Exosome-mediated antiviral testing system and identification of Punicalagins and Anthocyanidins as promising antiviral agents against SARS-CoV-2

Emerging viral infections like SARS-CoV-2 (SCV-2) highlight the need for effective antiviral therapies. We aimed to establish a biosafety level 2 (BSL-2) compatible screening platform using an exosome-polyethylenimine based gene delivery matrix (EPM) to evaluate plant-derived polyphenols for their antiviral potential against SCV-2. EPM platform, facilitates the transfection of plasmid DNA encoding SCV-2 spike, nucleocapsid, and RNA-dependent RNA polymerase (RdRp) proteins into the HEK293T cells, enabling the screening of plant polyphenols for their antiviral activity. Punicalagins (PC), anthocyanidins (Anthos), delphinidin, and cyanidin, completely inhibited viral gene expression. These compounds protected Vero E6 cells from SCV-2 induced cytopathic effects with EC₅₀ values of 18.42 μM, 72.9 μM, and 72.54 μM, respectively. Docking studies revealed strong binding affinities of PC to SCV-2 spike, nucleocapsid, angiotensin-converting enzyme 2 (ACE2) and RdRp proteins (-7.1, -8.0, -10.3 and -10.3 kcal/mol, respectively). In vivo, PC provided dose-dependent protection in K18-hACE2 mice infected with SCV-2. Viral titers in nasal turbinates and lungs reduced by 30-40 % at 6 mg/kg and by 80 % at 12 mg/kg after 5 and 10 days of treatment. These findings support the utility of EPM as a screening platform and establish PC and Anthos as promising antiviral candidates against SCV-2 and other viruses utilizing similar entry mechanism.