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

Multiple steps in the HIV-1 life cycle are inefficient or inhibited in resting CD4 Ï© T cells. It is now well established that a major inefficiency exists in reverse transcription in these cells (4, 24, 35, 38, 39) . A paucity of substrates for reverse transcriptase is in part responsible for this block, since the inefficiency can be partially overcome by supplementing the resting cells with exogenous deoxyribonucleosides (23) . Yet additional inefficiencies must exist downstream of reverse transcription, FIG. 5 . Sensitivity and specificity of the assay. (A) The plasmid pIIIB contains a full-length molecular clone of HIV-1 IIIB and no human DNA (in particular, no Alu sequences). One-way preamplification with the Alu primer alone (orange) produces a kinetic PCR signal comparable to the signal from a matched dose of nonpreamplified plasmid (green). Signals roughly fivefold higher arise from either one-way preamplification with the gag primer alone (black) or bidirectional preamplification with both Alu and gag primers. (B) One-way preamplification of the IS using either the Alu primer alone (orange) or the gag primer alone (black) produces roughly a fivefold amplification relative to a matched sample of nonpreamplified IS (green). In contrast, the kinetic PCR signal obtained from the IS following bidirectional Alu-gag preamplification (blue) is shifted roughly 12 threshold cycles to the left, corresponding to a 4-log amplification over the nonpreamplified control. (C) Twelve hours after spinoculation of CEM-SS-R5 cells with HIV-1 YU-2 (1,000 ng of p24 Gag /ml), proviruses comprise only a minority of HIV-1 DNA. The integration signal from the acutely infected cells (red) falls within the linear range of the preamplified IS (blue curves, corresponding to 3,300, 1,000, 330, 100, 30, and 12 proviruses per reaction). One-way preamplification with the gag primer alone (black) generates a signal that is similar to the signal obtained when the infection is performed in the presence of a 10 M dose of the integrase inhibitor L-731,988 (gray). This signal can be subtracted from the Alu-gag signal. It is produced via linear amplification of both integrated and unintegrated HIV-1 DNA. (D) Integration is not detected 12 h after spinoculation of CEM-SS-R5 cells with the HIV-1 YU2 -derived integrase-defective mutant virus D64A. Instead, Alu-gag preamplification results in a fivefold amplification, similar to one-way gag amplification. on April 1, 2020 by guest http://jvi.asm.org/ since progeny virions are not released by these latently infected cells in the absence of immune activation (4, 7, 10), even when their nucleoside pools are exogenously enhanced (23) . The quantitative integration assay we have developed should prove useful in mechanistic studies to dissect these additional inefficiencies. It should also prove applicable to clinical and epidemiologic studies of viral integration and assessments of the effectiveness with which future antiretroviral treatments eradicate latent reservoirs of HIV-1.