| Dengue virus (DV) is a member of mosquito-borne flaviviruses and possesses a positive-sense RNA genome. Infection with DV can result in classical dengue fever (DF) and/or dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in the tropics and subtropics worldwide. Vascular leak is the hallmark of dengue haemorrhagic fever, which implies dysfunction of the vascular endothelium. The replication of DV in vitro in human endothelial cells has been demonstrated by viral titers and immunofluorescent antibody studies. We therefore suggest that VEC is not only an important effective cell in DHF, but also a target for DV proliferation in vivo.Microfilaments play a role in changes of both shape and function of VEC. Microfilaments , also called fibrous-actin (F-actin), are filaments aggregated by global-actin(G-actin). These bundles usually form a dynamic network distributed in the cortical area beneath the membrane, associate with the cellular shape, migration, etc. Bacteria and virus particles that infect eukaryotic cells can take advantage of the actin cytoskeleton to penetrate, translocate, or release. Two kinds of drugs are commonly used to perturb the actin treadmilling cycle: one binds to the barbed ends of the actin filaments inhibiting the polymerization of subunits, such as cytochalasin, latrunculin A; the other enhances actin polymerization by inhibiting the depolymerization of actin filaments, such as jasplakinolide, phalloidin. In the present study, we used jas and cyto D to investigate whether actin filaments may play a role in the dengue virus serotype 2 (DV2) infection of ECV304 cell line, which was derived from human umbilical vein endothelial cells.?RESULTS DV-induced actin depolymerization and morphologic changes in human endothelial This work was partially supported by grants no. 30170848 and no. 30300303 from the National Science Foundation of China (NSFC)cells. Untreated ECV304 cells exhibited strong peripheral F-actin, as well as stress fiber formation in a large portion of the cells. However, the addition of DV caused a rapid depolymerization of actin within the cell at 5 min after infection. The depolymerization was accompanied by polymerization of short F-actin at 10min after infection. Few stress fibers were still visible at 30min after infection and F-actin "dots" and "rings" were observed at this time. Some actin staining was along the cell edge, indicative of cortical actin fibers, and recovered at 1 hr after infection. F-actin networks in some cells depolymerized again at 24, 48, 72 and 120 hr after infection, and might due to the accumulation of viral proteins. The distributions of some F-actin and some "actin tails"-like structure were highly related to the viral proteins. These results showed that the actin cytoskeleton could be induced to depolymerize, polymerize and rearrange after the DV infection. This indicates actin may play a role in DV infection, and the involement of actin may contribute to VEC dysfunction after DV infection. However, the DV antigen aggregation doesn't necessarily cause the actin rearrangement. In some viral antigen-positive cells, the actin networks were still intact. While in others, where the distribution of viral antigens were usually "flame"-like, some stress fibers depolymerized and short F-actins were polymerized near the viral proteins. This indicated that the actin cytoakeleton may play a role in viral assembly and/or transportation. The rearrangements of actin cytoskeleton in HUVEC after DV infection were similar to those in ECV304.Effects of cyto D and jas on DV replicationIn certain experiments, cells were incubated with cyto D and jas respectively just after the DV infection, lasting for 5 days. Without drug treatments, the virus titer increased at day 2, reached to peak at day 3, then slightly reduced thereafter. Though the virus titer also reached to the peak at day 3, but they decreased about 35% after treatment by 2 drugs. This indicated that jas and cyto D could interrupt the propagations of DV to some extent. S...
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