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Letters
November 26, 2003

Inhibition of SARS-Associated Coronavirus Infection and Replication by RNA Interference

Author Affiliations
 

Letters Section Editor: Stephen J. Lurie, MD, PhD, Senior Editor.

JAMA. 2003;290(20):2665-2666. doi:10.1001/jama.290.20.2665

To the Editor: A novel coronavirus has been identified as the etiologic agent of severe acute respiratory syndrome (SARS),1-3 for which there is no specific treatment. Small interfering RNAs (siRNAs) are double-stranded RNAs that direct sequence-specific degradation of messenger RNA in mammalian cells.4 It is also possible, however, that siRNAs could specifically interfere with viral RNA.

Methods

We designed six 21-mer SARSis (siRNAs [GENSET SA Ltd, Paris, France] targeting different sites of the replicase 1A region of the SARS coronavirus [SARS-CoV] genome; siRNA sequences in the senses strands: GUGAACUCACUCGUGAGCUCdTdT [SARSi-1]; GUACCCUCUUGAUUGCAUCdTdT [SARSi-2]; GAGUCGAAGAGAGGUGUCUdTdT [SARSi-3]; GCACUUGUCUACCUUGAUGdTdT [SARSi-4]; CCUCCAGAUGAGGAAGAAGdTdT [SARSi-5]; and GGUGUUUCCAUUCCAUGUGdTdT [SARSi-6]). We then performed 3 in vitro experiments to test their antiviral effects. In the first, we transfected monkey kidney cells (FRhk-4) with 1 of the 6 siRNAs. In addition to these 6 groups of cells, we also created 2 groups of control cells—1 transfected with an unrelated siRNA targeting luciferase (GL2i),5 and the other with the medium. OligoFectamine (Invitrogen Corp, Carlsbad, Calif) was the transfection reagent. All groups of cells were incubated for 8 hours before infection with SARS virus GZ50 strain. Thirty-six hours after viral infection, cytopathic effects were judged with phase-contrast microscopy. The cells were then fixed with –20°C ethanol for 10 minutes and immunostained with a SARS-CoV–specific antibody isolated from acute covalent sera of confirmed SARS patients. The coronavirus antigens were detected by indirect immunofluorescence assay using a fluoroscein isothiocyanate–coagulated antibody1,2 (Inova Diagnostic Inc, San Diego, Calif). To quantify the viral genomic RNA, real-time polymerase chain reaction was performed as described previously.2

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