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

To standardize this assay, one must first determine the HIV-1 copy number in the nonpreamplified IS-that is, from IS DNA that has not been subjected to the initial nonkinetic Alu-gag PCR. To do so, we used graded doses of accurately counted ACH-2 cells (with two proviruses per genome). Two separate samples of this cell suspension were counted in trypan blue dye; four hemacytometer counts were performed on each sample, and then the eight cell counts were averaged. A kinetic PCR amplification for HIV-1 LTR sequences (Fig. 2B) was performed on this ACH-2 standard and on dilutions of the nonpreamplified IS. Because all of the HIV-1 DNA in the IS is integrated (above, Fig. 1C) , the known HIV copy numbers in the ACH-2 standard can be used to measure the apparent integrated HIV-1 DNA content of the IS (Fig. 2C ). IS contained a mean of 1.4 Ï® 0.3 proviruses per cell.