Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence.
ABSTRACT The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5' cyclization sequence (5'CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an "in vivo-ready" version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
- [show abstract] [hide abstract]
ABSTRACT: RNA interference (RNAi) is a process that is induced by double stranded RNA and involves the degradation of specific sequences of mRNA in the cytoplasm of the eukaryotic cells. It has been used as an antiviral tool against many viruses, including flaviviruses. The genus Flavivirus contains the most important arboviruses in the world, i.e., dengue (DENV) and yellow fever (YFV). In our study, we investigated the in vitro and in vivo effect of RNAi against YFV. Using stable cell lines that expressed RNAi against YFV, the cell lines were able to inhibit as much as 97% of the viral replication. Two constructions (one against NS1 and the other against E region of YFV genome) were able to protect the adult Balb/c mice against YFV challenge. The histopathologic analysis demonstrated an important protection of the central nervous system by RNAi after 10 days of viral challenge. Our data suggests that RNAi is a potential viable therapeutic weapon against yellow fever.Virus Genes 02/2009; 38(2):224-31. · 1.77 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy revealed that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5' and 3' CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5' and 3' untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5' and 3' UAR (upstream AUG region). In order to investigate the functional role of 5'-3' UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5' or 3' UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.Journal of Virology 07/2005; 79(11):6631-43. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Flavivirus replication is mediated by interactions between complementary ssRNA sequences of the 5'- and 3'-termini that form dsRNA cyclisation stems or panhandles, varying in length, sequence and specific location in the mosquito-borne, tick-borne, non-vectored and non-classified flaviviruses. In this manuscript we manually aligned the flavivirus 5'UTRs and adjacent capsid genes and revealed significantly more homology than has hitherto been identified. Analysis of the alignments revealed that the panhandles represent evolutionary remnants of a long cyclisation domain that probably emerged through duplication of one of the UTR termini.Virology 10/2007; 366(1):8-15. · 3.37 Impact Factor
JOURNAL OF VIROLOGY, Oct. 2011, p. 10154–10166
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 19
Inhibition of Dengue Virus Infections in Cell Cultures and in
AG129 Mice by a Small Interfering RNA Targeting a
Highly Conserved Sequence?
David A. Stein,1Stuart T. Perry,2Michael D. Buck,2Christopher S. Oehmen,3Matthew A. Fischer,1
Elizabeth Poore,1Jessica L. Smith,1Alissa M. Lancaster,1Alec J. Hirsch,1Mark K. Slifka,1
Jay A. Nelson,1Sujan Shresta,2* and Klaus Fru ¨h1*
Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon1; La Jolla Institute for
Allergy & Immunology, La Jolla, California2; and Pacific Northwest National Laboratories, Richland, Washington3
Received 2 June 2011/Accepted 18 July 2011
The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically
distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in
tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently
available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small
interfering RNA (siRNA) (DC-3) targeting the highly conserved 5? cyclization sequence (5?CS) region of the
DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype
in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an “in vivo-ready” version of DC-3 was
synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of
infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the
control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight
loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups
in several tissues at various time points postinfection. These results suggest that exogenously introduced
siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent
severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has
potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
A variety of genetically distinct virus strains within four
antigenically distinguishable serotypes (dengue virus type 1
[DENV-1], DENV-2, DENV-3, and DENV-4) comprise the
DENV species, a member of the genus Flavivirus of the family
Flaviviridae (7, 55, 66). Dengue viruses are transmitted to hu-
mans primarily by Aedes aegypti mosquitoes and represent a
considerable threat to public health in tropical and subtropical
regions worldwide. Infection can be asymptomatic or can cause
a spectrum of clinical syndromes ranging from self-limiting
febrile illness to life-threatening severe dengue disease. Annu-
ally, hundreds of thousands of cases of clinical dengue disease
are reported by clinicians to the WHO, with a case-fatality rate
of ?0.5 to 5.0% (23, 24, 27, 67). DENV genomic sequences can
vary up to approximately 19% between strains in a single
serotype and up to approximately 34% between strains of
different serotypes (24, 66). Infection by various strains from
each serotype can cause severe disease in humans, and all four
serotypes now circulate globally (24, 27).
The DENV genome is a ?10.7-kb positive-sense single-
stranded RNA consisting of a single open reading frame
(ORF) flanked by a 5? untranslated region (5?UTR) and a
3?UTR. The ORF is translated into a single polyprotein that is
co- and posttranslationally cleaved to produce three structural
(C, prM/M, and E) and seven nonstructural (NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5) proteins. The viral ge-
nome features a 5? type 1 cap structure but lacks a 3? poly(A)
tail and serves both as mRNA and as a template for replication
via the production of a minus-strand intermediate (40). During
viral replication, a number of long-range RNA-RNA cis inter-
actions are necessary for efficient minus-strand synthesis (re-
viewed in references 32 and 65). The 3?UTR of all mosquito-
vectored flaviviruses contains a region of at least 8
nucleotides (nt), termed conserved sequence 1 (CS1), which
is both highly conserved and complementary to the 5? cycli-
zation sequence (5?CS) located in the N-terminal region of
the capsid gene (C) coding sequence (9, 26). A base-pairing
interaction between the CS1 and 5?CS sequences of DENV
and other flaviviruses was shown to promote the cyclization
of the genome and contribute to efficient minus-strand syn-
thesis (1, 2, 20, 65, 75, 76, 78).
Although many advances have been made recently in the
development of vaccines and antiviral drugs to address DENV
(reviewed in references 17, 23, 47, and 63), to date there is no
antiviral intervention formally approved to prevent or treat
DENV infections. To warrant commercial development, a pro-
spective antiviral drug against DENV would likely require
demonstrated efficacy against infections by representative
strains from each of the four DENV serotypes. The targeting
of flaviviral RNA with nucleic acid-based agents has provided
* Corresponding author. Mailing address for Klaus Fru ¨h: Vaccine
and Gene Therapy Institute, Oregon Health and Science University,
Beaverton, OR 97006. Phone: (503) 418-2735. Fax: (503) 418-2701.
E-mail: email@example.com. Mailing address for Sujan Shresta: Division
of Vaccine Discovery, La Jolla Institute for Allergy & Immunology,
9420 Athena Circle, La Jolla, CA 92037. Phone: (858) 752-6944. Fax:
(858) 752-6987. E-mail: firstname.lastname@example.org.
?Published ahead of print on 27 July 2011.
insight into virus biology and transmission and may provide
novel interventional approaches (57, 61). RNA interference
(RNAi) is a cellular process induced by double-stranded RNA
(dsRNA) capable of producing the RNase-mediated degrada-
tion of specific mRNA. The activation of RNAi machinery via
the introduction of a small dsRNA targeted against viral RNA
has been used with experimental success to inhibit various
RNA virus infections (18, 30, 44). Recently, several studies
have described the antiviral efficacy of chemical-, plasmid-, or
virus-vectored small dsRNAs in both cell cultures and mouse
models infected with various flaviviruses such as West Nile
virus (WNV) (4, 5, 37, 48, 71), Japanese encephalitis virus
(JEV) (37, 41, 46, 53), and yellow fever virus (YFV) (49).
