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

Equal amounts of each DNA preparation (20 g/lane) were applied to the lanes of an agarose gel (Fig. 1B) . Southern blotting revealed integrated, linear, and circular forms of HIV-1 DNA in the acutely infected cells (Fig. 1C, left lane) . However, only integrated HIV-1 DNA could be detected in the IS (Fig. 1C, center lane) . The signals on the blot were HIV-1 specific, since they were absent from uninfected CEM-SS cells The number of copies of HIV-1 DNA in dilutions of the nonpreamplified IS are shown over the corresponding amplification curves. These numbers were determined using the ACH-2 copy number standard (Fig. 3B ). Since 100% of the HIV-1 DNA in the IS is integrated (Fig. 1) , these numbers reflect the total number of proviruses in each reaction. (B) The number of proviruses per reaction in the nonpreamplified IS was assigned to the kinetic PCR signals from the preamplified IS, and a second standard line was constructed. These amplifications were performed in duplicate, with consistently close reproducibility. (C) Logarithmic regression of the preamplified IS signals. As few as eight proviruses per sample could be reliably measured in this run. (Fig. 1C, right lane) . To ensure that all of the HIV-1 cDNA in the IS was integrated, the IS lysate was prepared after prolonged G418 exposure, thereby expanding a subpopulation of cells that expressed the neomycin resistance phenotype efficiently. Although this selection should reduce the randomness of the integration sites represented in the selected population to some degree, there should be no selection pressure in favor of Alu-gag distances of any specific length.