Mechanism of Neuroinvasion and Interferon Response in Human Choroid Plexus Organoids during Japanese Encephalitis Virus Infection
Background: Orthoflaviviruses are major arthropod-borne pathogens that cause significant disease burden globally. Some of these viruses can cause neurological infections, requiring a clear understanding of how they enter the central nervous system (CNS). The Choroid Plexus (ChP), a cerebrospinal fluid (CSF)-producing tissue which reside in brain ventricles can form blood-cerebrospinal fluid (CSF) barrier. This is one of the crucial peripheral-CNS interfaces acting as a physical defense against neuroinvasive pathogens. While this tissue is increasingly recognized as a potential entry site for hematogenous pathogens, the precise mechanism by which orthoflaviviruses exploit the blood-CSF barrier and the corresponding intrinsic immune response in an immunocompetent human model remain critically undefined.  Methods: This study aims to investigate the potential of ChP to serve as a neuroinvasive site for orthoflaviviruses. ChP organoids derived from human inducible pluripotent stem cells were established and determined for susceptibility to a panel of orthoflaviviruses, namely Japanese encephalitis virus (JEV), Zika virus (ZIKV) and Dengue virus (DENV). Transcriptional responses and barrier permeability were assessed in the ChPOs infected with JEV.   Results: ChPOs exhibited differential susceptibility to orthoflaviviruses, with the pathogenic JEV strain being the most efficient. Transcriptomic analysis confirmed a robust interferon (IFN) response to JEV infection. Functional studies further showed that type-I IFNs dominate the antiviral defense and is essential for protection, overriding the epithelial-restricted type-III IFNs. Critically, despite productive JEV release into the CSF-like fluid within the ChP lumen, selective barrier integrity was maintained, confirming a non-destructive transcellular neuroinvasion mechanism across the blood-CSF barrier.  Conclusion: This study utilizes an immunocompetent human ChPO model to define the susceptibility, cellular response, and transcellular neuroinvasion mechanism of medically important orthoflaviviruses. This mechanistic understanding of how these viruses interact with the blood-CSF barrier is essential for the rational design of effective prevention and therapeutic strategies against neurotropic orthoflaviviruses entering the CNS.