DENV infection and transmission could be suppressed in mos-
quitoes transgenically engineered to produce DENV-targeted
small interfering RNA (siRNA) (21). A synthetic siRNA tar-
geting prM coding sequence inhibited the infection of
DENV-1 in mosquito cell cultures (69). The peptide-mediated
delivery of siRNA targeting a highly conserved sequence in the
DENV envelope (E) gene decreased DENV-2 replication in
human monocyte-derived dendritic cells and macrophages in
culture (62). Finally, adeno-associated virus (AAV) vectors
containing short hairpin RNA (shRNA) targeted to the CS1
region inhibited DENV-2 infections in mammalian cell cul-
tures (79). While RNAi has been shown to have efficacy against
DENV infections in vitro, there have not been any reports of
antiviral efficacy in in vivo models.
Chemically synthesized siRNAs are typically 19 to 21 bp in
length with 2-nt 3? overhangs and can be delivered into cell
cultures with lipid-based transfection reagents. To design a
synthetic siRNA suitable for use against one or more serotypes
of DENV, we employed two approaches. In one approach we
used genomic sequences from specific strains of DENV to
program a commercial siRNA design algorithm. In a second
approach we used a comparative genome analysis to identify
siRNA target sequences that are highly conserved across the
four serotypes of DENV. The siRNAs were then tested for
activity against DENV infections in cell cultures and in mice.
The AG129 mouse model has been used to investigate the
biology of DENV disease and to evaluate vaccine and antiviral
drug candidates (reviewed in references 68 and 72). A recent
version of this in vivo model system used DENV-2 strain S221
in the presence of an anti-prM antibody to mimic the antibody-
dependent enhancement (ADE)-mediated disease thought to
occur for a significant portion of cases of severe dengue disease
in humans (77). AG129 mice infected in this model exhibit
some of the same pathological features observed for humans
with severe dengue disease, including vascular leakage, intes-
tinal hemorrhage, elevated cytokine levels, low platelet counts,
and elevated hematocrit levels. We found that an siRNA tar-
geting a highly conserved sequence in the 5?CS region of the
DENV capsid gene was a powerful inhibitor of representative
strains of all four DENV serotypes in Huh7 cell cultures and
DENV-2 in this mouse model.
MATERIALS AND METHODS
Cells and viruses. Huh7 cells (a human hepatoma-derived cell line) and Vero
cells were obtained from F. Chisari (Scripps Research Institute) and the ATCC,
respectively. All cells were propagated in complete growth medium consisting of
Dulbecco’s modification of Eagle’s medium (DMEM; Cellgro) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 units/ml
penicillin and 100 g/ml streptomycin). All cell culture incubations were carried
out at 37°C in a humidified atmosphere containing 5% CO2. DENV-1 (strain
West-Pac; GenBank accession number DVU88535) was obtained from Robert
Putnak (Walter Reed Army Research Institute). DENV-2 strain New Guinea C
(NGC) (GenBank accession number AF038403), DENV-3 (strain H87;
GenBank accession number M93130), and DENV-4 (strain H241; GenBank
accession number AY947539) were obtained from the ATCC. The WNV strain
used (strain 385-99; GenBank accession number AY842931) was previously
described (70). Viral stocks were prepared and titers were determined as previ-
ously described (59). WHO primary seed lot 213/77 yellow fever virus (YFV)
17D passage 237 (GenBank accession number NC_002031) was propagated on
Vero cells. For in vivo experiments, DENV-2 strain S221, a triple-plaque-purified
clone isolated from mouse-passaged DENV-2 strain D2S10, was prepared and
quantified as previously described (58).
siRNA design and bioinformatics. To design siRNAs optimized to target the
individual genomes of each of the four DENV strains used in this study, we used
the Dharmacon algorithm (Dharmacon siRNA Design Center) (6, 54). This
analysis generated a list of sequences scored hierarchically with respect to their
likelihood to represent effective siRNA targets (data not shown). Two nonover-
lapping high-scoring sequences per serotype were selected from these lists (al-
gorithm siRNA; sequences D1-1 through D4-2) (Table 1).
To design an siRNA targeted against a sequence that is highly conserved
across the DENV virome (conserved siRNA), full-length genomic sequences of
the NCBI reference strains of the four serotypes of DENV were aligned (Table
1) by using TCoffee (http://www.tcoffee.org). This analysis revealed six regions
(sequences DC-2 through DC-7) (Table 1) of 19 contiguous nucleotides contain-
ing a 0- or 1-base difference between the four reference strains. A 20-nt region
having perfect conservation between the GenBank reference sequences for
DENV-1 to DENV-3 but with 2 mismatches to DENV-4 was also selected for
targeting (DC-1) (Table 1). Eight sequences (DC-1 through DC-7 and D2-2)
were aligned with a library of 2,754 full-length DENV genomes from GenBank
(GI numbers are available upon request). These genomes have representatives
from each of the four serotypes: 1,223 genomes of serotype 1, 852 genomes of
serotype 2, 587 genomes of serotype 3, and 92 genomes of serotype 4. NCBI
BLAST was used to align each of the candidate sequences against all of the
genomes in the library by using the blastn program, setting the number of results
reported to 3,000, with a gap-opening penalty of 2, a gap extension penalty of 1,
a nucleotide mismatch penalty of ?1, and a nucleotide match reward of ?1, with
an E value cutoff of 10.0. Multiple alignments for any query/genome pair were
eliminated, and only the top alignment was considered. Exact matches were
defined as BLAST alignments of the full length of the query (20 for DC-1 and 19
for all others) with 100% identities. Single mismatches were defined as BLAST
alignments of a length of query of ?1 (19 for DC-1 and 18 for all others) with
100% identities or full-length alignments with 1 mismatch.
Considerations for the design of siRNAs targeting WNV and YFV (sequences
WNV-1 and YFV-1, respectively) (Table 1) are described below in Results. The
WNV and YFV sequences were compared to libraries of WNV and YFV from
the NCBI nucleotide database having 284 and 18 complete genomes, respec-
tively. The same run-time parameters as those for the DENV calculations were
used to calculate blastn alignments between the WNV-1 and YFV-1 siRNA
queries and the corresponding libraries.
