2.5. Antiviral Assay in Vero E6 and Calu-3 Cells

Vero E6 cells were seeded at a density of 1.3 × 10⁵ cells/well in 12-well plates and Calu-3 cells were seeded at a density of 2 × 10⁵ cells/well in 24-well plates. The following day, cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) 1 and treated with increasing concentrations of resveratrol and pterostilbene or the equivalent volumes of EtOH corresponding to the highest concentration of compound for 2 h at 37 °C. Infection was done in 250 µL DMEM 2% FBS (v/v) medium. After infection, virus inoculum was removed, cells were washed twice with plain DMEM media, and fresh DMEM 10% FBS (v/v) containing the compound or the equivalent volumes of EtOH was added after which incubation was continued. Cell supernatant was collected at 8 hpi, centrifuged to clarify from cell debris, and the viral titer was determined using plaque assay. For the durability assay, Vero E6 cells were infected with SARS-CoV-2 at MOI 0.01 and treated with 150 µM resveratrol or 60 µM pterostilbene or the equivalent volume of EtOH as indicated above. Supernatants were collected at 16, 24, 40, and 60 hpi and analyzed as above.

Vero E6 cells were seeded and infected as described above for the infectivity assay. At 8 hpi, cells were harvested and fixated with 4% PFA for 10 min at 4 °C. Cells were washed, centrifuged, and subsequently stained with mouse anti-SARS-CoV-2 NSP8 monoclonal antibody (Cat no: GTX632696, GeneTex, Irvine, CA, USA) at a 1:1000 dilution in FACS buffer for 30 min at 4 °C. Upon incubation, cells were again washed, centrifuged, and incubated with rabbit anti-mouse- Alexa647 (Thermofisher, Waltham, MA, USA) at a 1:1000 dilution for 30 min in the dark at 4 °C. Upon incubation, cells were washed, centrifuged, and resuspended in FACS buffer. Cells were analyzed with the NovoCyte Quanteon Flow Cytometer (Agilent Technologies, Santa Clara, CA, USA). Data were analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA).

Vero E6 cells were seeded and infected as described above for the infectivity assay. At the indicated times post-infection, cells were harvested and lysed with 350 µL RLT buffer 1% β-ME. Viral RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. cDNA synthesis from viral RNA was performed using Omniscript RT kit (Qiagen, Hilden, Germany) with the reverse primer CARATGTTAAASACACTATTAGCATA. Next, qPCR was performed by using the Qiagen Hot star taq polymerase kit in combination with the forward primer GTGARATGGTCATGTGTGGCG, reverse primer CARATGTTAAASACACTATTAGCATA, and RdRp_SARSr-P2 (5′FAM/3′BHQ) probe CAGGTGGAACCT CATCAGGAGATGC.

DNA amplification was done at 50 °C 120 s, 95 °C 900 s, and subsequently 40 cycles of 95 °C 15 s, 60 °C 60 s.