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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.

SUBMITTER: Stein DA

PROVIDER: S-EPMC3196423 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence.

Stein David A DA Perry Stuart T ST Buck Michael D MD Oehmen Christopher S CS Fischer Matthew A MA Poore Elizabeth E Smith Jessica L JL Lancaster Alissa M AM Hirsch Alec J AJ Slifka Mark K MK Nelson Jay A JA Shresta Sujan S Früh Klaus K

Journal of virology 20110727 19

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' cyc ...[more]

PMID: 21795337

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Project description:BackgroundThe sequence diversity of dengue virus (DENV) is one of the challenges in developing an effective vaccine against the virus. Highly conserved, serotype-specific (HCSS), immune-relevant DENV sequences are attractive candidates for vaccine design, and represent an alternative to the approach of selecting pan-DENV conserved sequences. The former aims to limit the number of possible cross-reactive epitope variants in the population, while the latter aims to limit the cross-reactivity between the serotypes to favour a serotype-specific response. Herein, we performed a large-scale systematic study to map and characterise HCSS sequences in the DENV proteome.MethodsAll reported DENV protein sequence data for each serotype was retrieved from the NCBI Entrez Protein (nr) Database (txid: 12637). The downloaded sequences were then separated according to the individual serotype proteins by use of BLASTp search, and subsequently removed for duplicates and co-aligned across the serotypes. Shannon's entropy and mutual information (MI) analyses, by use of AVANA, were performed to measure the diversity within and between the serotype proteins to identify HCSS nonamers. The sequences were evaluated for the presence of promiscuous T-cell epitopes by use of NetCTLpan 1.1 and NetMHCIIpan 3.2 server for human leukocyte antigen (HLA) class I and class II supertypes, respectively. The predicted epitopes were matched to reported epitopes in the Immune Epitope Database.ResultsA total of 2321 nonamers met the HCSS selection criteria of entropy < 0.25 and MI > 0.8. Concatenating these resulted in a total of 337 HCSS sequences. DENV4 had the most number of HCSS nonamers; NS5, NS3 and E proteins had among the highest, with none in the C and only one in prM. The HCSS sequences were immune-relevant; 87 HCSS sequences were both reported T-cell epitopes/ligands in human and predicted epitopes, supporting the accuracy of the predictions. A number of the HCSS clustered as immunological hotspots and exhibited putative promiscuity beyond a single HLA supertype. The HCSS sequences represented, on average, ~ 40% of the proteome length for each serotype; more than double of pan-DENV sequences (conserved across the four serotypes), and thus offer a larger choice of sequences for vaccine target selection. HCSS sequences of a given serotype showed significant amino acid difference to all the variants of the other serotypes, supporting the notion of serotype-specificity.ConclusionThis work provides a catalogue of HCSS sequences in the DENV proteome, as candidates for vaccine target selection. The methodology described herein provides a framework for similar application to other pathogens.

Dataset Information

Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence.

Publications

Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence.

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