A random sequence negative-control siRNA (sequence NC) (Table 1) was
also prepared to control for off-target effects of the siRNA chemistry. To reduce
the chances of unintentional hybridization events, all siRNAs were further ana-
lyzed with blastn against all human transcript sequences and relevant siRNAs
against mouse transcript sequences.
siRNA transfections of cell cultures. For cell culture experiments, reverse
transfection in 48-well plates was employed. For each sample, the appropriate
amount of a 1 or 10 ?M stock of siRNA (Dharmacon Inc., Lafayette, CO) and
1 ?l of RNAiMAX (Invitrogen) were mixed into 100 ?l of Opti-MEM for 30 min
at room temperature, after which 300 ?l of DMEM (with 2% FBS and no
antibiotics) containing 3 ? 104Huh7 cells was added and mixed gently. Two days
later, the transfection-reagent-containing medium was removed, and the cells
were infected with virus (multiplicity of infection [MOI] of 0.2 or 0.5, as indi-
cated) in 200 ?l of DMEM (containing 2% FBS and antibiotics) for 2 h with
gentle rocking, after which the virus-containing medium was removed. The cells
were washed twice and replenished with 250 ?l DMEM (containing 2% FBS and
antibiotics). When an MOI of 0.5 was used, supernatants were collected at 24 h
(WNV), 48 h (DENV), or 72 h (YFV) postinfection (p.i.) and stored at ?80°C
for subsequent plaque assay analysis. In addition, the cells were lysed with 200 ?l
of 1? SDS buffer (50 mM Tris [pH 8], 2% SDS, 10% glycerol) for 30 min and
then collected for immunoblotting. For growth curve experiments the same
VOL. 85, 2011INHIBITION OF DENGUE VIRUS INFECTIONS BY siRNA10155
protocol as that described above was employed, except that an MOI of 0.2 and
a p.i. volume of 400 ?l DMEM (containing 2% FBS and antibiotics) were used,
with 50 ?l of the supernatant collected daily for 3 or 5 days, as indicated starting
at 1 day p.i., for subsequent analysis by plaque assays.
Immunoblots. Lysed cell samples were heated to 95°C for 5 min and subjected
to 10% SDS-PAGE before transfer onto a polyvinylidene difluoride (PVDF)
membrane (Millipore). Membranes were blocked for 1 h in PBST (1% phos-
phate-buffered saline [PBS] with 0.1% Tween 20) and 10% instant nonfat dry
milk, after which two monoclonal antibodies were added, one to detect the
envelope (E) protein of DENV and WNV (mouse monoclonal antibody 4G2-4-
15; ATCC) at 1 ?g/ml and one to detect glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) (used at a 1:7,500 dilution; Santa Cruz BioTechnology), for
2 h of incubation. After 2 rinses in PBST, secondary antibody consisting of
horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotech-
nology) at a 1:7,500 dilution was added in PBST and 10% milk as described
above for 1 h before PBST rinsing and detection with SuperSignal West Pico
chemiluminescent substrate (Thermo Scientific).
Titer determination. Viral titers for DENV and WNV were determined on
Vero cells by the immunostaining of foci (plaque assay). Briefly, serial dilutions
of virus in DMEM (with 2% FBS and antibiotics) were used to infect confluent
monolayers of Vero cells for 90 min. The inoculation solution was then replaced
with DMEM containing 2% FBS, antibiotics, and 0.5% carboxymethylcellulose
sodium salt (CMC) (Sigma). The culture medium was removed 2 days (for
WNV) or 4 days (for DENV) later, and the cells fixed with 4% (wt/vol) para-
formaldehyde (PFA) (in 1? PBS) for 15 min before immunostaining, as follows.
After the removal of PFA the cells were washed twice with 1? PBS and then
blocked for 1 h in 1? PBS containing 2% (vol/vol) normal goat serum (NGS) and
0.4% (vol/vol) Triton X-100 (TX-100). Mouse monoclonal anti-E antibody, sus-
pended in 1? PBS with 2% NGS, was added to each well of the assay plates at
4 ?g/ml for 1 h, followed by two washes with 1? PBS. As a secondary antibody,
horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotech-
nology) at a 1:5,000 dilution in 1? PBS with 2% NGS was added, and the mixture
was incubated for 1 h, followed by two rinses with 1? PBS. Immunoreactive foci
were stained with a Vector VIP peroxidase substrate kit (Vector Labs), and the
numbers of PFU/ml were calculated. For YFV, viral titers were determined on
Vero cells by a plaque assay. Briefly, serial dilutions of virus in DMEM (with
10% FBS) were used to infect confluent monolayers of Vero cells for 60 min.
Following infection, the monolayers were overlaid 1:1 with 1% agar in double-
distilled water (ddH2O)–2? Eagle’s minimal essential medium (with 5% FBS
and antibiotics). At 3 days postinfection, an additional overlay with 0.015%
(wt/vol) neutral red in 1% agar was added. At 4 days postinfection, viral plaques
were counted and recorded.
Cell viability assay. Uninfected Huh7 cells were subjected to the indicated
treatment using the same reverse transfection and cell culture conditions as those
used for the antiviral assays described above (for siRNA transfections of cell
cultures), except that 96-well plates and appropriately scaled reagent volumes,
with 104cells per well, were used. Cell viability was measured at 48 h after plating
by using the CellTiter Glo (Promega) cell proliferation assay kit according to the
manufacturer’s instructions and a Turner Biosystems (Sunnyvale, CA) plate
reader with Veritas (Mountain View, CA) software. The absorbance values of
siRNA-treated cells were converted to percentages by comparison to mock-
treated samples containing no transfection reagent or siRNA, set at 100%.
Construction of Huh7-ISRE-luc cells and assay for siRNA induction of inter-
feron-stimulated gene expression. To investigate possible interferon (IFN)-stim-
ulated gene expression, Huh7 cells were transduced with two recombinant len-
tiviral vectors, one containing the interferon-stimulated response element
(ISRE) fused with the coding sequence for firefly luciferase (catalog number
CLS-008L-1; SABiosciences) and the other containing the coding sequence for
Renilla luciferase driven by the cytomegalovirus (CMV) promoter/enhancer (cat-
alog number CLS-RCL-1; SABiosciences), resulting in a cell line stable for the
inducible and constitutive expression of firefly and Renilla luciferases, respec-
tively. These Huh7-ISRE-luc cells were subjected to the indicated treatments
using reverse transfection and cell culture conditions similar to those used for the
antiviral assays described above (for siRNA and cell culture transfections), in a
96-well plate. The respective luciferase levels were measured at 8 or 48 h after
plating by use of the Dual-Glo luciferase assay system (Promega) according to
the manufacturer’s instructions, using the same equipment as that used for the
cell viability assays described above. As a positive control for this experiment, 500
U IFN-? (PBL InterferonSource, NJ) was used.
Evaluation of siRNA antiviral efficacy in AG129 mice. 129/Sv mice doubly
deficient in IFN-?/? and -? receptors (AG129 mice) were bred and housed at the
La Jolla Institute for Allergy & Immunology (LIAI) (La Jolla, CA). The mice
were 5 to 6 weeks old at the beginning of the experiments. Mice were allowed
food and water ad libitum throughout the studies, and all experimental proce-
dures were preapproved and performed according to the guidelines set by the
LIAI Animal Care and Use Committee. For in vivo studies, mice were infected
intravenously (tail vein) with 109genomic equivalents (GE) (equivalent to 20,000
PFU, as measured by a plaque assay on BHK-21 cells ) of S221 and admin-
istered 5 ?g monoclonal antibody 2H2 by intraperitoneal injection on day 0. In
addition, mice received a retro-orbital intravenous administration of either PBS
or a mixture of Silencer In Vivo Ready siRNA (10 mg/kg of body weight/dose;
Ambion) and Invivofectamine 2.0 reagent (Invitrogen), combined as recom-
mended by the manufacturers, at 24 h before and 24 h and 72 h after DENV
infection. For survival studies, all mice were weighed daily and euthanized when
moribund or at the first signs of paralysis. Animals whose weight fell below 80%
of the weight at the start of the experiment were considered moribund. For tissue
analysis, mice were euthanized by isoflurane inhalation at 24 and 72 h postin-
fection, and blood and tissues were harvested as described previously (51).
