A Synthetic Bioactive Peptide Derived from the Asian Medicinal Plant Acacia catechu Binds to Dengue Virus and Inhibits Cell Entry

Dengue virus (DENV) infection has become a critically important globally prevalent infectious disease, especially in tropical and subtropical countries. Since neither currently exists, there is an urgent need for an effective vaccine to prevent, and a specific drug to treat DENV infection. Therapeutic peptides represent an attractive alternative for development into anti-DENV drugs due to their safety and their diverse biological and chemical properties. We recently reported novel bioactive peptides extracted from the Asian medicinal plant Acacia catechu that efficiently inhibited all four DENV serotypes. In this study, we investigated the anti-DENV activity of a synthetic bioactive peptide derived from this plant. The most effective peptide (designated Pep-RTYM) inhibited DENV infection with a half-maximal inhibition concentration value of 7.9 μM. Time-of-addition study demonstrated that Pep-RTYM interacted with DENV particles and inhibited cellular entry. Pep-RTYM at 50 μM significantly reduced DENV production in Vero-kidney epithelial cells about 1000-fold, but it could decrease the virus production in Huh7 hepatocyte cells approximately 40-fold. Binding of Pep-RTYM to DENV particles may prevent virus interaction with cellular receptor and subsequent virus entry. This finding suggests a potential role of Pep-RTYM in the development of a novel anti-DENV drug.     

Cell death gene expression profile: role of RIPK2 in dengue virus-mediated apoptosis

Dengue virus (DENV) is a major emerging arthropod-borne pathogen, which infects individuals in both subtropical and tropical regions. Patients with DENV infection exhibit evidence of hepatocyte injury. However, the mechanisms of hepatocyte injury are unclear. Therefore we examined the expression of cell death genes during DENV-infection of HepG2 cells using real-time PCR arrays. The expression changes were consistent with activation of apoptosis and autophagy. Expression of the up-regulated genes, including RIPK2, HRK, TGF-β, PERK, and LC3B, was confirmed by quantitative real-time PCR. RIPK2 belongs to the receptor-interacting protein family of serine/threonine protein kinases, which is a crucial mediator of multiple stress responses that leads to the activation of caspase, NF-κB and MAP kinases including JNK and p38. RIPK2 activity is inhibited by the p38 MAPK pathway inhibitor SB203580. The effect of SB203580 on RIPK2 expression and DENV-induced apoptosis was tested in DENV-infected HepG2 cells. The inhibition of RIPK2 expression by SB203580 significantly reduced apoptosis. SB203580 also significantly reduced DENV capsid protein (DENVC)-mediated apoptosis. Suppression of endogenous RIPK2 in DENV-infected HepG2 cells by small interfering RNA (siRNA) significantly decreased apoptosis suggesting for the first time that RIPK2 plays a role in DENV-mediated apoptosis.     

Role of CD61+ cells in thrombocytopenia of dengue patients

Although hematological disorders with salient features of thrombocytopenia have been well documented in dengue patients, the role of CD61-expressing platelets and the megakaryocytic cell lineage in the pathogenesis of dengue virus (DENV) infection remains largely unexplored. A prospective observational study was performed using blood samples and PBMCs from dengue-confirmed patients, as well as from rhesus monkeys (RM) experimentally infected with DENV. Immunohistochemical staining and FACS techniques were applied to evaluate the frequencies of CD61⁺ cells that contained DENV antigen. Highly enriched population of CD61⁺ cells was also isolated from acute DENV-infected RM and assayed for DENV RNA by quantitative RT-PCR. Results revealed that DENV antigen was found in small vesicles of varying size, and more frequently in anucleated cells associated with platelets in dengue patients. The DENV antigen-containing cells were CD61⁺ and appeared to share characteristics of megakaryocytes. Kinetic profiles of CD61⁺ cells from DENV-infected RM revealed a transient increase in CD61⁺CD62P⁺ cells early after DENV infection. DENV RNA in a highly enriched population of CD61⁺ cells from the infected RM was observed during acute stage. Our results indicate that virus containing CD61⁺ cells may be directly linked to the platelet dysfunction and low platelet count characteristics of dengue patients.