Selective Tropism of Dengue Virus for Human Glycoprotein Ib

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.

Figure 1

Tropism of CD42b, CD41 and CD41a in dengue virus. (A) CD42b is the marker of mature megakaryocytes and platelets. CD41 is the marker of early megakaryocytes. (B–G) MEG-01 cells were infected with DENV (MOI = 0.5) for 2 hours and washed with PBS. The cultures were maintained in fresh medium for another 7 days before being double immunostained. (B,D,F) Representative two-dimensional plot of surface platelet receptors and intracellular DENV ((B) CD42b, (D) CD41,  (F) CD41a). (C,E,G) Percentage of DENV⁺ cells without and with expressing platelet receptors ((C) CD42b (n = 4),  (E) CD41 (n = 7), (G) CD41a (n = 5)). Error bars represent mean ± SD. Statistical significance was determined by using the two-tailed Mann–Whitney test.

Figure 2

Specificity of CD42b, CD41 and CD41a in dengue virus infection. The cells from Fig. 1 were further analyzed. (A) Intracellular DENV⁺ cells were firstly gated (black eclipse) and characterized with surface platelet receptors ((B) CD42b, (C) CD41, (D) CD41a). The percentage of DENV⁺ cells after this gating was represented as a relative DENV infection. (E) The relative DENV infection in cells without expressing CD42b (n = 4), CD41 (n = 7) and CD41a (n = 5). (F) The relative DENV infection in cells with expressing CD42b (n = 4), CD41 (n = 7) and CD41a (n = 5). Error bars represent mean ± SD. Statistical significance was determined by using one-way ANOVA test.

Figure 3

Requirement of CD42b in dengue virus infection. MEG-01 cells were infected with DENV (MOI = 0.5) for 2 hours and washed with PBS. The cultures were maintained in fresh medium for another 7 days before being triple immunostained. Flow cytometry standard data files were analyzed in three-dimension using custom-written software in MATLAB. All the fluorescence intensities were shown as log10 of the actual data. (A) Representative three-dimensional plot of surface CD42b, surface CD41 and intracellular DENV. (B) The relative DENV infection in CD42b⁻CD41^−/+ and CD42b⁺CD41^−/+ subpopulation (n = 4). (C) The relative DENV infection in CD42b⁻CD41⁺, CD42b⁻CD41⁻, CD42b⁺CD41⁺ and CD42b⁺CD41⁻ subpopulation (n = 4). Error bars represent mean ± SD. Statistical significance was determined by using the two-tailed Mann–Whitney test. (D) Proposed model for the requirement of CD42b in dengue virus infection.

Figure 4

Dependence of dengue virus infection on CD42b. The cells from Fig. 3 were further analyzed. Intracellular DENV⁺ cells were firstly gated ((A) black eclipse) and then plotted in two-dimension of intracellular DENV and surface platelet receptors ((B) CD41, (C) CD42b). The cells were further gated to exclude the non-specific signal (black square). (D) Single-cell virus-protein analysis of intracellular DENV and surface CD41 (E) Single-cell virus-protein analysis of intracellular DENV and surface CD42b. r = Pearson correlation coefficient. p = p-value. n = the number of analyzed cells. Statistical significance was determined by using Fisher transformation test.

Figure 5

Dengue virus replicates itself in CD42b⁺ cells and eventually kills those cells. MEG-01 cells were infected with DENV (MOI = 0.5) for 2 hours and washed with PBS. The cultures were maintained in fresh medium before performing RT-PCR and apoptosis assays. (A) RT-PCR of the culture medium of DENV-infected Vero cells, the culture media of uninfected and DENV-infected MEG-01 cells. dpi = days post infection. Infected-1 and -2 experiments were 2 independent experiments. (B) Apoptosis assays of uninfected and DENV-infected MEG-01 cells at 7 dpi. Early apoptosis means Annexin V⁺ PI⁻ cells and late apoptosis means Annexin V⁺ PI⁺ cells. Data are presented as mean ± SEM of four independent experiments. The Mann-Whitney U test was used to assess the significance of differences between the observed data. *p < 0.05.

Figure 6

Proposed pathogenesis of platelet destruction and recovery in dengue patients. DENV might attack CD42b⁺ platelets and CD42b⁺ mature megakaryocytes leading to the rapid destruction of platelets. DENV could not attack CD42b⁻ early megakaryocytes explaining the characteristic delay of platelet recovery during the differentiation from early to mature megakaryocytes for 3 days. All new CD42b⁺ megakaryocytes could start producing platelets resulted in the rapid recovery of platelets.

Figure 7

Proposed the strategy to stop the disease progression to severe dengue. Aedes aegypti inject DENV into human skin. Dendritic cells phagocytose and amplify DENV leading to viremia causing fever^,,. DENV in bloodstream may infect CD42b⁺ mature megakaryocytes and platelets leading to thrombocytopenia causing hemorrhage. DENV may also infect CD42b⁺ endothelial cells leading to plasma leakage causing shock. Inhibiting the binding of DENV to CD42b would be able to stop the disease progression from dengue fever to severe dengue.