Quantitative reverse transcription (RT)-PCR was performed to detect DENV
genomes and cellular 18S RNA as previously described (52). Viral loads are
TABLE 1. siRNAs and their sequences and target locations
siRNAsiRNA target sequence Virus(es) targeted
Positions of siRNA target location in viral
genome (genome region)a
NA (negative control)
1–20 (5? UTR)
aBased on the following GenBank accessions numbers: NC_001477 for DENV-1, NC_001474 for DENV-2, NC_001475 for DENV-3, NC_002640 for DENV-4,
DQ211652 for WNV, and NC_002031 for YFV. The genome region containing the siRNA target is shown in parentheses. For siRNAs targeting multiple DENV
serotypes, the target nucleotide positions correspond to DENV-2 (accession number NC_001474). NA, not applicable.
10156STEIN ET AL.J. VIROL.
expressed as GE per ml in serum or GE normalized to copies of 18S RNA in
TNF measurements. Serum from infected animals was analyzed by use of the
tumor necrosis factor alpha (TNF-?) enzyme-linked immunosorbent assay
(ELISA) Ready-Set-Go kit (eBioscience) according to the manufacturer’s in-
Statistical analysis. Cell culture data were analyzed with the Student t test,
with the exception of growth curves, where a repeated-measures analysis of
variance (ANOVA) (SAS, version 9.2) followed by contrast tests was used for
day-by-day comparisons of virus growth under the two treatments. For in vivo
data, Kaplan-Meier survival curves were analyzed by a log rank test. The viral
load in mouse tissues and TNF levels determined by ELISA were analyzed with
the Student t test. Results with error bars are expressed as the standard errors of
the means (SEM). Unless noted otherwise, statistical analyses were performed
with Prism 5 (GraphPad Software).
Design and antiviral activity of algorithm and conserved
siRNAs against four DENV serotypes. All siRNAs used in this
study are defined in Table 1. To design siRNAs against DENV
genomic sequences, we used two different strategies. In one
strategy, we applied a predictive algorithm and selected two
high-scoring siRNA target sequences for each strain of DENV
that we planned to use in our experiments (one strain from
each of the four serotypes [for strain designations, see Mate-
rials and Methods]) (sequences D1-1 through D4-2; algorithm
siRNA). In a second strategy, we identified seven regions that
were highly conserved across the GenBank reference se-
quences (listed in Table 1) for the four serotypes of DENV
(DC-1 through DC-7; conserved siRNA) and encompassed at
least 19 nt, as required for effective siRNA targeting. The
sequences of the algorithm siRNA were not well conserved
across serotypes (data not shown), with the exception of D2-2,
which showed a moderately high level of conservation com-
pared with the reference sequences (Table 2). None of the
conserved-siRNA targets were present in the list of potential
siRNA targets determined with the predictive algorithm, and
we reasoned that conserved siRNAs could therefore benefit
from empirical comparisons to the algorithm siRNAs for the
ability to inhibit different strains of DENV.
To determine the antiviral efficacy of the algorithm and
conserved siRNAs, we used Huh7 cells, as they are both easily
infected by DENV and easily transfected with siRNA and are
derived from human liver, an organ that has been shown to be
infected by DENV in vivo (33, 50). The growth of four DENV
strains, each one representing a different serotype, was moni-
tored in Huh7 cells after transfection with the conserved
siRNA and the algorithm siRNA D2-2 as well as with the
appropriate strain-specific algorithm siRNA. A negative-con-
trol siRNA (NC) and a no-treatment sample (containing no
siRNA or transfection reagent) were included in each experi-
ment. Cells were reverse transfected with 100 nM siRNA 2
days before infection with 0.5 MOIs of DENV. To assess the
impact of each siRNA on viral protein production, we used
Western blotting to detect viral envelope glycoprotein (anti-E
antibody) in lysates collected at 2 days after infection. Cellular
GAPDH was detected simultaneously as a loading control and
cell viability indicator. A noninfected control is present in
three of the four Western blots shown in Fig. 1, demonstrating
the specificity of the two antibodies used. The reduction of
infectious viral particle release was monitored with an immu-
nostain-based plaque assay of the cell culture supernatants at 2
days postinfection. The viruses used in this study varied to
some extent in growth characteristics, with DENV-2 titers
reaching approximately an order of magnitude higher in peak
titer than those of the other three viruses. The results of the
Western blots were generally consistent with those of the
plaque assays. A 10- to 100-fold reduction of infectious virus
corresponded to a marked reduction in viral protein levels
(Fig. 1), with viral protein being detectable only upon the
prolonged exposure of Western blots (not shown). With the
exception of D4-2, all the algorithm siRNAs markedly sup-
pressed virus protein production and the amount of infectious
virus produced (Fig. 1). This result is consistent with the pre-
dictive power of the design methodology, and we therefore
used the activity of select algorithm siRNAs as benchmarks to
evaluate the efficacy of conserved siRNA. The DC-5 and DC-6
sequences were generally ineffective, despite a perfect agree-
ment between siRNAs and target sequences for all four strains
(with the exception of a single-base mismatch in the DC-5
target for DENV-3) (Table 2). Of note, the DC-6 siRNA
contains the DENV CS1 sequence (CAGCAUAUUG), a
functional and highly conserved motif in mosquito-borne fla-
viviruses (26, 35, 45, 65, 76). DC-4 showed some activity against
DENV-3 and DENV-4, whereas DC-7 had some activity
against DENV-2 and DENV-3. The algorithm siRNA D2-2
was active not only against DENV-2 but also against DENV-3
and DENV-4. D2-2 has 0, 1, 2, and 3 mismatches with the
TABLE 2. Sequence agreement between conserved siRNA and DENV strains
No. of base differences
DC-1DC-2 DC-3DC-4DC-5DC-6 DC-7 D2-2
DENV-1 (Ref Seq)
DENV-2 (Ref Seq)
DENV-3 (Ref Seq)
DENV-4 (Ref Seq)
aStrain of DENV used in this study (see Materials and Methods).
bRef Seq, GenBank reference sequence (see Table 1 for accession numbers).
cNA, not applicable. The 5?-terminal nucleotides of this GenBank entry may be somewhat inaccurate (see Results).
