKL1607, which exhibited efficient replication and superior binding in rhabdomyosarcoma and neuroblastoma cells compared to other clades. In one-day-old mice, subcutaneous infection with 12MYKL1607 (A2) resulted in consistent AFM-like paralysis by 4 to 5 days post-infection. Tissue viral load and histopathological analysis identified that brown adipose tissue may serve as an initial replication site, followed by robust replication in limb skeletal muscle. The resulting high viral load likely facilitates entry into the spinal cord via the neuromuscular junction, with occasional progression to the brainstem. Immunohistochemical analysis confirmed the localization of viral antigens within the motor neurons of the spinal cord anterior horns, correlating with the onset of limb paralysis. We speculate that the virus travels along somatic motor nerves via retrograde axonal transport, consistent with other neurotropic enteroviruses. Our findings demonstrate that neurovirulence is not clade-specific, as contemporary clade A2 isolates closely recapitulate human AFM. This experimental model provides a robust platform for evaluating potential prophylaxis or treatment interventions, and highlights the necessity for broad molecular surveillance beyond clade B to mitigate the ongoing threat of enteroviral neurological disease.