Yanping Tian

Inhibitory effects of glutathione on dengue virus production.

Reduced glutathione (GSH) is the most powerful intracellular antioxidant and also involved in viral infections. The pathogenesis of dengue virus (DV) infection has not been completely clarified. This study investigated the relationship between DV serotype 2 (DV2) infections and host intracellular GSH content. Results showed infection with DV2 resulted in a decrease in intracellular GSH, which caused NF-kappaB activation and increased DV2 production. Supplemental GSH significantly inhibited activation of NF-kappaB, resulting in a decreased production of DV2 in HepG2 cells. Furthermore, high activity of NF-kappaB and increased production of DV2 was observed in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. In conclusion, DV2 infection could reduce host intracellular GSH concentration and benefited from this process. Supplemental GSH could inhibit viral production, indicating GSH might be valuable in the prevention and treatment of DV2 infection.

Inhibitory Effect of Glutathione on Oxidative Liver Injury Induced by Dengue Virus Serotype 2 Infections in Mice

The pathogenesis of dengue virus (DV) infection has not been completely defined and change of redox status mediated by depletion of glutathione (GSH) in host cell is a common result of viral infection. Our previous study has demonstrated that DV serotype 2 (DV2) infection alters host intracellular GSH levels, and exogenous GSH inhibits viral production by modulating the activity of NF-κB in HepG2 cells. GSH is the most powerful intracellular antioxidant and involved in viral infections. Thus, this study was to investigate whether DV2 infection can induce alteration in redox balance and effect of GSH on the disease in HepG2 xenografts SCID mice. Our results revealed that mice infected with DV2 showed alterations in oxidative stress by increasing the level of malondialdehyde (MDA), an end product of lipid peroxidation, and GSSG/GSH ratio. DV2-infected mice also showed a decrease in the activity of catalase (CAT) and total superoxide dismutase (T-SOD) in the serum and/or observed organs, especially the liver. Moreover, DV2 infection resulted in elevated serum levels of the cytokines tumor necrosis factor-α and interlukin-6 and obvious histopathological changes in the liver. The administration of exogenous GSH significantly reversed all of the aforementioned pathological changes and prevented significant liver damage. Furthermore, in vitro treatment of HepG2 cells with antioxidants such as GSH inhibited viral entry as well as the production of reactive oxygen species in HepG2 cells. These results suggest that GSH prevents DV2-induced oxidative stress and liver injury in mice by inhibiting proinflammatory cytokine production, and GSH and may be a promising therapeutic agent for prevention of oxidative liver damage during DV infection.

Vimentin is required for dengue virus serotype 2 infection but microtubules are not necessary for this process

The present study investigated the effect of microtubules (MTs) and vimentin during dengue virus serotype 2 (DV2) infection. Immunostaining showed that DV2 infection induced MT and vimentin reorganization. Colocalization of DV2 antigens with MTs or vimentin were often observed in ECV304 cells. MT-disrupting agents could enhance DV2 release but did not affect other steps of virus replication. In contrast, disruption of vimentin inhibited DV2 infection. Our results suggest that an MT-dependent mechanism may not be necessary for DV2 infection, and MT disruption may promote DV2 release. However, vimentin is required for DV2 infection.

ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus

Our previous study showed that dengue virus 2 (DENV2) infection induces rearrangement of vimentin into dense structures at the perinuclear area. However, the underlying mechanism of this phenomenon is poorly characterized. In the present work, we found that vimentin and Ser71 phosphorylated vimentin display similar distributions in DENV2-infected cells. DENV2 infection also induced ROCK activation and phosphorylation of vimentin at Ser71 as the DENV2 infection progressed. Furthermore, Ser71 phosphorylation and vimentin rearrangement induced by DENV2 infection were blocked by the ROCK inhibitor Y-27632. In addition, DENV2 led to endoplasmic reticulum (ER) redistribution in the perinuclear region of the host cells, which was partially blocked by pretreatment with Y-27632. Together, these data support indicate that ROCK may have a role in governing regulating vimentin and ER rearrangement during DENV2 infection. We hypothesize that DENV2 infection, via ROCK activation, induces both vimentin rearrangement and ER redistribution around the perinuclear region, which may play a structural role in anchoring DENV2 to replication sites.

