Antiviral Mechanisms of Justicidin B and Tuberculatin from <i>Phyllanthus brasiliensis</i> against Oropouche Virus Infection

Oropouche virus (OROV) has emerged as a growing public health concern in Latin America, with an unprecedented surge in Oropouche fever cases since 2023. Previously considered a sporadic and neglected arboviral infection, Oropouche fever now represents one of the most rapidly expanding arboviral threats in the region. Despite its increasing incidence, no approved vaccines or antiviral therapies are available, highlighting the urgent need for effective interventions. Natural compounds, particularly those derived from Phyllanthus brasiliensis, have shown promising antiviral properties against several RNA viruses. This study investigated the anti-OROV activity and mechanism of action of two lignans isolated from P. brasiliensis, justicidin B and tuberculatin. Cytotoxicity was evaluated by MTT assay, and antiviral efficacy was quantified by plaque-forming assays. Mechanistic analyses included virucidal assays and evaluations at distinct stages of the viral replication cycle. Both lignans exhibited potent anti-OROV activity, with selectivity indices of 2307 (justicidin B) and 1153 (tuberculatin). Chloroquine was used as a positive control and was found to be 47.7 and 30.1 times less potent than justicidin B and tuberculatin, respectively. Treatments initiated within 6 hpi reduced viral titers by >99.99% (>4log10), and by ∼2log10 when added at 12 hpi. These results demonstrate that justicidin B and tuberculatin act mainly in intracellular processes occurring after viral entry. Molecular coupling analysis corroborated these findings, as both demonstrated a propensity to interact with residues of the OROV Gc glycoprotein, highlighting their strong therapeutic potential as lead candidates for antiviral development against Oropouche fever.