BackgroundIncreased vascular permeability is a hallmark feature in severe dengue virus (DV) infection, and dysfunction of endothelial cells has been speculated to contribute in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Rho-family GTPase Rac1 is a significant element of endothelial barrier function regulation and has been implicated in the regulation of actin remodeling and intercellular junction formation. Yet there is little evidence linking Rac1 GTPase to alteration in endothelial cell function induced by DV infection.Methods and FindingsHere, we showed that actin is essential for DV serotype 2 (DV2) entry into and release from ECV304 cells, and Rac1 signaling is involved these processes. At early infection, actin cytoskeleton rearranged significantly during 1 hour post infection, and disrupting actin filament dynamics with jasplakinolide or cytochalasin D reduced DV2 entry. DV2 entry induced reduction of Rac1 activity within 1 hour post infection. The expression of dominant-negative forms of Rac1 established that DV2 entry is negatively regulated by Rac1. At late infection, actin drugs also inhibited the DV2 release and induced accumulation of viral proteins in the cytoplasm. Meanwhile, the activity of Rac1 increased significantly with the progression of DV2 infection and was up-regulated in transfected cells expressing E protein. Confocal microscopy showed that DV2 E protein was closely associated with either actin or Rac1 in DV2-infected cells. The interaction between E protein and actin was further confirmed by co-immunoprecipitation assay.ConclusionsThese results defined roles for actin integrity in DV2 entry and release, and indicated evidence for the participation of Rac1 signaling pathways in DV2-induced actin reorganizations and E-actin interaction. Our results may provide further insight into the pathogenesis of DHF/DSS.
Caveolae- and clathrin-mediated endocytosis are major internalization pathways used by several pathogens; however, their distinctive roles in dengue virus (DV) entry have not been addressed. In this study, we compared the involvement of caveolae- and clathrin-mediated endocytosis in the infectious entry of DV serotype 2 (DV2) into human endothelial-like ECV304 cells. Confocal microscopy study on DV2-infected cells showed that viral antigens were co-localized with clathrin heavy chains, epidermal growth factor pathway substrate clone 15 (Eps15), and adaptin-alpha, but not with caveolin-1. Treatment with chlorpromazine, which inhibits clathrin-dependent endocytosis, led to reduced virus entry into cells, whereas treatment with nystatin, a caveolae inhibitory agent, did not. Furthermore, gene silencing of Eps15 resulted in an average of 75% reduced infection of ECV304 cells by DV2. Our results demonstrated that DV2 enters ECV304 cells by clathrin-dependent endocytosis, not by caveolae-dependent endocytosis.
Background: Genetic diseases represent a significant public health challenge in China that will need to be addressed by a correspondingly large number of professional genetic counselors. However, neither an official training program for genetic counseling, nor formal board certification, was available in China before 2015. In 2009, a genetic counseling training program based on role-playing was implemented as a pilot study at the Third Military Medical University to train third-year medical students. Methods: Questionnaires on participant attitudes to the program and role-playing were randomly administered to 324 students after they had finished their training. Pre-and post-training instructional tests, focusing on 42 key components of genetic counseling, were administered randomly to 200 participants to assess mastery of each component. Finally, scores in final examinations of 578 participants from 2009 to 2011 were compared to scores obtained by 614 non-participating students from 2006 to 2008 to further assess program efficacy.
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.
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