VOL. 85, 2011 INHIBITION OF DENGUE VIRUS INFECTIONS BY siRNA 10157
FIG. 1. DENV-specific siRNAs reduce the production of viral protein and infectious virus in Huh7 cells. Cell cultures received a single transfection
with 100 nM the indicated siRNA at 48 h before infection with the respective strains from each of the serotypes of DENV (for strain designations see
Materials and Methods) at an MOI of 0.5. After a 2-h adsorption period the virus was removed and replaced with medium containing no siRNA. At 48 h
postinfection, supernatants were collected for focus-forming (plaque) assays (A, C, E, and G), and cell lysates were prepared for Western blotting (B,
A sample of an uninfected-cell lysate was included in the Western blots for DENV-1 to -3. A few of the treatments that did not appear to reduce virus
protein levels by Western blotting were omitted from the plaque assays for DENV-4. Each experiment was repeated at least twice, with similar results.
A representative Western blot is shown, and histograms represent mean values of triplicate plaque assays ? SEM. Asterisks indicate a significant
difference relative to the negative-control siRNA-treated group (*, P ? 0.05, Student t test).
DENV-2, -4, -3, and -1 strains used in this study, respectively
(Table 2). The most consistent results against all strains were
obtained with DC-1 and DC-3. The DC-1 sequence has no
disagreement with the strains of DENV-1 to -3 used in this
study but uncertain agreement with strain H241 of DENV-4
used in this study, due to uncertainty regarding the accuracy of
the GenBank sequence annotation at the H241 5?-terminal
region (M. Schreiber, Novartis Institute for Tropical Diseases,
personal communication). DC-3 suppressed the titers of all
four strains robustly, by more than 98% compared to control
siRNA (Fig. 1). DC-3 has perfect agreement with the DENV-2
and -4 strains and 1 mismatched base with the DENV-1 and -3
strains used here (and similarly with the GenBank reference
sequences) (Table 2). These data show that at least some of the
siRNAs (DC-1 and DC-3) designed solely on the basis of their
conservation across GenBank reference sequences were active
against representative strains from each of the four serotypes.
To rule out that the cytotoxicity of siRNA reagents had
contributed to the inhibition of DENV recovery observed in
Fig. 1, we measured the viability of uninfected Huh7 cells
subjected to siRNA under the conditions used in the antiviral
assays. Huh7 cells were transfected with 100 nM NC, DC-1,
DC-3, D2-2, WNV-1, and YFV-1 siRNAs under the same
conditions as those used in the experiments described above
for Fig. 1 and incubated for 48 h. When ATP utilization was
measured as an indicator of cellular metabolism, we did not
observe a significant effect on cell viability for any of the
siRNAs combined with the amount of RNAiMAX used for the
transfections in this study (1 ?l per transfection in a total
volume of 400 ?l). However, a higher concentration of
RNAiMAX (5 ?l) did cause a ?60% diminishment of cell
viability (data not shown). These results indicate that the
siRNA and transfection conditions used in this study did not
impact cell viability.
Next, we sought to determine the level of conservation of the
conserved siRNAs against all full-length DENV sequences pres-
ent in the GenBank database. We aligned full-length sequences
of 2,754 strains of DENV from the various serotypes and deter-
mined the percentages of sequences that had a 0- or 1-base
and 92% (Fig. 2A). However, the target sequences for DC-1 and
D2-2 were each conserved in only 15 to 16% of the sequences
analyzed (Fig. 2A). Importantly, the target sequence for DC-3
FIG. 2. Conservation of sequence between different strains of DENV at 8 siRNA target sites. Shown are the fractions of DENV strains with
a 0- or 1-base mismatch to various siRNAs. (A) Total numbers of best alignments with an exact match or 1 mismatch as defined in Materials and
Methods for each query/target pair identified by blast divided by 2,754, the total number of DENV strains in the library. (B) Total number of best
alignments having an exact match or 1 mismatch in each DENV serotype divided by the number of strains in each serotype (defined in Materials
VOL. 85, 2011 INHIBITION OF DENGUE VIRUS INFECTIONS BY siRNA10159
was found to be highly conserved in more than 99% of all DENV
sequences analyzed (Fig. 2A). A similar disposition was observed
when these full-length sequences were subdivided into individual
serotypes. Whereas most of the conserved-siRNA targets showed
lower conservation across strains of at least one of the four
DENV serotypes, the DC-3 target sequence was highly conserved
in all four serotypes (Fig. 2B). Thus, our empirical approach
yielded at least one panspecific siRNA (DC-3) that was highly
active in cell cultures against representative strains from all four
serotypes and conserved in almost all DENV strains annotated to
Panspecific siRNA DC-3 inhibits multiple rounds of DENV
infection. To further characterize the activity of DC-3, we
evaluated its activity over several rounds of viral replication, in
multistep growth curves. Huh7 cells were transfected with
siRNA at 2 days before infection, and supernatants were col-
lected daily for evaluations of virus production. At an input
MOI of 0.2, the level of recovery of DENV-1, -3, and -4
steadily increased over a period of 5 days, consistent with
several rounds of infection (Fig. 3). At each of these time
points, DC-3 reduced the recovery of infectious virus for each
of these strains by 90 to 99%. DC-3 thus delayed the accumu-
lation of extracellular virus by 1 to 2 days for up to 5 days
postinfection, a full week after the single transfection.
DENV-2 strain NGC grew more quickly and to a higher titer,
resulting in noticeable cell death beginning at day 3 postinfec-
tion. Taken together, these data suggest that DC-3 was active
over several days, reducing virus production during sequential
rounds of infection.
Comparison of the inhibitory concentrations of an algo-
rithm siRNA and those of a conserved siRNA against DENV-2
infection. The conserved siRNA DC-3 and the algorithm
siRNA D2-2 each reduced DENV-2 production by more than
2 logs at a concentration of 100 nM (Fig. 1C). To further
compare the activities of these two siRNAs, we tested them
against DENV-2, the most robustly growing virus, in a dose-
response format. Cells were transfected with different concen-
trations of siRNA at 2 days before infection, and virus titers in
the supernatant were measured at 2 days postinfection. As
shown in Fig. 4, viral release was suppressed by more than
10-fold when D2-2 was present at under 2 nM, generating an
90% inhibitory concentration (IC90) (the concentration of drug
producing a 90% reduction in the amount of virus compared to
mock treatment) of 1.4 nM. DC-3 was less potent, with an IC90
of 9.8 nM. Thus, at a concentration of approximately 10 nM,
D2-2 reduced DENV-2 production 7-fold more than did DC-3.
However, both siRNAs reduced viral titers by more than 100-
fold at 50 nM.
siRNA targeting of the 5?CS inhibits WNV moderately and
YFV potently. Since the DC-3 target includes 5 of the 10 nt of
the DENV 5?CS (Fig. 5A), we tested whether 5?CS-directed
siRNA would also be effective against other mosquito-borne
flaviviruses. Although the 5?CS regions of all mosquito-borne
flaviviruses share 8 nt of sequence, the adjacent sequences vary
between species. We therefore designed species-specific
siRNAs to target the 5?CS region of WNV and YFV. For
WNV, we designed an siRNA (WNV-1) to target the 11-nt
5?CS and the adjacent 8 nt in the 3? direction, as the sequence
FIG. 3. Multistep growth curves of 4 DENV serotypes after DC-3 siRNA treatment. Huh7 cell cultures received a single transfection with 100
nM DC-3 or NC siRNA at 48 h before infection with a strain from each serotype of DENV as indicated, at an MOI of 0.2. After a 2-h adsorption
period the virus was removed and replaced with medium containing no siRNA. Supernatants were collected daily for 3 to 5 days after infection.
