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

Stefania Vogiatzia¹Gianni Gori Savellini²Chiara Terrosi²Marta Trevisan¹Gabriele Anichini²Letizia Rizzo²Giulia Alessandri²Emanuela Dal Molin¹Camilla Lucca¹Luisa Barzon¹Maria Grazia Cusi^2,3*

• ¹Universita degli Studi di Padova, Padua, Italy
• ²University of Siena, Siena, Italy
• ³Azienda Ospedaliera Universitaria Senese, Siena, Italy

Toscana virus (TOSV) is a sandfly-borne virus of the Phlebovirus genus, representing a common cause of meningitis and meningoencephalitis in Mediterranean countries during summer months. In this study, we investigated in vitro the pathobiological features of two Toscana virus (TOSV) strains, SI-1812 and INMI, both associated with meningoencephalitis in patients, but with different course: self-limited disease and persistent infection lasting over three months, respectively. Three human neural systems were used for infection modelling, DBTRG.05MG glioblastoma cells, embryonic stem cell-derived neurons, and human brain organoids (hBO). In both neurons and hBOs, the INMI strain replicated less efficiently and induced less cytopathic effect than the SI-1812 strain, allowing persistent infection in hBOs for up to 21 days titre. SI-1812 infection triggered a robust IFN-β response even at low viral titres, whereas INMI induced a modest innate immune response in the early stages of infection, likely supporting its persistence in hBOs. The timing of viral non-structural NSs protein expression and whole-genome sequencing revealed differences between the two TOSV strains, suggesting distinct mechanisms in RIG-I activation and inflammatory response modulation. Understanding these mechanisms offers new insights into TOSV pathogenesis and persistence, providing a foundation for future therapeutic strategies.