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Letters Section Editor: Stephen J. Lurie, MD, PhD, Senior Editor.
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.
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
In the second experiment, we transfected FRhk-4 cells with a combination
of 2 or 3 SARSis (SARSi-2/3, SARSi-2/4, SARSi-3/4, SARSi-1/2/4, and SARSi-2/3/4),
using GL2i as a control. In each combination, an equal amount of individual
siRNA was used while the final concentration of total siRNAs remained the
same (10 nM).
In the third experiment, we tested the inhibitory effect of SARSi-2/3/4
on the infection and replication of 3 other SARS-CoV strains isolated from
SARS patients in Hong Kong and Guan Zhou (GZ34, HKR1, and HKR2 strains).
Compared with uninfected cells, cells infected with SARS-CoV exhibited
a marked morphologic change with cytopathic effects (Figure 1). The uninfected cells were flattened, whereas the SARS-CoV
infected cells became refractile and rounded. Judged by morphologic changes,
SARSi-2, SARSi-3, and SARSi-4 markedly inhibited the cytopathic effects caused
by viral infection and replication, whereas SARSi-1, SARSi-5, and SARSi-6
were less effective. The results were further confirmed by immunostaining
with antibody against SARS-CoV antigens. There was a consistent and marked
92.5%, 89.6%, and 85.8% reduction in the viral genomic RNA copies (as determined
by quantitative real-time polymerase chain reaction) in cells transfected
by SARSi-4, SARSi-2, and SARSi-3, respectively. The reduction was much less
marked in cells transfected by the other 3 siRNAs (only 50%-65%).
In the second experiment, the combinations of SARSi-2, SARSi-3, and
SARSi-4 also inhibited the infection and replication of different strains
of SARS-CoV. No obvious synergistic effects were observed, however, from any
of these combinations.
In the third experiment, we found that the efficacy of SARSi-2, SARSi-3,
or SARSi-4 in inhibiting the infection and replication of the 3 other SARS-CoV
strains were similar to that of the GZ50 strain. Transfection with SARSi alone
or GL2i did not show any change in cell morphology or viral genomic RNA copies
(data not shown).
siRNAs targeting the replicase 1A region of the SARS-CoV genome appear
to be effective in vitro against the SARS virus. Their clinical usefulness,
however, has yet to be demonstrated.
Funding/Support: This research was supported
by a University Grants Committee seed grant (Drs He and Kung), Competitive
Earmarked Research and Area of Excellence grants (Dr Kung) from the Research
Grant Council, and a grant from the Innovation and Technology Fund (Dr Lin).
Acknowledgment: Drs He and Zheng contributed
equally to this study.
He M, Zheng B, Peng Y, et al. Inhibition of SARS-Associated Coronavirus Infection and Replication by RNA Interference. JAMA. 2003;290(20):2665–2666. doi:10.1001/jama.290.20.2665
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