Deep well plate

Tick samples were homogenized in 600 μl of pre-cooled PBS using the TissueLyser system (Qiagen, Hilden, Germany). After a short centrifugation step, 400 μl of the supernatant were transferred to a Deepwell plate (Eppendorf, Hamburg, Germany), 60 μl of glycerin were added per well and the plates stored at -80 °C for further use. 100 μl of the supernatant were used for nucleic acid extraction.

The virucidal activity of disinfectants was assessed by adding 200 µl virus/soil mixture to 800 µl of the diluted disinfectants mentioned above achieving 70 and 40% final concentrations for ethanol, 0.5 and 0.05% for NaOCl, and 0.2 and 0.02% for PAA, and incubating for 30 s. Subsequently, 50 µl of the virucidal test was inoculated directly into 25 cm² flasks to determine residual infectivity after treatment. Another 50 µl were added to 450 µl of cell culture media (to dilute ethanol to a non-working concentration) or cell culture media supplemented with neutralizer (1% sodium thiosulfate to neutralize NaOCl) or cell culture media supplemented with both neutralizer (0.25% sodium thiosulfate) and additional 25 mM of HEPES (to neutralize and buffer PAA; Gibco, Fischer Scientific, Reinach, Switzerland) and diluted tenfold in a deep well plate (Eppendorf, Schönenbuch, Switzerland). 150 µl of each dilution was then inoculated on Vero E6 cells (median passage number 42) in a 24-well plate (Techno Plastic Products, TPP, Trasadingen, Switzerland) and incubated at RT for 1 h. After incubation, 1 ml of overlay media, 2% MEM + 1% methylcellulose 90 H G 4000 cP (Sigma Aldrich, Buchs, Switzerland), was added on top and cells incubated at 37 °C without CO₂ for 7 days.