Modelling Enterovirus-D68 Infection in hESC-Derived Brain Organoids: Insights into Acute Flaccid Paralysis

Abstract

Background: As poliovirus nears eradication owing to worldwide vaccination efforts, focus has shifted to Enterovirus D68 (EV-D68), which induce polio-like paralysis illness, now referred to as acute flaccid paralysis (AFM). Although EV-D68 has become a major cause of AFM, the exact processes behind its ability to infect nerve cells, damaging human brain tissues, are still not fully understood. Methods: Human brain organoids derived from stem cells were produced and utilized for modeling EV-D68 infectivity. EV-D68 were cultured in A549 cells, was used to infect organoids. Viral kinetics assay was measured using RT-qPCR. Immunofluorescence was conducted for pluripotency markers, EV-D68 viral protein 1, double stranded RNA (dsRNA), GFAP, NEUN and the entry receptor MFSD6 using organoids sections post-transfection. Infected organoids morphology was evaluated up to 30 days post-infection (dpi). A nine-gene panel (CX3CR1, IFNa2, IFNb1, CXCL10, IFNy1, IFNy2/3, IkBa, MCP1, NFkB) was longitudinally profiled in infected organoids at 1, 3, 6, and 30 dpi. Results: Generated brain organoids showed NEUN-positive neurons and GFAP-positive astrocytes, confirming the present of brain neural cells in these organoids. We also found MFSD6 positive cells in these organoids. Immunostaining of dsRNA further confirmed the EV-D68 induced infection in these organoids, with intracellular viral RNA reaching its peak at dpi-6 and extracellular viral shedding remained raised from dpi-6 to dpi-10 before declining by dp-30. VP1 and dsRNA were identified in infected organoids; dsRNA co-localized with GFAP, indicating possible active replication in infected astrocytes. Infected organoids exhibited localised membrane damage and structural distorted at dpi-30. Gene expression analysis further showed inflammation enrichments in infected organoids characterised by predominant type I interferon induction and transient upregulation of CXCL10/MCP1, IkBa, IFNb1, IFNy2/3, CX3CR1, and IFNa2. Conclusion: Brain organoids captured aspects of EV-D68 CNS infection. Our data further support the utility of human brain organoid platform for dissecting the mechanism of EV-D68 neurotropism and innate immunopathology associated with AFM.

Date of Award	2024
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences