Antiviral Activity of Microbial Metabolites Monensin and Brefeldin A Against Toscana Virus: In Vitro Evaluation and Mechanistic Insights

Toscana virus (TOSV), a negative-sense RNA phlebovirus transmitted by <i>Phlebotomus</i> sandflies and endemic in Mediterranean regions, is an emerging pathogen capable of causing diseases ranging from mild febrile illness to severe central nervous system involvement. With no licensed vaccines or specific antiviral therapies available, the identification of novel therapeutic approaches is urgently needed. Microbial secondary metabolites have recently attracted attention for their broad-spectrum antiviral activities. Among them, monensin and brefeldin A have shown antiviral efficacy against a variety of viruses, often by disrupting viral protein trafficking and inducing Golgi-associated stress responses. However, their potential activity in the context of TOSV infection has not yet been explored. This study aimed to evaluate the in vitro antiviral activity of monensin and brefeldin A against TOSV and to gain mechanistic insights into their effects at the cellular level. Vero cells were infected with TOSV and treated with monensin (1.5-25 µM) or brefeldin A (10.9-175 nM) at different time points (4, 6, 12, 24 h). Cytotoxicity was assessed using MTT and hemolysis assays. Antiviral activity was measured via plaque reduction assays and quantitative real-time PCR targeting the viral <i>L</i> gene. Western blot analysis was performed to assess TFE3 expression, a transcription factor associated with the Golgi stress response. Monensin exhibited rapid antiviral activity, achieving IC₅₀ values of 2.7 µM and 2.5 µM at 4 and 6 h post-treatment, respectively, with dose-dependent suppression of viral <i>L</i> gene expression. Brefeldin A displayed a delayed effect, with maximal inhibition after 12 h (IC₅₀ = 66.9 nM). Monensin treatment induced a concentration-dependent upregulation of TFE3, while brefeldin A caused only a modest increase, suggesting differential activation of the Golgi stress response during TOSV infection. These findings support the potential of microbial metabolites as therapeutic candidates for emerging arboviral infections in the Mediterranean region.