ABSTRACT
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ABSTRACT
Since the hemorrhage in severe dengue seems to be primarily related to the defect of the platelet, the possibility that dengue virus (DENV) is selectively tropic for one of its surface receptors was investigated. Flow cytometric data of DENV-infected megakaryocytic cell line superficially expressing human glycoprotein Ib (CD42b) and glycoprotein IIb/IIIa (CD41 and CD41a) were analyzed by our custom-written software in MATLAB. In two-dimensional analyses, intracellular DENV was detected in CD42b+, CD41+ and CD41a+ cells. In three-dimensional analyses, the DENV was exclusively detected in CD42b+ cells but not in CD42b- cells regardless of the other expressions. In single-cell virus-protein analyses, the amount of DENV was directly correlated with those of CD42b at the Pearson correlation coefficient of 0.9. Moreover, RT- PCR and apoptosis assays showed that DENV was able to replicate itself and release its new progeny from the infected CD42b+ cells and eventually killed those cells. These results provide evidence for the involvement of CD42b in DENV infection.
Subject(s)
Dengue Virus/physiology , Platelet Glycoprotein GPIb-IX Complex/physiology , Blood Platelets/metabolism , Cell Line , Cells, Cultured , Dengue/virology , Dengue Virus/immunology , Dengue Virus/pathogenicity , Flow Cytometry/methods , Humans , Megakaryocytes/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Membrane Glycoprotein IIb/physiology , Severe Dengue/metabolism , Tropism , Viral Tropism/physiologyABSTRACT
Understanding dengue virus (DENV)-induced hemorrhage remains a challenging jigsaw puzzle with many pieces missing to understand the complex interactions between DENV and blood coagulation system. To use flow cytometry studying the interactions between DENV and human platelet aggregation receptor, glycoprotein IIb/IIIa (gpIIb/IIIa), directly conjugated fluorochrome monoclonal antibody (mAb) is essential to facilitate multifluorochrome immunostaining. However, the obstacle was that no directly conjugated fluorochrome-anti-DENV mAb had been commercially available. To overcome, we directly conjugated fluorochrome to a primary anti-DENV mAb using a LYNX rapid conjugation kit. Flow cytometry analysis showed that this conjugated antibody and anti-gpIIb/IIIa mAb were able to detect DENV and CD41a simultaneously. Fluorescence microscopy analysis further demonstrated CD41a superficially and DENV intracellularly. Potentially, this strategy can facilitate virologists for directly conjugating any virus-specific primary antibodies, which are not commercially available with fluorochrome, to study the infectivity in any surface marker-specific hosts through flow cytometry. Together, DENV can interact with both human gpIIb/IIIa- and gpIIb/IIIa+ cells revealed by flow cytometry and fluorescence microscopy for the first time.