Error bars indicate the SEM of data from triplicate plaque assays. The growth of each virus was significantly different with the two treatments at
each day postinfection (P ? 0.05, contrast test), except for DENV-2 at day 3.
10160STEIN ET AL. J. VIROL.
located 5? to the 5?CS is thought to be part of a highly ordered
region and, therefore, perhaps less accessible to siRNA (16, 65,
78). WNV-1 was 100% conserved across all 284 complete ge-
nomes against which it was tested by comparative alignment.
Interestingly, the sequences targeted by DC-3 and WNV-1 are
similar, with 12 bases targeted by DC-3 being present in WNV
and 17 of the 19 bases targeted by WNV-1 being present in
DENV-2 (Fig. 5A). Up to 18 nt in a sequence of 19 contiguous
nucleotides are predicted to comprise the 5?CS region for YFV
(65), and we therefore chose that entire region for siRNA
targeting. All 18 YFV genomes analyzed had 100% conserva-
tion with the YFV-1 sequence.
The antiviral activities of the 5?CS-targeting siRNAs against
WNV and YFV were examined in a dose-response manner.
The WNV 5?CS-directed siRNA (WNV-1) reduced the pro-
duction of WNV by only about 5- to 20-fold when present at 50
and 100 nM, respectively (Fig. 5B). We note that WNV grows
quickly and to high titers (more than 109PFU/ml under the
conditions used here) in Huh7 cells, and for that reason we
measured the infectious virus produced at 24 h p.i. In contrast,
the YFV 5?CS-directed siRNA (YFV-1) reduced viral release
by several logs when used at low-nanomolar concentrations
As a further control for the sequence specificity of the
siRNA in this study, Huh7 cells were transfected with 100 nM
NC, WNV-1, and YFV-1 siRNAs and then infected with 0.5
MOI of DENV-2 under the same conditions as those used for
the antiviral experiments described in the legend of Fig. 1.
WNV-1 produced a 3- to 4-fold, and YFV-1 or NC produced
a negligible, reduction in the titer of DENV-2 (data not
shown). Likewise, the DC-3 siRNA, which was potently inhib-
itory against DENV (Fig. 1 and 4), was not significantly active
against WNV or YFV (Fig. 5B and C).
These results suggest that although 5?CS-targeting siRNA
must be designed species specifically, the 5?CS region in gen-
FIG. 4. DENV-specific siRNAs reduce the production of infectious
virus in Huh7 cells in a dose-responsive manner. Cell cultures received
a single transfection with the indicated concentration of DC-3 (A) or
D2-2 (B) siRNA at 48 h before infection with DENV-2 at an MOI of
0.5. After a 2-h adsorption period the virus was removed and replaced
with medium containing no siRNA. At 48 h postinfection, superna-
tants were collected. Each experiment was carried out at least twice,
with similar results, and the mean values from triplicate plaque as-
says ? SEM are shown.
FIG. 5. Comparison of genomic sequences and siRNA targets in
DENV-2 and WNV at the 5?CS region and dose-response efficacy of
siRNAs targeting the 5?CS of WNV or YFV in Huh7 cell cultures.
(A) Alignment of the 5?CS regions of DENV-2 (GenBank accession
number NC_001474) bases 131 to 158 (top) and WNV (GenBank
accession number DQ211652) bases 136 to 161 (bottom). The 5?CS (10
nt for DENV-2 and 11 nt for WNV ) of each virus is shown in
boldface type, and the targets of DC-3 (in DENV-2) and WNV-1 (in
WNV) are underlined. The aligned sequence in common between
DENV-2 and WNV is shaded. (B and C) Cells received a single
transfection with the indicated concentration of the indicated siRNAs
at 48 h before infection with WNV (B) or YFV (C) at an MOI of 0.5.
After a 2-h adsorption period the virus was removed and replaced with
medium containing no siRNA. At 24 h (WNV) or 72 h (YFV) postin-
fection, supernatants were collected for plaque assays. Each experi-
ment was carried out at least twice, with similar results, and the his-
tograms show the mean values of triplicate plaque assays ? SEM.
Asterisks indicate a significant difference relative to that of the nega-
tive-control siRNA-treated group (*, P ? 0.05, Student t test).
VOL. 85, 2011 INHIBITION OF DENGUE VIRUS INFECTIONS BY siRNA10161
eral represents a sensitive site for the targeting of mosquito-
borne flaviviruses with exogenous siRNA.
Antiflavivirus siRNAs do not induce interferon-stimulated
gene expression. It was reported previously that siRNA can
produce innate immune stimulation in a sequence-specific
manner (34, 56). To rule out the possibility that the stimulation
of interferon production had contributed to the antiviral activ-
ity observed in this study, we tested select siRNAs at 100 nM
on uninfected Huh7-ISRE-luc cells, which contain a stably
integrated interferon-stimulated response element (ISRE)
fused to the luciferase coding sequence. Under the conditions
used in the antiviral experiments described above, none of the
siRNAs generated a significant stimulation of interferon, mea-
sured at either 8 h (optimal time for a firefly luciferase readout
with these cells) or 48 h (matching the time point at which cells
were infected in our antiviral assays), whereas the beta inter-
feron positive control induced a high level of reporter signal at
both time points (data not shown).
DC-3 inhibits DENV-2 infection and disease in AG129 mice.
To investigate if siRNA sequences could interfere with DENV
infection in vivo, we utilized a model of ADE-mediated disease
in AG129 mice. In this model, AG129 mice were infected with
DENV-2 strain S221 in the presence of DENV-1- to -4-reac-
tive antibody (anti-prM, clone 2H2), which mediates severe
pathology and death between days 4 and 8 (77). We chose to
test three siRNAs, a negative control (NC); DC-3, as it pos-
sessed high levels of activity against all 4 representative sero-
types; and D2-2, as it consistently produced higher antiviral
activity against DENV-2 than any other siRNA (Fig. 1 and 4).
