Multiploid CD61+ Cells Are the Pre-Dominant Cell Lineage Infected during Acute Dengue Virus Infection in Bone Marrow

Abstract

Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.

Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing

“…Consistent with previous studies (21,32), we also observed a significant decrease in circulating total platelets, i.e., thrombocytopenia, another hallmark of DENV infection in humans. Thrombocytopenia is fre- quently observed among dengue fever patients, and two mechanisms have been proposed to explain it: antibody-mediated depletion of platelets from peripheral blood (45)(46)(47)(48)(49) and deficient platelet production by megakaryocytes in the bone marrow (25,50). The latter hypothesis is supported by a study that demonstrated that human megakaryocytes from the bone marrow were highly permissive for DENV infection in vitro, suggesting that megakaryocytes may also be targeted in vivo, thus providing a plausible mechanism for thrombocytopenia during acute infection in humans (50).…”

“…Thrombocytopenia is fre- quently observed among dengue fever patients, and two mechanisms have been proposed to explain it: antibody-mediated depletion of platelets from peripheral blood (45)(46)(47)(48)(49) and deficient platelet production by megakaryocytes in the bone marrow (25,50). The latter hypothesis is supported by a study that demonstrated that human megakaryocytes from the bone marrow were highly permissive for DENV infection in vitro, suggesting that megakaryocytes may also be targeted in vivo, thus providing a plausible mechanism for thrombocytopenia during acute infection in humans (50). Furthermore, a recent study on DENV in humanized mice showed that DENV infection inhibited the production of human megakaryocytes and their progenitor cells in the bone marrow of NSG mice, resulting in depletion of circulating human platelets (25).…”

Transient Monocytosis Subjugates Low Platelet Count in Adult Dengue Patients

“…In general, immune cells with phagocytic capacity have been suggested to be infectable by DENV, especially cells with mannose-related receptors [15], DC-specific ICAM3-grabbing nonintegrin molecules [16], and CLEC5A [14] on the surface. However, recent evidence suggests that other immune cells, such as platelets and its precursor cells, megakaryocytes, are a favored target of DENV as well [17,18,19,20,21,22]. The latter may partially explain the dysfunction of platelets resulting in prolonged activated partial thromboplastin time (aPTT) during the early phase of dengue disease, and subsequent low platelet counts when the disease progresses to a critical stage [1].…”

Characterization of dengue virus 2 growth in megakaryocyte–erythrocyte progenitor cells

“…It was noted even in early studies that bone marrow resident cells change in morphology and frequency (Bierman and Nelson, 1965; Kho et al, 1972; La Russa and Innis, 1995; Nelson et al, 1964; Noisakran et al, 2012). Bone marrow-derived megakaryocyte–erythrocyte progenitor cells were permissive and yielded high DENV2 titers (1 × 10 5 FFU/mL and 1 × 10 8 genome copy number [GCN]/mL) (Basu et al, 2008; Clark et al, 2012; Nakao et al, 1989). Also, a recent publication reports a positive correlation between DENV titers in dengue fever patient plasma and circulating CD61 + (megakaryocyte marker) cell count numbers (Hsu et al, 2015).…”

“…While not conclusive, these observations suggest that CD61 + cells might contribute to DENV replication in vivo , since DENV can be propagated ex vivo from CD61 + cells isolated from bone marrow of infected animals (Noisakran et al, 2012). Studies have indicated that megakaryocytes stain positive for viral antigen and antigen positivity correlates with peak infectious titer and virus-like particle (VLP) production (Basu et al, 2008; Clark et al, 2012; Noisakran et al, 2012). However, despite an association of DENV2 with the megakaryocyte, the cell types that initially encountered and took up the virus in these experiments were uncertain because the effect could be due to infection of any of several cell types capable of differentiating into megakaryocytes.…”