Dengue Virus NS1 Exposure Affects von Willebrand Factor Profile and Platelet Adhesion Properties of Cultured Vascular Endothelial Cells.

Hematological abnormalities and altered vascular permeability are frequently encountered in Dengue virus infected patients, but the mechanisms that alter platelet-endothelium interactions remain incompletely understood. The DENV NS1 protein has been implicated in adverse disease outcomes. In the present study the role of NS1 protein in affecting the expression of vWF and platelet adhesion properties of endothelial cells was studied in vitro. The results suggest that vWF is down regulated in cultured endothelial cells 6 and 24 h after exposure with increase in vWF levels in culture supernatants at corresponding time points. Ultrastructural studies showed distinct evidence of endothelial cell activation morphology and degranulation of Weibel-Palade bodies in NS1 exposed cells that also showed increased platelet activation physiology. The findings suggest that changes in vWF production and secretion may be induced in endothelial cells exposed to DENV NS1 protein; and play a role in bleeding complications of severe DENV disease.

Dengue virus infection of SK Hep1 cells: inhibition of in vitro angiogenesis and altered cytomorphology by expressed viral envelope glycoprotein.

Dengue virus (DENV) infection of human endothelial cells has been implicated in the pathobiology of dengue hemorrhagic fever and dengue shock syndrome. However, the mechanisms by which DENV infections alter the functional physiology of endothelial cells remain incompletely understood. In the present study, we examined the susceptibility of a human liver sinusoidal endothelial cell line SK Hep1 to all four serotypes of DENV and studied the effect of the virus on in vitro angiogenesis. All four serotypes of DENV could infect the SK Hep1 cells, but showed variable cytopathic effects, the most pronounced being that of DENV-2. Electron microscopy of the infected cells showed significant ultrastructural changes. In vitro angiogenesis assays on DENV-2 exposed SK Hep1 cells in the matrigel system showed inhibition compared with the controls. Importantly, transfection and transient expression of the DENV-2 envelope glycoprotein (E) in these cells showed drastic alterations in cell shapes and the E protein could be localized by fluorescence microscopy in terminal knob-like structures. Therefore, SK Hep1, a human hepatic sinusoid-derived endothelial cell line, may constitute a potential model to study DENV-endothelial cell interactions in vitro, especially towards understanding the possible virus-induced changes in hepatic endothelium and its role in disease pathogenesis.

Direct imaging of pH1N1 2009 influenza virus replication in alveolar pneumocytes in fatal cases by transmission electron microscopy.

Human influenza virus pandemics constitute a major global public health issue. Although studies on autopsy specimens from the recent pandemic by the 2009 influenza A (H1N1) virus have revealed a broad spectrum of pathologic findings, direct electron microscopic studies of the lung tissue from influenza fatalities are few. In this study, we examined five well-preserved pulmonary necropsy specimens from fatal cases of laboratory-confirmed pH1N1 from India. The novel observations in comparison with earlier reports included direct imaging of influenza virus budding within dilated cisternae of pneumocytes, cell-free virus emerging from the cell membrane of a pneumocyte in the alveolar lumen, presence of polymorphonuclear cells with red blood cells as inflammatory exudates close to hyaline membranes and extensive cytoplasmic degeneration of epithelial cells of the alveolar lining. These observations are in consistent with the earlier findings and emphasize the possible role of this virus directly infecting cells of the lower respiratory tract as a key event in the rapid pathogenesis of pH1N1 disease process.

Dengue virus-induced autophagosomes and changes in endomembrane ultrastructure imaged by electron tomography and whole-mount grid-cell culture techniques.

The biogenesis events and formation of dengue virus (DENV) in the infected host cells remain incompletely understood. In the present study, we examined the ultrastructural changes associated with DENV-2 replication in three susceptible host cells, C6/36, Vero and SK Hep1, a cell line of human endothelial origin, using transmission electron microscopy, whole-mount grid-cell culture techniques and electron tomography (ET). The prominent feature in C6/36 cells was the formation of large perinuclear vacuoles with mature DENV particles, and on-grid whole-mount examination of the infected Vero cells showed different forms of DENV core structures associated with cellular membranes within 48 h after infection. Distinct multivesicular structures and prominent autophagic vesicles were seen in the infected SK Hep1 cells when compared with the other two cell lines. ET showed the three-dimensional organization of these vesicles as a continuous system. This is the first report of ET-based analysis of DENV-2 replication in a human endothelial cell line. These results further emphasizes the strong role played by intracellular host membranes-virus interactions in the biogenesis of DENV and strongly argues for the possibility of targeting compounds to block such structure formation as key anti-dengue agents.

Imaging the interaction between dengue 2 virus and human blood platelets using atomic force and electron microscopy.

Thrombocytopenia is frequently associated with dengue virus infection. Host factors such as anti-platelet immunopathogenic processes have been implicated in the origin of dengue-associated thrombocytopenia but the role of dengue virus in directly interacting with platelets and altering their hemostatic property remains incompletely understood. In the present study, we examined the effect of dengue 2 virus on the morphology and physiological activation profile of normal human platelets using atomic force microscopy, electron microscopy and flowcytometry. Platelets obtained from healthy donors were exposed to a cell culture-adapted 10(4) LD(50) dengue 2 virus isolate in vitro and the subsequent effect on morphology and activation biology studied. Our results show that dengue 2 virus exposure at doses comparable to natural viremic states in human infections can activate platelets with an increase in P-selectin expression and fibrinogen-binding property. Atomic force, scanning and transmission electron microscopy also showed typical activation-related morphological changes such as altered platelet membrane architecture, degranulation, presence of filopodia and dilatation of the open canalicular system in the dengue 2 virus-exposed platelets but not in the controls. Importantly, Japanese encephalitis virus exposure at the same dose did not activate platelets or show any morphological changes. Our findings suggest that dengue 2 virus may directly interact with and activate platelets - an event that might be important in the origin of dengue-associated thrombocytopenia. Detailed molecular characterization of this effect might provide key knowledge toward better prophylaxis of the hemostatic complications of dengue disease.

Dengue 2 virus inhibits in vitro megakaryocytic colony formation and induces apoptosis in thrombopoietin-inducible megakaryocytic differentiation from cord blood CD34+ cells.

Thrombocytopenia is frequently associated with dengue virus infection in humans. Although antiplatelet immunopathogenic processes have been implicated in the origin of dengue-associated thrombocytopenia, the effect of dengue viruses on megakaryocyte differentiation remains incompletely understood. In this study, we examined the effect of human dengue 2 virus isolates on the in vitro growth and differentiation of thrombopoietin-induced megakaryopoiesis of cord blood CD34+ cells. Dengue 2 viruses, but not Japanese encephalitis virus, showed a dose-dependent inhibition of CFU-Mk. Viral antigens could be detected by an immunohistochemical technique in 3-5% of the early megakaryocytic progenitors by the 5th postexposure day in liquid cultures with cell loss, increased annexin V binding and active caspase-3 expression. In summary, dengue 2 viruses can inhibit in vitro megakaryopoiesis, as well as infect and induce apoptotic cell death in a subpopulation of early megakaryocytic progenitors. These events might contribute towards the origin of thrombocytopenia in dengue disease.