Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro

Abstract

This report describes the application of real-time PCR for testing antivirals against highly pathogenic viruses such as Lassa virus, SARS coronavirus and Ebola virus. The test combines classical cell culture with a quantitative real-time PCR read-out. The assay for Lassa virus was validated with ribavirin, which showed an IC(50) of 9 micrograms/ml. Small-scale screening identified a class of imidazole nucleoside/nucleotide analogues with antiviral activity against Lassa virus. The analogues contained either dinitrile or diester groups at the imidazole 4,5-positions, and many of which possessed an acyclic sugar or sugar phosphonate moiety at the imidazole 1-position. The IC(50) values of the most active compounds ranged from 5 to 21 micrograms/ml. The compounds also inhibited replication of SARS coronavirus and Ebola virus in analogous assays, although to a lesser extent than Lassa virus.

Fig. 1

Real-time PCR-based assay for testing the effect of compounds on replication of Lassa virus, SARS-CoV, and Ebola virus. (A) Flowchart of the assay. (B) Growth kinetics of Lassa virus, SARS-CoV, and Ebola virus in Vero cells. The RNA concentration in supernatant was determined by real-time PCR as described in the text. (C) Validation of the Lassa virus assay using ribavirin. Inhibition of Lassa virus replication was tested by real-time PCR and immunofocus assay. Cell growth was measured by MTT test.

Fig. 2

Chemical structures of imidazole nucleoside/nucleotide analogues tested. Abbreviations: Et, ethyl; Ac, acetyl; Bz, benzoyl; Tol, toluoyl.

Fig. 3

Antiviral effects exerted by selected compounds shown in Fig. 2. The compound is indicated above each graph. (A) Inhibition of replication of Lassa virus as tested by real-time PCR and immunofocus assay. Cell growth was measured by MTT test. (B) Inhibition of replication of SARS-CoV replication as tested by real-time PCR. Cell growth was measured by MTT test. (C) Inhibition of replication of Ebola virus replication as tested by real-time PCR. Cell growth was measured by MTT test. Note the increased toxicity of the compounds due to the prolonged assay time of 3 days.