Mice received the selected siRNAs via retro-orbital intrave-
nous injection at ?24 h, ?24 h, and ?72 h in relation to
infection. Although the same siRNA sequences were used, in
vivo application required the use of a specialized siRNA (Si-
lencer In Vivo Ready siRNA; Ambion) and transfection agent
(Invivofectamine 2.0; Invitrogen). One group was treated with
PBS only, and the other three groups received the transfection
reagent mixed with NC, DC-3, or D2-2 siRNA. The time of
survival of animals treated with DC-3 was significantly ex-
tended (median survival time of 14 days) compared to that of
PBS- or NC-treated animals (median survival time of 5 days)
(Fig. 6A). In contrast to the efficacy observed in cultured cells,
D2-2 failed to increase the survival times of the infected mice
compared to NC- or PBS-treated mice. Seventeen out of 18
animals in the PBS-, NC-, and D2-2-treated groups succumbed
to early death, a phenotype indicated by hunched posture,
ruffled fur, and severe weight loss, within the first 8 days fol-
lowing infection. In contrast, the eight DC-3-treated mice
maintained normal appearance and behavior until at least day
11 and were euthanized between days 12 and 17 following signs
of hind-limb paralysis. Body weight measurements mirrored
the survival pattern and reflected disease severity, with DC-3-
treated mice retaining on average around 95% of their prein-
fection body weight for at least 11 days after infection (Fig.
6B). The siRNA dose of 10 mg/kg was chosen based on rec-
ommendations from the manufacturer, and the normal health
and body weight of DC-3-treated animals after infection indi-
cate that the dosing was both subtoxic and efficacious. How-
ever, it may be noteworthy that treatment with siRNA that did
not produce an antiviral effect in vivo (NC and D2-2) appeared
to be associated with accelerated weight loss in infected ani-
mals (Fig. 6B).
To examine the effects of the DC-3 siRNA treatment on
viral replication directly, the viral load was assessed in several
tissues by RT-PCR at 24 and 72 h postinfection. In the ADE
model of infection used here, initial viral replication occurs in
the spleen, followed by increasing viral loads in other tissues
during the several days following infection. By 24 h p.i., mice
treated with DC-3 possessed an 18-fold-lower viral load in the
spleen than either control group of animals (PBS- or NC-
treated group) (Fig. 6C). DC-3 treatment also generated a
statistically significant 20-fold reduction in viremia compared
to NC treatment; however, the 7-fold reduction of the viremia
in the DC-3-treated group compared to the PBS-treated group
was not significant. By 72 h postinfection, the viral loads in the
serum and spleen had increased in all sample groups regardless
of treatment, and the differences were less pronounced. The
level of viremia in DC-3-treated mice was 8-fold lower than
that in the NC-treated group and remained consistently 3-fold
lower than that in the PBS-treated group. The greatest effect
from DC-3 treatment at 72 h postinfection was observed for
the kidney, where the viral load was 23-fold and 117-fold lower
than those of the PBS-treated and NC-treated groups, respec-
tively (Fig. 6D). DC-3 treatment also significantly suppressed
levels of viral RNA in the small intestine at 72 h compared to
those of the PBS-treated or NC-treated group (Fig. 6D). It is
noteworthy that in this model death from S221 is typically
preceded by intestinal hemorrhage (77) and that gastrointes-
tinal bleeding is the most common type of severe hemorrhage
in patients with severe dengue disease (13). Together, these
data indicate that treatment with DC-3 siRNA suppressed the
accumulation of viral RNA in selected tissues, thereby promot-
ing prolonged survival.
Tumor necrosis factor (TNF) is a key mediator of severe
DENV disease in this mouse model (77). Measurements taken
FIG. 6. DC-3 siRNA limits the viral load and TNF response and protects mice from lethal ADE-DENV infection. AG129 mice were treated
with 10 mg/kg of the indicated siRNAs via retro-orbital intravenous injection at 24 h before and 24 h and 72 h after the tail vein administration
of 109genomic equivalents of DENV-2 (S221) and the intraperitoneal injection of 5 ?g of antibody 2H2 (anti-prM/M, DENV-1 to -4 reactive).
(A) Survival time of AG129 mice (PBS, n ? 5; NC, n ? 8; DC-3, n ? 8; D2-2, n ? 5). The curve shows the combined results from two survival
experiments carried out under similar conditions. Log rank analysis indicates that the difference between the DC-3-treated groups and the other
groups is statistically significant at a P value of ?0.0001. (B) Body weight measured daily and represented as a percentage of the animal weight
24 h prior to infection. (C and D) Viral loads in serum and spleen at 24 and 72 h p.i. (for 24 h p.i., n ? 10 for PBS, n ? 5 for NC, and n ? 10
for DC-3; for 72 h p.i., n ? 5 per group) (C) and in the liver, kidney, and small intestine at 72 h p.i. (n ? 5 per group) (D). Viral RNA was quantified
by RT-PCR as described in Materials and Methods (***, P ? 0.0005;**, P ? 0.005;*, P ? 0.05 [Student t test]). (E) Levels of TNF in serum
collected from naïve and infected mice at 24 h and 72 h p.i. (n ? 5 per group), detected by ELISA as described in Materials and Methods (**,
P ? 0.005;*, P ? 0.05 [Student t test]).
VOL. 85, 2011 INHIBITION OF DENGUE VIRUS INFECTIONS BY siRNA10163
at 72 h postinfection showed that PBS- and NC-treated mice
had significantly elevated serum TNF levels compared to those
of mice treated with DC-3 (Fig. 6E). This result indicates that
DC-3 treatment was associated with reduced levels of a cyto-
kine known to mediate the early-death phenotype in this ani-
In this study we demonstrate that a synthetic siRNA, DC-3,
targeting the highly conserved 5?CS region, inhibits represen-
tative strains from each of the four DENV serotypes in cell
cultures. Furthermore, we show that an analog of DC-3, chem-
ically modified for in vivo applications, inhibited pathogenic
progression in a murine model of severe dengue disease. DC-3
was identified by the systematic testing of the antiviral activities
of a panel of siRNAs designed against sequences that are
highly conserved in the genomes of genetically diverse dengue
viruses. The majority of siRNAs designed solely on the basis of
the targeting of highly conserved sequences were not efficient
at inhibiting the growth of various strains of DENV in cell
cultures, presumably because other parameters were not con-
sidered in their design. In contrast, most of the siRNAs de-
signed against individual strains of DENV by a commercial
algorithm, which factors in such considerations as G/C content
and secondary structure, were effective against their targeted
strains in cell cultures. The target specificity of DC-3 was
confirmed in part by challenge against heterologous viruses.
Cell viability, TNF, and interferon assays further ruled out that
the antiviral activity of DC-3 was due to off-target effects. We
conclude therefore that DC-3 acted via RNA interference tar-
geting the 5?CS region.
During the course of this study, two other reports docu-
mented the antiflaviviral efficacy of nucleic acid-based agents
targeting the 5?CS. DENV-2 replication in mosquito cell cul-
tures was suppressed by group 1 splicing introns targeted to the
5?CS region (8), and WNV and St. Louis encephalitis virus
infections were inhibited in vitro and WNV infection was in-
hibited in a mouse model by a 5?CS-targeting siRNA conju-
gated to a 41-mer peptide (73). We observed a potent inhibi-
tion of YFV and a modest inhibition of WNV by the targeting
of their respective 5?CS regions. Taken together, these studies
show that the flavivirus 5?CS region is susceptible to RNAi-
mediated interventional strategies and that it warrants further
exploration as a target for the development of nucleic acid-
based inhibitors of flavivirus infections.
