Lipid Droplets mediated Japanese Encephalitis Virus small Extracellular Vesicle Release

Lipid droplets (LDs) and small Extracellular Vesicles (sEVs) are classically known for lipid metabolism and intercellular communication, respectively. Here, we reveal a mechanistic connection between LD dynamics and sEV-mediated non-lytic release of Japanese Encephalitis Virus (JEV) from neuronal cells. Using Neuro2A, SH-SY5Y, N9 microglia, and primary cortical neurons, we show that JEV is packaged within sEVs (∼200 nm) through an ESCRT-independent, neutral sphingomyelinase 2 (nSMase2)/ ceramide-dependent pathway. Virions inside sEVs display a higher JEV Premature Membrane/Membrane protein (PrM/M) ratio compared to those released via the conventional secretory pathway. Although containing a higher proportion of premature virions than mature ones, sEV-associated JEV virions gain an evolutionary advantage by evading immune detection and delivering multiple virions to recipient cells, thereby increasing overall infection efficiency. Temporal profiling showed early cytoplasmic LD enrichment (from 6 hpi), followed by a surge in sEV release from 14 hpi, suggesting sequential roles for LDs and sEVs. nSMase2 inhibition decreased sEV-mediated egress without affecting viral replication, but increased cytoplasmic LD abundance, consistent with LD underutilization in multivesicular bodies (MVB) biogenesis. Our findings identify LDs as facilitators of MVB formation and nSMase2 as a key driver of sEV-mediated viral exit, revealing parallel yet coordinated pathways in JEV’s stealthy egress.