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody.

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.

Rab8, a Vesicular Traffic Regulator, Is Involved in Dengue Virus Infection in HepG2 Cells

Objective: The pathogenesis of dengue virus (DV) has not been completely clarified. Rab8 regulates vesicular traffic from Golgi to plasma membrane where DV is matured and then delivered by exocytosis. In this study, involvement of Rab8 in DV serotype 2 (DV2) infection was investigated in HpeG2 cells. Methods: Distributions of Rab8 and DV2, and the number of infection cells were observed by immunostaining. HepG2Rab8AM and HepG2Rab8DN cells were constructed to stably express a constitutively active mutant of Rab8 and a dominant negative mutant, respectively, which were assessed by flow cytometry. Production of infectious virions and the amounts of DV2 entry were detected by standard plaque assay. Viral RNA replication was detected by real-time RT-PCR. Results: Rab8 showed high co-localization with DV2 in HpeG2 cells and the amount of DV antigen-positive cells decreased in HepG2Rab8AM and HepG2Rab8DN cells. Also, progeny virus released from those cells was drastically reduced. Infectious virions produced in cells were also significantly reduced, while the viral RNA replication was down-regulated by a different level. Furthermore, viral entry into those cells was reduced by about 80%. Conclusions: Our data suggest that the function of Rab8 is important for DV2 infection, and Rab8 may be involved in DV2 infection.

Production of a Monoclonal Antibody Against Non-Structural Protein 3 of Dengue-2 Virus by Intrasplenic Injection

Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are highly infectious diseases caused by dengue virus (DV). DV non-structural protein 3 (NS3) is known to possess ATPase, helicase, and protease activity that is a constitutive part of the replication complex of DV. In this study, we discuss the cloning, expression, and purification of the DV-2 NS3 protein to immunize mice by intrasplenic injection and then to generate a monoclonal antibody (MAb). One MAb, named 4F5, was obtained and it was specific to NS3 of DV-2. Immunofluorescence show that 4F5 recognizes the native protein in infected ECV304 cells. Likewise, C6/36-infected lysates were used in Western blot analysis, and we observed the specific characteristic band that defines NS3. We conclude that MAb 4F5 may be a useful tool, not only to study the replicative process of DV, but also to generate specific diagnostic tools for DV infection.

Application of antibodies against nonstructural protein 2B of dengue serotype 2 virus induced by DNA immunisation or recombinant protein NS 2B immunisation in BALB/c mice.

To investigate the structure and function of nonstructural (NS) protein 2B of the dengue serotype 2 virus (DV2) during infection, polyclonal antibodies (Abs) against DV2 NS2B were prepared by immunisation with NS2B protein or by DNA immunisation. The full-length NS2B gene was cloned and inserted into the prokaryotic expression vector pQE31, resulting in a vector, named pQE-NS2B, or into the eukaryotic expression vector pCAGGS-P7, resulting in the vector pCAG-NS2B. The pQE-NS2B vector was transfected into Escherichia coli JM109, and recombinant NS2B protein was obtained by Ni(2+)-NTA agarose affinity chromatography. Vero cells transfected with pCAG-NS2B showed that NS2B protein can be expressed in eukaryotic cells. Finally, mice were immunised with the recombinant NS2B protein or pCAG-NS2B. Anti-NS2B sera from the immunised mice could specifically react with DV2 NS2B proteins, as visualised by fluorescence staining and Western blotting. Immunisation with NS2B protein induced a higher titre of the antibody than that induced by DNA immunisation. These data indicate that our antisera against DV2 NS2B can recognise both the natural and denatured NS2B protein. Based on these results, the polyclonal Abs could be used as a tool for studying the role of NS2B in the pathogenesis of DV2.