Avicel

One hour after infecting the cell monolayers with 30–50 plaque forming units of the virus in 1 ml of maintenance medium without trypsin, we removed the virus inoculum, covered the cells with 3 ml of the different overlay media and incubated cultures at 35°C in 5% CO₂ atmosphere. In the case of MC and Avicel overlays, care was taken not to disturb the plates during the incubation period in order to avoid formation of non-even plaques. After three days of incubation, we removed the overlays and fixed the cells. Agar overlay was removed using metal spatula; MC, Avicel, and liquid overlays were removed by suction. The cells were fixed with 4% paraformaldehyde solution in MEM for 30 min at 4°C and washed with PBS. All subsequent treatments of the cells were performed at room temperature. We permeabilized the cells and simultaneously blocked residual aldehyde groups by incubating the cells for 10–20 min with 1 ml/well of solution containing 0.5 % Triton-X-100 and 20 mM glycine in PBS. We immuno-stained virus-infected cells by incubating for 1 hr with monoclonal antibodies specific for the influenza A virus nucleoprotein (kindly provided by Dr. Alexander Klimov at Centers for Disease Control, USA) followed by 1 hr incubation with peroxidase-labeled anti-mouse antibodies (DAKO, Denmark) and 30 min incubation with precipitate-forming peroxidase substrates. Solution of 10% normal horse serum and 0.05% Tween-80 in PBS was used for the preparation of working dilutions of immuno-reagents. We washed the cells after the primary and secondary antibodies by incubating them three times for 3–5 min with 0.05% Tween-80 in PBS. As peroxidase substrates, we employed either ready to use True Blue™ (KPL) or solution of aminoethylcarbazole (AEC, Sigma) (0.4 mg/ml) prepared in 0.05 M sodium acetate buffer, pH 5.5 and containing 0.03% H₂O₂. Stained plates were washed with tap water to stop the reaction and dried. In the case of True Blue staining, which is relatively unstable in water solutions, plates were dried inverted in order to minimize bleaching. Stained plates were scanned on a flat bed scanner and the data were acquired by Adobe Photoshop 7.0 software.
As an alternative to immuno-staining, in some experiments we revealed plaques as areas of destroyed cells. To this end, after removing the overlays, we stained the cells with 1% crystal violet solution in 20% methanol in water.

We infected MDCK or MDCK-SIAT1 cell monolayers in 96-well plates with 50 μl/well of serial dilutions of the virus in the maintenance medium without trypsin. After 1 h incubation at 35°C in 5% CO2, we removed the virus, added 100 μl/well of either MC or Avicel overlay media, and incubated the cells for further 24 h to allow plaque formation. Fixation and immuno-staining were performed as described above for 6-well plates but using 50 μl of reagents per well.

Vero E6 cells and HuH7 cells were grown as described previously [35 (link)]. SARS-CoV-2 isolate Australia/VIC01/2020 (GenBank ID: MT007544.1 [34 (link)]) was derived from a positively testing nasopharyngeal swab in Melbourne, Australia, and was propagated twice in Vero/hSLAM cells, before being shared with other laboratories. In Leiden, the virus was passaged two more times at low m.o.i. in Vero E6 cells to obtain a working stock (p2 stock) that was used in all experiments. SARS-CoV isolate Frankfurt 1 [36 (link)] was used to compare growth kinetics and other features with SARS-CoV-2. Infection of Vero E6 cells was carried out in PBS containing 50 µg ml⁻¹ DEAE-dextran and 2 % FCS (Bodinco). The inoculum was added to the cells for 1 h at 37 °C, after which cells were washed twice with PBS and maintained in Eagle’s minimal essential medium (EMEM; Lonza) with 2 % FCS, 2 mM l-glutamine (PAA) and antibiotics (Sigma). Viral titres were determined by plaque assay in Vero E6 cells as described previously [37 (link)]. For plaque picking, plaque assays were performed using our p1 stock, while using an overlay containing 1 % of agarose instead of Avicel (RC-581; FMC Biopolymer). Following neutral red staining, small and large plaques were picked and used to inoculate a 10 cm² dish of Vero E6 cells containing 2 ml of EMEM-2%FCS medium, yielding p1 virus. After 48 h, 200 µl of the culture supernatant was used to infect the next dish of cells (p2), a step that was repeated one more time to obtain p3 virus. All work with live SARS-CoV and SARS-CoV-2 was performed in biosafety laboratory level 3 facilities at Leiden University Medical Center, the Netherlands.

Neutralization assay was conducted as described previously⁹ . Briefly, sera from vaccinated individuals were heat treated for 30 min at 56 °C. Sixfold serially diluted plasma, from 1:10 to 1:2430 were incubated with SARS-CoV-2 lineage A(USA-WA1/2020) for 1 h at 37 °C. The mixture was subsequently incubated with TMPRSS2-VeroE6 in a 12-well plate for 1 h, for adsorption. Then, cells were overlayed with MEM-supplemented NaHCO₃, 4% FBS 0.6% Avicel mixture. Plaques were resolved at 40 h post-infection by fixing in 10% formaldehyde for 1 h followed by staining in 0.5% crystal violet. All experiments were performed in parallel with baseline control sera, in an established viral concentration to generate 60–120 plaques/well.