Dried leaves of Artemisia plants grown in different countries and in different years were obtained as donations (see Addoitional file 1). Covid-Organics was purchased in Madagascar.
Distilled water (10 mL) was heated to 90 °C in an Erlenmeyer flask. Dried plant material (1 g) was added to the solvent and kept for two minutes at 90 °C, then 20 min at room temperature. The mixture was filtered using filter paper and solid material washed with fresh distilled water (20 × 2 mL). The solvent was removed by rotary evaporation and the solid material was stored at −10 °C prior to sample preparation. An ethanolic extract of A. annua var. CPQBA 1 was prepared by treating 50 g of dried leaves of A. annua with 250 mL of ethanol at 50 °C for 200 min.
Aqueous Extract: Dried extract (~ 135 mg) was warmed to room temperature before dimethylsulfoxide (DMSO, 3 mL) was added and the mixture heated (40 °C) to ensure solvation. The solution was filtered using a syringe filter and stored in a snap-close vial at −20 °C prior to use. Final concentration: ~ 45 mg/mL.
Dried extract (61 mg) was warmed to room temperature before dimethylsulfoxide (DMSO, 3 mL) was added and the mixture heated (40 °C) to ensure solvation. The solution was filtered using a syringe filter and stored in a snap-close vial at −20 °C prior to use. Final concentration: ~ 20 mg/mL.
African green monkey kidney VeroE6 cells (ATCC CRL-1586) and Crandell-Rees Feline Kidney (CRFK, ATCC CCL-94) cells were maintained at 37 °C with 5% CO2 in Minimum Essential Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (PAN Biotech), 100 IU/mL penicillin G and 100 µg/mL streptomycin (Carl Roth, Karlsruhe, Germany).
The SARS-CoV-2 BavPat 1 isolate (SARS-CoV-2/human/Germany/BavPat 1/2020) was provided by Dr. Daniela Niemeyer and Dr. Christian Drosten (Charité, Berlin, Germany) and obtained from an outbreak in Munich, Germany, in February 2020 (BetaCoV/Germany/BavPat1/2020). Feline coronavirus (ATCC VR-989, WSU 79-1683) was propagated and titrated on CRFK cells .
Plaque reduction antiviral assay
Tenfold dilutions of the compounds described above were prepared in cell culture medium. To determine the effect of the compounds, dilutions were incubated with 100 plaque forming units (PFU) of FCoV or SARS-CoV-2 for one hour at 37 °C. The compound-virus mix was incubated with CRFK or VeroE6 cells for 45 min at room temperature respectively. The cells were washed with PBS once, overlayed with 1.3% sample-free methylcellulose containing medium and plaque formation was assessed two days post infection. Plaques of FCoV were stained with specific antibodies (primary antibody: mouse anti-feline coronavirus nucleocapsid protein monoclonal antibody at 1 µg/mL, Bio-Rad; secondary antibody: Alexa 488-labeled goat anti-mouse IgG at a 1 µg/mL; ThermoFisher) and counted manually by fluorescence microscopy (Axio observer, Zeiss). SARS-CoV-2 plaques were stained using crystal violet.
Cell viability assays in CRFK and VeroE6 cells
To evaluate cytotoxic effects of the tested extracts, compounds and diluent (DMSO), cell viability was studied using Cell Counting Kit-8 (CCK8, Merck, Germany). The protocol was the same for CRFK and VeroE6 cells: cells were seeded at 10,000 cells per well of flat bottom 96-well plates (Thermo Fisher Scientific, Roskilde, Denmark). The next day, medium was exchanged containing specified concentrations of the samples. Each concentration or dilution was tested in three replicates; at least six nontreated control wells were included in the assay. After 45 min or 24 h of incubation at 37 °C and 5% CO2, CCK8 Reagent (10 µL) was added per well and plates were incubated for 1 h at 37 °C, prior to recording absorbance at 450 nm using a FLUOstar OPTIMA 96-well plate reader (BMG LABTECH, Offenburg, Germany). The viability of CRFK cells was only tested after 45 min incubation. Absorbance recorded in each well was related to the average absorbance of nontreated control wells to calculate the percentage of cell viability. 1% SDS was used as negative control. Sigmoidal dose response curves were fitted, and median cytotoxic concentration (CC50) values were calculated with GraphPad Prism 8.0.0.