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

In order to study the efficiency of integration in resting CD4 Ï© T cells, we required a fully quantitative method with improved sensitivity. In our hands, one-step PCR methods lacked the requisite sensitivity for use in primary T cells. Accordingly, a nested PCR strategy was implemented, using conditions where deoxynucleoside triphosphates (dNTPs), primers, and enzyme were not limiting in the nonkinetic preamplification of Alu-gag sequences. The preamplification is followed by a real-time PCR assay that quantitates HIV-1 long terminal repeat (LTR) sequences using molecular beacon detection (36) . In parallel, we developed an alternative integration standard (IS) cell line, wherein a large number of target T cells were infected with a high-titer, randomly integrated, replication-incompetent HIV-1 construct containing an antibiotic resistance cassette. Prolonged antibiotic selection was performed prior to preparation of genomic DNA from these IS cells. The latter step ensured that every cell in this population contained at least one provirus, and that all of the HIV-1 DNA within these cells was integrated. The large number of targets initially infected insured that these integration events were numerous, occurring at a large range of distances from genomic Alu elements.