Quantification of monkey bone marrow cells positive for dengue viral antigen^a.

a<p>values represent the percentage of surface marker positive or negative among 200 dengue positive (4G2+) cells with 3–5 histochemical stainings.</p><p>± standard deviation, P.I., post-infection, BDCA2, plasmacytoid dendritic cell antigen 2.</p

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

<div><p>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.</p> </div

Bone marrow cells from rhesus monkeys are permissive for dengue virus infection in vitro.

<p>Fresh whole BM cells were infected with dengue virus at an MOI = 0.1. Supernatant fluids were collected at the indicated times and analyzed by qRT-PCR and nonstructural protein 1 (NS1) ELISA as described in Methods. (A) Viral RNA in supernatants. (B) NS1 in supernatants. Varying degrees of susceptibility to dengue virus infection was noticed.</p

Viral particles are present in megakaryocytes from the human bone marrow.

<p>Sample preparations for EM studies were performed as described in the Methods. (A) Uninfected control. (B) Cellular vesicle containing viral particles (single particle, red arrow; cluster of viral particles, blue arrow) inside a diploid megakaryocyte on day one post-infection. (C) Large numbers of viral particles inside the cytoplasm of a multi-lobulated megakaryocyte on day three post-infection. (D) Cytoplasm containing many virus particles shedding off in a vesicle (red arrow). (E) A virion-containing vesicle (dash circle) at the vicinity of an activated mononuclear cell. (F) Virion containing vesicle (V) fusing with a monocyte (M). A zipper junction (blue arrow) is indicated. No viral particles were observed in the monocytes. A scale bar is 0.2 µM.</p

Infectivity of dengue virus in Colony Forming Unit cells picked from human bone marrow^a.

a<p>Cells were characterized based-upon their morphology and giemsa staining characteristics.</p>b<p>Day 0 means the time point 2 hours after adsorption, in which culture supernatants were extensively washed for unbound virus. The amount of residual virus in the culture supernatant was determined and used as the baseline.</p>c<p>Quantification determined by qRT-PCR (unadjusted copy number per 140 µlof the supernatant).</p>d<p>The fold increase relative to the viral titer at Day 0, supernatant at 2 hours post-infection, was calculated.</p

Megakaryocytes were likely the dominant dengue virus antigen positive cells in monkey bone marrow.

<p>Smears of bone marrow cells were prepared and immunohistochemical stainings were performed as described in Methods. Dengue antigen (4G2 positivity) is indicated by DAB staining (brown) (A) Dengue viral antigen in a diploid megakaryocyte. (B) Dengue antigen in a multi-lobulated megakaryocyte. (C), Dengue antigen in cellular debris. Red, PAS staining. Blue, hematoxylin staining. (D and E) Dengue viral antigen-containing vesicles engulfed by phagocytic cells. (F) Isotype control.</p

CD61+ cells were the early cells infected by dengue virus bone marrow.

<p>Freshly aspirated bone marrows at various time points from DV infected rhesus monkeys were stained with dengue viral specific monoclonal antibody (clone 3H5) and cell lineage markers CD41, CD61, and CD14, and subjected to FACS analysis. Results revealed that viral antigen was observed early in CD61+ cells with a decreasing trend while the opposite trend was evident in CD14+ monocytic cells.</p

Human bone marrow is more permissive than rhesus macaque bone marrow to dengue virus infection in vitro.

<p>(A) A comparison of peak virus genome copy number levels in human and monkey BM cultures. (B) Comparison of NS1 in the supernatant fluid of human and monkey BMs. The levels of viral RNA and NS1 in the supernatant fluid from infected human BM were significantly higher than that from the rhesus monkey.</p

Human bone marrow is permissive for dengue virus infection in vitro.

<p>Healthy human BM cells were obtained from the BM transplantation center at Emory University and infected with dengue virus as described in the Methods. Supernatant fluids were collected at the indicated times; viral RNA and NS1 were quantified as described in the Methods. (A) Viral RNA in supernatant fluids. (B) NS1 in supernatant fluids.</p

Megakaryocytes from human bone marrow contain dengue virus antigen.

<p>Bone marrow smears were prepared and fluorescent cell stainings were performed as described in the Methods. (A) Dengue viral E antigen (identified by 4G2) in tetraploid megakaryocyte in the process of shedding vesicles as evidenced by immunohistochemical staining in the presence of DAPI. Dengue viral antigen (red) and nucleus (blue) (B) Dengue NS1 antigen in a CD61+ megakaryocytic cell depicted by immunofluorescence staining. NS1 (green), CD61 (red) and nucleus (blue). Scale bar,10 µm.</p