Ted human bone marrow appear uninfected and activated. Infected bone marrows were processed for EM investigations as described in methods. (A and B) Activated and vacuole-loaded phagocytic cells, likely monocytes or macrophages. (C and D) Absence of discernible viral particles or replication complexes in vacuolated cytoplasm of activated monocytes or macrophages. The images were captured after one day of infection. (TIF) Figure S6 Phagocytic cell engulfs virion-containing vesicles. Images were captured by EM of human whole bone marrow on day 5 after infection. (A) A vesicle loaded with virusDengue Virus Nafarelin site infection in Bone Marrowlike particles (V) fusing with a monocyte or macrophage (M). (B) Zipper junction (circle) at the fusion point. (C) Virions transfering from the vesicle to the cytoplasm of the phagocytic cell. (D) Degenerated viral particles inside the cytoplasm of phagocytic cells on day 7 after infection. (TIF)Figure S7 The efficiency of colony formation in human bone marrow was inhibited by dengue virus in a dosedependent manner. Healthy human bone marrow was exposed to dengue virus at an MOI = 1 or 0.1 for two hours. Unbound virus was removed with three washes of media, and cells were cultured with CFU media according to the protocol suggested by the manufacture (StemCells Technologies Inc., Vancouver, Canada). Uninfected human bone marrow was used as control. (A) Fewer and smaller colonies are observed with increased MOI. (B) Quantification of colony formation in the presence and absence of dengue virus. Y-axis indicates the number of colonies per dish. Data was tabulated from three replicates performed on different days. There is a statistically significant inhibition of colony formation in human bone marrows exposed to dengue virus. (TIF) Figure S8 Multi-lobulated cells were the dominantexperimental groups described in Figure S9 from four monkeys: 2DEAB, bone marrow pre-treated with DEAB for two days before virus infection; WBM, DEAB-untreated and DENV-infected whole bone marrow; DEAB, DEAB added to culture immediately after dengue virus infection. The kinetic fold max increase in viral titer compared to that at time 0, or two hours after absorption, was calculated. The peak fold increase in viral titers is presented. Cells were cytospun onto slides and immunohistochemical staining for CD41a and dengue E antigen was performed as described in the Methods. (B) IgG2a Isotype K162 web control and CD41a. (C) Viral antigen observed in megakaryocyte that was ongoing vesicle-shedding. Dengue E antigen (brown), CD41 (blue) and nucleus (DAPI stained). 1. Noisakran S, Onlamoon N, Hsiao HM, Clark KB, Villinger F, et al. (2012) Infection of bone marrow cells by dengue virus in vivo. Exp Hematol 40: 250?59 e254. 2. Onlamoon N, Noisakran S, Hsiao HM, Duncan A, Villinger F, et al. 1527786 (2010) Dengue virus-induced hemorrhage in a nonhuman primate model. Blood 115: 1823?834. (TIF)AcknowledgmentsWe thank the veterinary and research staff of 11967625 the Yerkes National Primate Center and the staff of the Stem Cell Processing Laboratory of the Emory Center for Transfusion and Cellular Therapy Center for Bone Marrow Transplant at Emory staff for their excellent generosity in collecting the healthy monkey bone marrow and human morrows for this study. The authors would like to appreciate the help, guidance, suggestions and discussions provided by Dr. Tristram Parslow from the department of Pathology and Laboratory Medicine at Emory University School of Medicine. The au.Ted human bone marrow appear uninfected and activated. Infected bone marrows were processed for EM investigations as described in methods. (A and B) Activated and vacuole-loaded phagocytic cells, likely monocytes or macrophages. (C and D) Absence of discernible viral particles or replication complexes in vacuolated cytoplasm of activated monocytes or macrophages. The images were captured after one day of infection. (TIF) Figure S6 Phagocytic cell engulfs virion-containing vesicles. Images were captured by EM of human whole bone marrow on day 5 after infection. (A) A vesicle loaded with virusDengue Virus Infection in Bone Marrowlike particles (V) fusing with a monocyte or macrophage (M). (B) Zipper junction (circle) at the fusion point. (C) Virions transfering from the vesicle to the cytoplasm of the phagocytic cell. (D) Degenerated viral particles inside the cytoplasm of phagocytic cells on day 7 after infection. (TIF)Figure S7 The efficiency of colony formation in human bone marrow was inhibited by dengue virus in a dosedependent manner. Healthy human bone marrow was exposed to dengue virus at an MOI = 1 or 0.1 for two hours. Unbound virus was removed with three washes of media, and cells were cultured with CFU media according to the protocol suggested by the manufacture (StemCells Technologies Inc., Vancouver, Canada). Uninfected human bone marrow was used as control. (A) Fewer and smaller colonies are observed with increased MOI. (B) Quantification of colony formation in the presence and absence of dengue virus. Y-axis indicates the number of colonies per dish. Data was tabulated from three replicates performed on different days. There is a statistically significant inhibition of colony formation in human bone marrows exposed to dengue virus. (TIF) Figure S8 Multi-lobulated cells were the dominantexperimental groups described in Figure S9 from four monkeys: 2DEAB, bone marrow pre-treated with DEAB for two days before virus infection; WBM, DEAB-untreated and DENV-infected whole bone marrow; DEAB, DEAB added to culture immediately after dengue virus infection. The kinetic fold max increase in viral titer compared to that at time 0, or two hours after absorption, was calculated. The peak fold increase in viral titers is presented. Cells were cytospun onto slides and immunohistochemical staining for CD41a and dengue E antigen was performed as described in the Methods. (B) IgG2a Isotype control and CD41a. (C) Viral antigen observed in megakaryocyte that was ongoing vesicle-shedding. Dengue E antigen (brown), CD41 (blue) and nucleus (DAPI stained). 1. Noisakran S, Onlamoon N, Hsiao HM, Clark KB, Villinger F, et al. (2012) Infection of bone marrow cells by dengue virus in vivo. Exp Hematol 40: 250?59 e254. 2. Onlamoon N, Noisakran S, Hsiao HM, Duncan A, Villinger F, et al. 1527786 (2010) Dengue virus-induced hemorrhage in a nonhuman primate model. Blood 115: 1823?834. (TIF)AcknowledgmentsWe thank the veterinary and research staff of 11967625 the Yerkes National Primate Center and the staff of the Stem Cell Processing Laboratory of the Emory Center for Transfusion and Cellular Therapy Center for Bone Marrow Transplant at Emory staff for their excellent generosity in collecting the healthy monkey bone marrow and human morrows for this study. The authors would like to appreciate the help, guidance, suggestions and discussions provided by Dr. Tristram Parslow from the department of Pathology and Laboratory Medicine at Emory University School of Medicine. The au.
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