FIG 4.

The effect of therapy initiation delays on intracellular viral production and spread as well as EIDD-1931 metabolism. Tissue culture flasks seeded with ACE2 expressing A549 cells were infected with SARS-CoV-2 at an MOI = 0.03. EIDD-1931 was added to cell monolayers at concentrations equivalent to 0.648 μg/mL and 3.24 μg/mL at 0 h, 24 h, 48 h, or 72 h postinfection. Control flasks that did not receive drugs served as a negative control. Viral supernatants and cells were harvested daily beginning 24 h posttherapy initiation for up to 120 h postinfection. (A) The concentration of extracellular infectious viral burden was determined by performing plaque assays on viral supernatant samples. (B) Flow cytometry was performed on a portion of harvested cells to determine the amount of cell-to-cell viral spread over time. Cells were fixed and stained with an antibody specific to the SARS-CoV-2 nucleocapsid (NP) protein. (C) The intracellular infectious viral burden was determined by performing a plaque assay on a portion of the harvested cells. The intracellular viral burden is reported as log₁₀ PFU per antigen (NP) positive cells. (D) The ratio of EIDD-1931 triphosphate (TP) to CTP (CYT-TP) was determined from the remaining harvested cells. Cells were inactivated with a mixture of acetonitrile and methanol for 30 min and frozen at −80°C. Intracellular levels of EIDD-1931-TP and CYT-TP were determined via ultraperformance liquid chromatography-tandem mass spectrometry. All data points represent the mean of two biological replicates and two independent studies were conducted.