All SARS-CoV-2 experiments were performed in a laboratory of biosafety level 3.
SARS-CoV-2 isolates from respiratory specimens of four different patients: Alpha variants SARS-CoV-2/hu/Germany/Jena-vi005159/2020 [isolate 5159] (MW633322.1), SARS-CoV-2/hu/Germany/Jena-vi005587/2020 [isolate 5587] (MW633323.1) and SARS-CoV-2/hu/Germany/Jenavi005588/2020 [isolate 5588] (MW633324.1) [44 (link)] and Delta variant SARS-CoV-2/hu/Germany/Jena-0114749/2021 [isolate 4749] (ON650061) were used for infection experiments. The isolate 5159 belongs to the B.1-lineage, the isolates 5587 and 5588 to the B.55-lineage and the isolate 4749 to the AY.126-lineage.
For plaque assay, Vero-76 cells were seeded in 6-well plates and infected with serial dilutions of the supernatants in PBS supplemented with 1 mM MgCl2, 0.9 mM CaCl2, 0.2% BSA and 100 U mL−1 Pen/Strep (Sigma-Aldrich, Taufkirchen, Germany) for 90 min at 37 °C. After aspiration, the cells were incubated with 2 mL MEM supplemented with 0.9% agar (Oxoid, Wesel, Germany), 0.01% DEAE-Dextran (Pharmacia Biotech, Freiburg im Breisgau, Germany), 0.2% BSA and 0.2% NaHCO3 (Sigma-Aldrich, Taufkirchen, Germany) at 37 °C and 5% CO2 for 3 days. To visualize the plaques, a staining with neutral red solution (Sigma-Aldrich, Taufkirchen, Germany) in PBS was performed and the number of infectious particles (pfu mL−1) was determined by counting.
SARS-CoV-2 isolates from respiratory specimens of four different patients: Alpha variants SARS-CoV-2/hu/Germany/Jena-vi005159/2020 [isolate 5159] (MW633322.1), SARS-CoV-2/hu/Germany/Jena-vi005587/2020 [isolate 5587] (MW633323.1) and SARS-CoV-2/hu/Germany/Jenavi005588/2020 [isolate 5588] (MW633324.1) [44 (link)] and Delta variant SARS-CoV-2/hu/Germany/Jena-0114749/2021 [isolate 4749] (ON650061) were used for infection experiments. The isolate 5159 belongs to the B.1-lineage, the isolates 5587 and 5588 to the B.55-lineage and the isolate 4749 to the AY.126-lineage.
For plaque assay, Vero-76 cells were seeded in 6-well plates and infected with serial dilutions of the supernatants in PBS supplemented with 1 mM MgCl2, 0.9 mM CaCl2, 0.2% BSA and 100 U mL−1 Pen/Strep (Sigma-Aldrich, Taufkirchen, Germany) for 90 min at 37 °C. After aspiration, the cells were incubated with 2 mL MEM supplemented with 0.9% agar (Oxoid, Wesel, Germany), 0.01% DEAE-Dextran (Pharmacia Biotech, Freiburg im Breisgau, Germany), 0.2% BSA and 0.2% NaHCO3 (Sigma-Aldrich, Taufkirchen, Germany) at 37 °C and 5% CO2 for 3 days. To visualize the plaques, a staining with neutral red solution (Sigma-Aldrich, Taufkirchen, Germany) in PBS was performed and the number of infectious particles (pfu mL−1) was determined by counting.
Free full text: Click here
