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.
References
[1]
Gubler DJ, Kuno G, editors (1997) Dengue And Dengue Hemorrhagic Fever. First ed. Wallingford, UK: CABI.
[2]
Nakao S, Lai CJ, Young NS (1989) Dengue virus, a flavivirus, propagates in human bone marrow progenitors and hematopoietic cell lines. Blood 74: 1235–1240.
[3]
Halstead SB, O'Rourke EJ, Allison AC (1977) Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection. J Exp Med 146: 218–229.
[4]
Rigau-Perez JG, Vorndam AV, Clark GG (2001) The dengue and dengue hemorrhagic fever epidemic in Puerto Rico, 1994–1995. Am J Trop Med Hyg 64: 67–74.
[5]
Bierman HR, Nelson ER (1965) Hematodepressive Virus Diseases of Thailand. Ann Intern Med 62: 867–884.
[6]
Kho LK, Wulur H, Himawan T (1972) Blood and bone marrow changes in dengue haemorrhagic fever. Paediatr Indones 12: 31–39.
[7]
Na-Nakorn S, Suingdumrong A, Pootrakul S, Bhamarapravati N (1966) Bone-marrow studies in Thai haemorrhagic fever. Bull World Health Organ 35: 54–55.
[8]
Tsai J-J, Liu L-T, Chang K, Wang S-H, Hsiao H-M, et al. (2012) The importance of hematopoietic progenitor cells in dengue. Therapeutic Advances in Hematology 3: 59–71.
[9]
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: 250–259 e254.
[10]
Institute of Laboratory Animal Research CoLS, National Research Council (1996) Guide for the Care and Use of Laboratory Animals. Washington, DC: The National Academic Press.
[11]
Russell PK, Nisalak A, Sukhavachana P, Vivona S (1967) A plaque reduction test for dengue virus neutralizing antibodies. J Immunol 99: 285–290.
[12]
Onlamoon N, Noisakran S, Hsiao HM, Duncan A, Villinger F, et al. (2010) Dengue virus-induced hemorrhage in a nonhuman primate model. Blood 115: 1823–1834.
[13]
Murgue B, Cassar O, Guigon M, Chungue E (1997) Dengue virus inhibits human hematopoietic progenitor growth in vitro. J Infect Dis 175: 1497–1501.
[14]
Basu A, Jain P, Gangodkar SV, Shetty S, Ghosh K (2008) Dengue 2 virus inhibits in vitro megakaryocytic colony formation and induces apoptosis in thrombopoietin-inducible megakaryocytic differentiation from cord blood CD34+ cells. FEMS Immunol Med Microbiol 53: 46–51.
[15]
Rothwell SW, Putnak R, La Russa VF (1996) Dengue-2 virus infection of human bone marrow: characterization of dengue-2 antigen-positive stromal cells. Am J Trop Med Hyg 54: 503–510.
[16]
Butthep P, Bunyaratvej A, Bhamarapravati N (1993) Dengue virus and endothelial cell: a related phenomenon to thrombocytopenia and granulocytopenia in dengue hemorrhagic fever. Southeast Asian J Trop Med Public Health 24 Suppl 1246–249.
[17]
Noisakran S, Gibbons RV, Songprakhon P, Jairungsri A, Ajariyakhajorn C, et al. (2009) Detection of dengue virus in platelets isolated from dengue patients. Southeast Asian J Trop Med Public Health 40: 253–262.
[18]
Wong KF, Chan JK, Chan JC, Lim WW, Wong WK (1991) Dengue virus infection-associated hemophagocytic syndrome. Am J Hematol 38: 339–340.
[19]
Jacobs MG, Brook MG, Weir WR, Bannister BA (1991) Dengue haemorrhagic fever: a risk of returning home. Bmj 302: 828–829.
[20]
Hathirat P, Isarangkura P, Srichaikul T, Suvatte V, Mitrakul C (1993) Abnormal hemostasis in dengue hemorrhagic fever. Southeast Asian J Trop Med Public Health 24 Suppl 180–85.
[21]
Nelson ER, Bierman HR (1964) Dengue Fever: A Thrombocytopenic Disease? JAMA 190: 99–103.
