Ance to the guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council and the Health andDengue Virus Infection in Bone MarrowFigure 2. Supernatant fluids early post infection contain infectious virus. All in all, 15 BM samples from healthy rhesus monkeys were studied as described in Figure 1. (A) Kinetics of viral replication in 15 bone marrow cell cultures. Red line indicates RNA titers and green line indicates NS1 protein levels. (B) Infectious virus recovery from supernatant fluids. Supernatants from days 2 (red line) and 5 (green line) were cultured with Vero cells. Approximately equal amounts of viral RNA from 5 randomly chosen monkey BM supernatant fluids were utilized in these culture experiments. Supernatant from day 2 contained infectious dengue virus. doi:10.1371/journal.pone.0052902.gHuman Services [10]. In addition, Yerkes is a facility fully Licochalcone-A cost accredited with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animals were fed species specific monkey chow (Purina) ad libitum, with daily supplementation with fresh fruit, and cared for by the Yerkes Veterinary Division. Animals are monitored at least twice daily for clinical and psychological health and provided social enrichment. Animals which develop clinical conditions that cannot be relieved therapeutically are euthanized using the recommended procedures by the American Veterinary Medical Association. Healthy human BM samples that would otherwise be discarded were obtained from the Stem Cell Processing Laboratory of the Emory Center for Transfusion and order Met-Enkephalin Cellular Therapy. The experiments were conducted following appropriate approval by the Emory IRB (Institutional Ethics Committee) with approval protocol #00046063. All patients gave written informed consent for the study.were washed X3 with media to remove unbound virus. The infected cells were then re-suspended in 2 ml of culture media and incubated in suspension without shaking and 400 ml of the cell suspension was removed at different time points as indicated in the text. BM smears were prepared by pelleting cells at low speed and applying them to slides.Infectious Virus Analysis of BM Supernatant in Vero CellsMonkey BM cells were similarly infected with an MOI of 0.1 and the supernatant fluids collected on days 2 and 5 following infection. These supernatant fluids were added to Vero cells at 80 confluency. Subsequently supernatant fluids from these Vero cells were collected at the indicated time points and immediately stored at 280uC until real time (RT)-PCR analysis. Focus forming unit assays were performed by infecting a monolayer of Vero cells in 96-well plates with serial dilutions of supernatant fluids in MEM media collected from human bone marrow cultures. After a two-hour absorption, the cells were overlayed with 1 methylcellulose in EMEM (with 2 mM LGlutamine, 1 mM sodium pyruvate, 2 FBS, HEPES). Cells were incubated for 3 days and fixed with 3.7 paraformaldehyde. Cells 11967625 were permeabilized with 1 triton-X for 10 minutes. Cells were washed 5 times with PBS and incubated for 1 hour at 37uC with a predetermined optimum concentration of the monoclonal antibody clone 4G2. Cells were washed 3 times and then incubated with HRP-conjugated rabbit anti-mouse IgG (Dako) for 1 hour at 37uC. Cells were washed 3 times and incubated with diaminobenzidine for 10 minutes.In vitro Infection of th.Ance to the guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council and the Health andDengue Virus Infection in Bone MarrowFigure 2. Supernatant fluids early post infection contain infectious virus. All in all, 15 BM samples from healthy rhesus monkeys were studied as described in Figure 1. (A) Kinetics of viral replication in 15 bone marrow cell cultures. Red line indicates RNA titers and green line indicates NS1 protein levels. (B) Infectious virus recovery from supernatant fluids. Supernatants from days 2 (red line) and 5 (green line) were cultured with Vero cells. Approximately equal amounts of viral RNA from 5 randomly chosen monkey BM supernatant fluids were utilized in these culture experiments. Supernatant from day 2 contained infectious dengue virus. doi:10.1371/journal.pone.0052902.gHuman Services [10]. In addition, Yerkes is a facility fully accredited with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animals were fed species specific monkey chow (Purina) ad libitum, with daily supplementation with fresh fruit, and cared for by the Yerkes Veterinary Division. Animals are monitored at least twice daily for clinical and psychological health and provided social enrichment. Animals which develop clinical conditions that cannot be relieved therapeutically are euthanized using the recommended procedures by the American Veterinary Medical Association. Healthy human BM samples that would otherwise be discarded were obtained from the Stem Cell Processing Laboratory of the Emory Center for Transfusion and Cellular Therapy. The experiments were conducted following appropriate approval by the Emory IRB (Institutional Ethics Committee) with approval protocol #00046063. All patients gave written informed consent for the study.were washed X3 with media to remove unbound virus. The infected cells were then re-suspended in 2 ml of culture media and incubated in suspension without shaking and 400 ml of the cell suspension was removed at different time points as indicated in the text. BM smears were prepared by pelleting cells at low speed and applying them to slides.Infectious Virus Analysis of BM Supernatant in Vero CellsMonkey BM cells were similarly infected with an MOI of 0.1 and the supernatant fluids collected on days 2 and 5 following infection. These supernatant fluids were added to Vero cells at 80 confluency. Subsequently supernatant fluids from these Vero cells were collected at the indicated time points and immediately stored at 280uC until real time (RT)-PCR analysis. Focus forming unit assays were performed by infecting a monolayer of Vero cells in 96-well plates with serial dilutions of supernatant fluids in MEM media collected from human bone marrow cultures. After a two-hour absorption, the cells were overlayed with 1 methylcellulose in EMEM (with 2 mM LGlutamine, 1 mM sodium pyruvate, 2 FBS, HEPES). Cells were incubated for 3 days and fixed with 3.7 paraformaldehyde. Cells 11967625 were permeabilized with 1 triton-X for 10 minutes. Cells were washed 5 times with PBS and incubated for 1 hour at 37uC with a predetermined optimum concentration of the monoclonal antibody clone 4G2. Cells were washed 3 times and then incubated with HRP-conjugated rabbit anti-mouse IgG (Dako) for 1 hour at 37uC. Cells were washed 3 times and incubated with diaminobenzidine for 10 minutes.In vitro Infection of th.
