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

Early HIV-1 integration assays utilizing Alu repeat elements as "anchors" within genomic DNA were sensitive but were not strictly quantitative, since they lacked real-time reaction mon-itoring and polyclonal standards. In particular, most of these studies used genomic DNA prepared from graded doses of persistently infected cell lines such as 8E5 (34) , U1 (10), ACH-2 (10, 29) , or mixtures of such lines (37) as their standards. The problem with these cell lines is that they are clonal and contain one (19) , two (18) , or two (11) nonrandom HIV-1 integration sites, respectively. Standards prepared from these lines are likely to under-or overestimate the number of integration events in nearly all types of unknowns, since the proviruses these cell lines harbor are integrated at specific, nonvariable distances from the Alu repeats within their genomes. Single integration events can be detected in these clonal DNA standards, presumably arising from integration sites relatively close to the nearest Alu element. However, in assigning numbers to these signals, an implicit assumption must be made that all integration events in the unknowns must also be detectable by PCR (7) . Due to the random nature of both HIV-1 integration and Alu retroposition, each Alu-gag integration site in a nonclonal collection of infected cells will be amplified with a different efficiency. It is likely that significant (and unknown) numbers of proviruses are inserted too far away from an Alu site to be efficiently amplified. A corollary to this assertion is that the frequencies of integration within resting CD4 Ï© T cells that were previously determined using clonal PCR standards are likely to have been underestimated.