Phenotypic characterization of two neuroinvasive Toscana virus strains clinically associated with self-limited and persistent infections in human neural cells and brain organoids.

Background: Toscana virus (TOSV), a sandfly-borne phlebovirus, is a frequent cause of viral meningitis and meningoencephalitis in Mediterranean countries during summer months. Clinical outcomes vary from self-limited disease to prolonged persistent infection lasting over 3 months, but the mechanisms underlying these differences remain unclear. In this study, we compared the pathobiological features of two clinical TOSV strains, SI-1812 (associated with self-limited disease) and INMI (associated with persistent infection), using human neural models. Methods: Three human neural systems, DBTRG.05MG glioblastoma cells, embryonic stem cell-derived neurons, and human brain organoids (hBOs), were infected with the two TOSV strains. Viral replication, cytopathic effect, innate immune response, and viral protein expression were assessed. Furthermore, whole-genome sequencing was performed to identify strain-specific differences. Results: The INMI strain showed reduced replication and cytopathic effect compared to SI-1812, supporting persistent infection in hBOs for up to 21 days. SI-1812 infection triggered a strong interferon-β response even at early stages of infection and low viral titers, whereas INMI induced a modest innate immune response in the early stages of infection, likely supporting its persistence in hBOs. The timing of viral NSs protein expression differred between the two viral strains suggesting distinct mechanisms in RIG-I activation and inflammatory response modulation. Conclusions: Distinct host-pathogen interactions underlie the divergent clinical courses of TOSV strains. The findings provide insights into mechanisms of viral persistence in the human brain and may guide future therapeutic strategies.