An Intracerebrally‐Infected Mouse Model of Enterovirus A71 Demonstrates Restricted Inter‐Neuronal Spread Within the Brain Parenchyma Despite Strong SCARB2 Expression

ABSTRACT Aims Enterovirus A71 (EV‐A71) can cause fatal encephalomyelitis, but the mechanisms of its spread within the central nervous system (CNS) remain unclear. This study aimed to investigate the pathways of EV‐A71 dissemination after direct intracerebral inoculation and to assess the role of the murine Scavenger Receptor Class B Member 2 (mSCARB2) receptor in this process. Methods A mouse‐adapted EV‐A71 strain (MAVS) was intracerebrally inoculated into the thalamus/hypothalamus or pons/medulla of 2‐week‐old ICR mice. Tissues were harvested and analysed by histopathology and viral titration at 24, 48 and 72 h post‐infection (hpi). The infectivity of MAVS was also tested on N1E115 mouse neuroblastoma cells. Results Viral antigens at the injection sites diminished over time, with restricted centrifugal inter‐neuronal spread. From 48 hpi, viral antigens increased in distant motor‐related neurons of the brainstem and spinal cord. There was a poor correlation between mSCARB2 expression and sites of infection; despite high mSCARB2 expression in the brain, spread was limited, while skeletal muscle, which lacks mSCARB2, showed severe infection. Direct infection of N1E115 cells was inefficient, but viral RNA transfection resulted in robust replication. Conclusions The findings suggest a circuitous dissemination route: viral leakage from the CNS leads to viraemia and peripheral muscle infection, followed by retrograde axonal transport back into the brainstem and spinal cord. This pathway appears to be the dominant mode of CNS invasion, independent of mSCARB2 distribution. Alternative receptor pathways likely play a critical role in EV‐A71 neuropathogenesis.