2.7. Antiviral activity assay for SARS-CoV-2

Vero E6 cells at a density of 50,000 cells/well were cultured in 100 μl of MEM supplemented with 10% FBS, 1% GlutaMAX, and 1% penicillin/streptomycin antibiotics on a 96-well flat-bottomed microtiter plate for 24 h at 37°C. The following day, SARS-CoV-2 was pre-treated with 1% v/v of Salix bark extract of clone 5 or P-16 by preparing a virus–extract mix in 2% MEM and incubating it for 1 h at 34°C. Handling of the virus was carried out at the BSL-3 facility at the University of Helsinki, Finland. The virus titer in the virus–extract mix was 20 PFU/ml. After the incubation, the virus–extract mix was added to cells (MOI-0.00002) for 2 h at 34°C. Following the incubation, the cells were aspirated, fresh media was added, and they were incubated for 3 days at 34°C. Finally, the supernatant solution from the cells was collected and transferred to a new 96-microtiter plate for the extraction of viral RNA. The extraction was done using a Chemagic Viral RNA/DNA Kit (PerkinElmer, Turku, Finland). Once the viral RNA was extracted, we performed a real-time reverse transcriptase polymerase chain reaction (RT-qPCR) to qualitatively detect viral nucleic acid. This was performed using a SARS-CoV-2 RT-qPCR reagent kit (PerkinElmer, Turku, Finland). To compare the relative amounts of RNA in samples, we make use of the fact that 1 difference in Cq (cycle quantification) value means ~2 × difference in RNA amount. An equation (RNA difference = 0.9646e^0.6948x, x is the difference in the Cq values between the mean of test samples and the mean of virus control] was deduced by using Cq differences down from 10 (10 cycle difference meaning ~1000 difference in relative RNA amount). This was used in our calculations to gain a value for RNA difference, of which a log value was then calculated to describe the difference in virus amounts.