In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

Abstract

We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8 +/- 1.2 microM) was significantly lower than its cytostatic activity; CC50 (261.3 +/- 14.5 microM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.

Fig. 1

SARS-CoV-infected Vero E6 cells were incubated for three days in the presence of 0, 0.8, 4, 20, and 100 μM chloroquine. Data are mean values ± SE of four replicates. White bars indicate the effect of chloroquine on viability of cells infected with SARS-CoV. Black bars show the effect of chloroquine on viability of mock-infected cells. The concentration of chloroquine that results in 50% inhibition of the viral cytopathic effect, IC₅₀, is 8.8 ± 1.2 μM. IFN β-1a was used as a positive control for the antiviral assay and yielded an IC₅₀ of 2550 IU/mL (data not shown).

Fig. 2

Dose–response of inhibitory effect of chloroquine on the virus yield. SARS-CoV-infected Vero E6 cells were incubated for one and three days in the presence of 0, 2, 4, 8, 16, 32, 64, 128, or 256 μM chloroquine. Cell supernatants were used for viral RNA extraction and subjected to real-time RT-PCR. cRNA standards were used for absolute quantification of the genome equivalents of SARS-CoV. These results are from a single experiment, representative of two independent experiments.

Fig. 3

Time-of-drug-addition experiments performed using quantitative RT-PCR on viral RNA extracted from the cell supernatant. The data represent mean values ± SE for three separate experiments. Virus replication was calculated as percentage of SARS-CoV genome equivalents comparing treated with untreated infected cells.