The 10-nt DENV 5?CS is highly conserved in dengue viruses
and complementary to the CS1 sequence (corresponding to nt
135 to 144 and 10618 to 10627, respectively, in DENV-2
[GenBank accession number NC_001474]). The base-pairing
interaction between the 5?CS and CS1 has been shown to be
important for the efficient replication of DENV (20, 35, 64,
76), WNV (42, 78), and YFV (14). The importance of the
interaction between the 5?CS and CS1 regions may render
these sites particularly vulnerable to RNAi, especially when the
flavivirus genome is present in a noncyclized form prior to
minus-strand synthesis. Alvarez et al. showed previously that
while the disruption of base pairing between the 5?CS and CS1
reduced DENV RNA synthesis, the replacement of base pairs
with different but complementary nucleotide sequences res-
cued viral RNA production. This result suggested that com-
plementarity rather than the specific nucleotide sequence of
the CS interaction was essential for viral replication (1). Thus,
DENV mutants with simultaneous replacements of comple-
mentary bases at the 5?CS and CS1 sequences could conceiv-
ably escape DC-3-mediated inhibition. However, the require-
ment for multiple mutations in disparate regions of the
genome lowers the likelihood of such a scenario. The fact that
the 5?CS and the CS1 regions are highly conserved across the
dengue viruses suggests that mutations in either region would
likely result in viruses with lower fitness in vivo.
The DENV CS1 region was previously found to be a pro-
ductive target site for sequence-specific antiviral inhibition by
peptide-conjugated morpholino oligomers (PPMO) (29, 36,
60) and shRNA (79). However, the DC-6 siRNA, designed
against the CS1 region, was consistently inactive. While a pos-
sible explanation for the discrepancy between results obtained
with the PPMO and those obtained with siRNAs could be their
differing mechanisms of action (steric blockade versus the
RNase-mediated cleavage of the target, respectively), the dif-
ference in results with AAV-vectored shRNA compared to
those with lipid-transfected siRNA remains unexplained. In-
terestingly, an siRNA designed against the first 20 nt of the
DENV genome (DC-1) was highly active against a DENV
strain from each of the four serotypes. The DC-1 target site is
of functional significance for various aspects of the DENV life
cycle, including translation (12, 28), RNA synthesis (19, 20, 31,
43), and RNA 5? cap methylation (15), and was also an effec-
tive target site for PPMO (29, 36, 60). The efficacy of DC-1 was
somewhat unexpected considering that its target is part of the
stable hairpin structure formed by the first 70 nt of the DENV
5?UTR (22, 45) and suggests that highly ordered RNA second-
ary structures in flaviviral genomes can be susceptible to
siRNA targeting. Although the DC-1 target sequence is not as
highly conserved across the DENV virome as the other con-
served siRNAs (Fig. 2), the 5?-terminal region of the genome
also appears to warrant further investigation as a target for
nucleic acid-based inhibitors.
In addition to producing multiple-log reductions of viral
titers in cell cultures, the DC-3 siRNA proved to be highly
effective against a lethal DENV infection in vivo. Compared to
two control groups, AG129 mice receiving DC-3 showed re-
duced TNF levels and suppressed viral RNA levels in the
spleen at 24 h p.i. and in kidney and small intestine at 72 h p.i.
DC-3-treated mice were protected from lethality for at least 2
weeks, in contrast to the other groups, in which most of the
mice died by day 5. Furthermore, DC-3-treated mice appeared
to eventually succumb to neurologic symptoms rather than to
the vascular pathology known to be a hallmark of the demise of
untreated AG129 mice infected with DENV strain S221 (77),
indicating robust antiviral activity early in the infection pro-
cess. The lack of antiviral efficacy of two other in vivo-ready
siRNAs (NC and D2-2) along with the lack of interferon stim-
ulation by DC-3 in vitro (data not shown) suggest that the
antiviral activity of DC-3 in vivo is most likely a result of
sequence-specific inhibition and not an off-target effect. The
ADE mouse model used in this study constitutes a severely
pathogenic DENV challenge, and the efficacy exhibited by the
DC-3 siRNA treatment suggests that the activation of endog-
10164STEIN ET AL.J. VIROL.
enous mammalian RNAi machinery by exogenous siRNA may
be sufficient to thwart a natural DENV infection.
In contrast to DC-3, D2-2 was unable to prevent DENV-2-
mediated disease in vivo despite the fact that D2-2 inhibited
DENV-2 more potently than DC-3 in vitro. The reason for
D2-2’s lack of efficacy in vivo is currently unknown. Although
different strains of DENV-2 were used in the in vitro and in
vivo arms of the study, both strains have identical sequences in
the D2-2 target region. It is possible that during replication
in vivo, viral mutants arose that were resistant to D2-2, which
targets a sequence which is less conserved and perhaps less
constrained than the 5?CS targeted by DC-3. Alternatively, it is
also possible that the stabilities of the two compounds differed
or that the siRNA sequence had some other effect on the
respective antiviral efficacy of each compound in vivo, although
neither of these possibilities has been addressed experimen-
tally. As the DC-3 treatment regimen employed here was un-
able to fully eradicate the virus, future studies will include in
vivo dose responsiveness, an extended time of treatment, and
postinfection-only dosing and will explore the potential gener-
ation and characterization of escape mutants.
To date, few compounds have been documented to extend
the time of survival of AG129 mice challenged with a lethal
DENV infection. A small-molecule adenosine nucleotide an-
alog was reported previously to completely protect S221-in-
fected mice for up to 11 days, while untreated mice succumbed
to primary infection by day 4 (11, 74), in a mortality pattern
similar to that reported here. In two other studies, one using a
PPMO targeting the 5?-terminal region of the genome and/or
the CS1 region (roughly equivalent in targeting to the DC-1
and DC-6 siRNAs here) (60) and the other using a heparin
sulfate mimetic (39), the average survival time of AG129 mice
receiving a lethal challenge of mouse brain-adapted DENV-2
strain New Guinea C was extended for about a week. Together,
the PPMO-, self-splicing intron-, and RNAi-based studies sug-
gest that the targeting of regions of highly conserved DENV
RNA with antisense-based methodologies may represent a
productive strategy for the development of antiflaviviral com-
The study here represents a further advancement in the
development of siRNA inhibitors of DENV infections. The
targeting of pathogenically relevant RNA is valuable for mech-
anistic analyses and is being developed clinically against vari-
ous human diseases (3, 10, 25, 38, 44). Further study will be
necessary to investigate whether this technology could be de-
veloped as the basis for a therapeutic system to address DENV
infections in humans.
This work was supported by the NIAID Pacific Northwest Regional
Center of Excellence for Biodefense and Emerging Infectious Diseases
Research (grant U54 AI 081680), National Center for Research Re-
sources support for the Oregon National Primate Research Center
(grant RR00163), and National Institutes of Health grants UO1
AI082196 and R44 AI079898 (M.K.S.). M.A.F. and J.L.S. were sup-
ported by OHSU training grants T32 A1074494 and T32 AI07472.
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