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

Cell lines, plasmids, and viruses. The CD4 ϩ T-lymphoblastoid cell lines CEM-SS (27) and ACH-2 (11) were maintained at densities of 10 5 to 10 6 per ml and 5 ϫ 10 4 to 5 ϫ 10 5 per ml, respectively. The culture medium was RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 25 mM HEPES, pH 7.4, with 1ϫ penicillin-streptomycin (Invitrogen Life Technologies). In order to prepare the IS cell line, a rev-deficient, G418 resistance-conferring proviral vector, pIIIB/⌬R/N, was derived from pIIIB/⌬ 12Rev/N (25) by repairing the nef gene and U3 sequences and inserting a 5Ј-internal ribosome entry site downstream of the neomycin resistance cassette. 293T cells were then cotransfected with three plasmids, namely, (i) pIIIB/⌬R/N, (ii) pcRev, and (iii) pVSV G (pHIT/G) (20) . Twenty-four hours after transfection, the supernatants containing pseudotyped virus were centrifuged at 500 ϫ g for 10 min, treated with 30 g of RNase-free DNase I (Roche)/ml for 30 min at room temperature in the presence of 10 mM MgCl 2 , and then sterile filtered through 0.2-m-pore-size syringe filters (Acrodisc, HT Tuffryn, Pall Gelman).