[22]
Levine RF (1980) Isolation and characterization of normal human megakaryocytes. Br J Haematol 45: 487–497.
[23]
Bessman JD (1984) The relation of megakaryocyte ploidy to platelet volume. Am J Hematol 16: 161–170.
[24]
Larramendy ML, Nylund SJ, Wessman M, Ruutu T, Knuutila S (1994) Ploidy in bone marrow cells from healthy donors: a MAC (morphology antibody chromosomes) study. Br J Haematol 86: 203–206.
Halstead SB, O'Rourke EJ (1977) Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J Exp Med 146: 201–217.
[27]
Auffray C, Sieweke MH, Geissmann F (2009) Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Annu Rev Immunol 27: 669–692.
[28]
Marianneau P, Flamand M, Deubel V, Despres P (1998) Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited. Clin Diagn Virol 10: 113–119.
[29]
Josefsson EC, James C, Henley KJ, Debrincat MA, Rogers KL, et al. (2011) Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets. J Exp Med 208: 2017–2031.
[30]
Fink K, Ng C, Nkenfou C, Vasudevan SG, van Rooijen N, et al. (2009) Depletion of macrophages in mice results in higher dengue virus titers and highlights the role of macrophages for virus control. Eur J Immunol 39: 2809–2821.
[31]
Marchette NJ, Halstead SB, Falkler WA Jr, Stenhouse A, Nash D (1973) Studies on the pathogenesis of dengue infection in monkeys. 3. Sequential distribution of virus in primary and heterologous infections. J Infect Dis 128: 23–30.
[32]
Mosquera JA, Hernandez JP, Valero N, Espina LM, Anez GJ (2005) Ultrastructural studies on dengue virus type 2 infection of cultured human monocytes. Virol J 2: 26.
[33]
Tsai JJ, Jen YH, Chang JS, Hsiao HM, Noisakran S, et al. (2011) Frequency alterations in key innate immune cell components in the peripheral blood of dengue patients detected by FACS analysis. J Innate Immun 3: 530–540.
[34]
Cassel DL, Keller MA, Surrey S, Schwartz E, Schreiber AD, et al. (1993) Differential expression of Fc gamma RIIA, Fc gamma RIIB and Fc gamma RIIC in hematopoietic cells: analysis of transcripts. Mol Immunol 30: 451–460.
[35]
Wu Z, Markovic B, Chesterman CN, Chong BH (1996) Characterization of Fc gamma receptors on human megakaryocytes. Thromb Haemost 75: 661–667.
[36]
Fuhrken PG, Chen C, Miller WM, Papoutsakis ET (2007) Comparative, genome-scale transcriptional analysis of CHRF-288-11 and primary human megakaryocytic cell cultures provides novel insights into lineage-specific differentiation. Exp Hematol 35: 476–489.
[37]
Carballal G, Cossio PM, Laguens RP, Ponzinibbio C, Oubina JR, et al. (1981) Junin virus infection of guinea pigs: immunohistochemical and ultrastructural studies of hemopoietic tissue. J Infect Dis 143: 7–14.
[38]
Carballal G, Rodriguez M, Frigerio MJ, Vasquez C (1977) Junin virus infection of guinea pigs: electron microscopic studies of peripheral blood and bone marrow. J Infect Dis 135: 367–373.
[39]
Dalton AJ, Law LW, Moloney JB, Manaker RA (1961) An electron microscopic study of a series of murine lymphoid neoplasms. J Natl Cancer Inst 27: 747–791.
[40]
Boukour S, Masse JM, Benit L, Dubart-Kupperschmitt A, Cramer EM (2006) Lentivirus degradation and DC-SIGN expression by human platelets and megakaryocytes. J Thromb Haemost 4: 426–435.
[41]
Zucker-Franklin D, Cao YZ (1989) Megakaryocytes of human immunodeficiency virus-infected individuals express viral RNA. Proc Natl Acad Sci U S A 86: 5595–5599.
[42]
Kim JA, Jung YJ, Seoh JY, Woo SY, Seo JS, et al. (2002) Gene expression profile of megakaryocytes from human cord blood CD34(+) cells ex vivo expanded by thrombopoietin. Stem Cells 20: 402–416.
