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Phylogenetic analysis suggested that the DENV1 strains isolated in the DF outbreak in Guangdong province in 2006 were likely to be imported from Southeast Asian. Specifically, viruses isolated from Shantou and Chaozhou were imported from Singapore; viruses isolated from Guangzhou, Yangjiang, and Foshan were imported from Thailand/Vietnam. Therefore, importation of DENV1 from Southeast Asia was an important contributory factor of the 2006 DF outbreak in Guangdong province., (Copyright © 2011 Elsevier B.V. All rights reserved.)

RNA interference can be mediated by fully complementary siRNA or partially complementary miRNA. siRNAs are widely used to suppress viral replication and the fully complementary siRNA bound Ago-2 in the RISC is known to degrade the target RNA. Although other argonaute proteins lacking slicer activity can also bind oligonucleotides with both si and miRNA structures, whether they can also contribute to antiviral effects is not entirely clear. We tested si and miRNA structured oligos for target repression in dual luciferase assays as well as for inhibition of Dengue and West Nile virus replication in ES cells expressing individual Ago proteins. In luciferase assays, both fully complementary and partially complementary oligos effectively repressed their targets in all individual Ago expressing cell lines, although the efficacy with fully complementary oligos was higher in Ago-2+ cells. However, partially complementary oligos had no effect on virus replication in any cell line, while fully complementary siRNAs were highly effective in Ago-2 expressing, but not in cells expressing other Ago proteins. This occurred irrespective of whether the target sequences were located in the coding region or 3'UTR of the virus. We conclude that Ago-2 slicer activity is essential for anti-viral efficacy of siRNAs and miRNA-mediated translational repression/transcript destabilization is too weak to suppress the abundantly expressed flaviviral proteins.

The role of the 3'untranslated region (UTR) of the dengue virus (DENV) genome during viral translation remains to be elucidated. We assessed the contribution of well-defined RNA elements in the 3'UTR of DENV-2 to viral translation using a virus-induced reporting gene system and deoxyribozymes (DRzs) targeting the 3'UTR of the DENV-2 genome. Results show that mRNAs carrying a deletion of repeated conserved sequence (RCS2)-CS2 are translated less efficiently than wild type mRNAs. However, mRNAs with a deletion of CS1-stem loop (SL) are translated more efficiently. Thus, CS1-SL and RCS2-CS2 may have different effects on translational regulation. Additionally, the translation-suppressing effect of CS1-SL or the SL element is further confirmed in DENV-2-infected cells using DRzs. Mutagenesis studies show that, rather than the secondary structure, nucleotides 10663-10677 and 10709-10723 are responsible for translational suppression of SL. Overall, our results demonstrate that sequences and elements within the DENV-2 3'UTR regulate viral translation.

In the present study, the domain IIIs of all four dengue virus (DENV) serotypes were connected sequentially to construct the tandem domain III. The resulting DNA fragment was then cloned into pBAD/Topo ThioFusion plasmid. After induction, the Escherichia coli expression protein was purified and used to immunize BALB/c mice by subcutaneous route. The sera from mice immunized with the purified protein were confirmed to contain specific high antibody titers against DEN1, DEN2, and DEN4, and moderate antibody titer against DEN3. In suckling mouse model, 70% of the mice challenged with DEN1, DEN2, and DEN4 in combination with sera from mice immunized with the purified protein were protected, and 18% of the mice challenged with DEN3 in combination with the same sera were protected. Our data suggest that the tandem domain III of the envelope protein can be used as a potential tetravalent dengue vaccine based on a single antigen.