Determination of daily reproduction numbers of SARS-CoV2 based on death cases suggests more rapid initial spread in Italy and the United States

Armin Ensser; Klaus Ueberla

Preparation of the IS cell line. CEM-SS cells were infected with pseudotyped virus (described above) by spinoculation. Briefly, 2 ϫ 10 7 CEM-SS cells were mixed with 10 ml of viral stock (45 ng of p24 Gag /ml) and placed into all six wells of a flat-bottom six-well tissue culture plate (1.6 ml of suspension per well). The plates were sealed in plastic bags and centrifuged in microplate carriers at 1,200 ϫ g for 2 h at 25°C. Cells were collected and washed once with 50 ml of ice-cold culture medium (RPMI 1640 with 10% heat-inactivated fetal calf serum, 10 mM HEPES [pH 7.4], 1ϫ penicillin-streptomycin). Infected cells were then cultured for 2 days before the addition of 300 g of G418 (Geneticin; Invitrogen Life Technologies)/ml. Upon initial addition of G418, approximately 50% of the cells died. The cells were then selected with G418 for an additional 4 weeks. After selection, cells were lysed at a density of 10 6 /ml in the following buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.2 mM CaCl 2 , 0.001% sodium dodecyl sulfate, 0.001% Triton X-100, 1 mg of proteinase K (Sigma)/ml. Lysates were digested overnight at 58°C, and then the protease was heat inactivated for 15 min at 95°C. Aliquots of IS (10 6 /ml) were stored at Ϫ80°C. Prior to assay, serial dilutions of the IS genomic DNA were made in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.001% sodium dodecyl sulfate, 0.001% Triton X-100 supplemented with 1 g of poly(rA)/ml, to reduce nonspecific adsorption of DNA to the walls of the reaction vessel.