Inhaled and systemic heparin as a repurposed direct antiviral drug for prevention and treatment of COVID-19

Heparin inhibits SARS-CoV-2 plaque formation. a) 800,000 Vero E6 cells were seeded in a 12-well plate the day before infection to result in a 100% confluent cell monolayer. The next day, medium was removed, cells were washed once with PBS and medium containing heparin (Sigma-Aldrich) was added. Cells were then inoculated with SARS-CoV-2 isolate BetaCoV/Netherlands/01/NL/2020 (#010V-03903; European virus archive – global) in 500 μl and incubated for 2 h at 37°C with shaking every 15 to 30 min. Next, cells were overlaid with 1.5 ml of 0.8% Avicel RC-581 (FMC Corporation) in medium containing heparin and incubated for 3 days. Cells were fixed, stained with crystal violet and imaged. b) Virus-induced plaques shown in Fig 1a were counted. c) Cytopathic effect (CPE) was quantified using a custom ImageJ macro as the percentage of non-stained area within one well. Raw images were converted to greyscale, regions of interest (= one well) defined, automatic thresholding (Minimum algorithm) applied and total/white pixel areas calculated. Values represent means of two technical replicates ± sd.