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Characterization of coxsackievirus B3 replication in human umbilical vein endothelial cells

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After successful invasion of susceptible hosts, systemic distribution of coxsackievirus B3 (CVB3) most likely requires interactions with the endothelial system. Thereby, infection of endothelial cells occurs directly or viruses and/or virus-infected leukocytes migrate through the endothelial barrier. Many of these processes have not been studied so far. In order to analyze viral replication in the endothelium, human umbilical vein endothelial cells (HUVEC) were isolated and infected with CVB3. Time-course experiments revealed maximal viral replication at 10–24 h and viral RNA persistence up to 120 h post-infection (p. i.) without the induction of obvious general cytopathic effects or the loss of cellular viability. However, the application of the EGFP-expressing recombinant virus variant CVB3/EGFP revealed shrinkage and death of individual cells. Using infectious center assays, a noticeable CVB3 replication occurred on an average of 20 % of HUVEC at 10 h p. i. This may be in part due to a higher coxsackievirus/adenovirus receptor expression in a small subgroup of HUVEC (5–7 %) as analyzed by flow cytometry. Interestingly, CVB3 replication escalated and cellular susceptibility increased significantly after reversal of cell cycle arrest caused by serum deprivation indicating that reactivation of cellular metabolism may help to promote CVB3 replication. Finally, CVB3-infected HUVEC cultures revealed increased DNA fragmentation, and inhibition of caspase activity caused an accumulation of intracellular virus particles indicating that apoptotic processes are involved in virus release mechanisms. Based on these observations, it is assumed that CVB3 replicates efficiently in human endothelial cells. But how this specific infection of the endothelium may influence viral spread in the infected host needs to be investigated in the future.

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  1. 1.

    Cooper LT Jr (2009) Myocarditis. N Engl J Med 360(15):1526–1538

  2. 2.

    Why HJ, Meany BT, Richardson PJ, Olsen EG, Bowles NE, Cunningham L, Freeke CA, Archard LC (1994) Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89(6):2582–2589

  3. 3.

    Dotta F, Censini S, van Halteren AG, Marselli L, Masini M, Dionisi S, Mosca F, Boggi U, Muda AO, Prato SD, Elliott JF, Covacci A, Rappuoli R, Roep BO, Marchetti P (2007) Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients. Proc Natl Acad Sci USA 104(12):5115–5120

  4. 4.

    Filippi C, von Herrath M (2005) How viral infections affect the autoimmune process leading to type 1 diabetes. Cell Immunol 233(2):125–132

  5. 5.

    Magnani JW, Dec GW (2006) Myocarditis: current trends in diagnosis and treatment. Circulation 113(6):876–890

  6. 6.

    Henke A, Jarasch N, Wutzler P (2003) Vaccination procedures against coxsackievirus-induced heart disease. Expert Rev Vaccines 2(6):791–801

  7. 7.

    Henke A (2002) DNA immunization—a new chance in vaccine research? Med Microbiol Immunol 191(3–4):187–190

  8. 8.

    Huber SA, Haisch C, Lodge PA (1990) Functional diversity in vascular endothelial cells: role in coxsackievirus tropism. J Virol 64(9):4516–4522

  9. 9.

    Huber SA, Job LP, Woodruff JF (1984) In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo. Infect Immun 43(2):567–573

  10. 10.

    Carson SD, Hobbs JT, Tracy SM, Chapman NM (1999) Expression of the coxsackievirus and adenovirus receptor in cultured human umbilical vein endothelial cells: regulation in response to cell density. J Virol 73(8):7077–7079

  11. 11.

    Funke C, Farr M, Werner B, Dittmann S, Uberla K, Piper C, Niehaus K, Horstkotte D (2010) Antiviral effect of Bosentan and Valsartan during coxsackievirus B3 infection of human endothelial cells. J Gen Virol 91(Pt 8):1959–1970

  12. 12.

    Conaldi PG, Serra C, Mossa A, Falcone V, Basolo F, Camussi G, Dolei A, Toniolo A (1997) Persistent infection of human vascular endothelial cells by group B coxsackieviruses. J Infect Dis 175(3):693–696

  13. 13.

    Zanone MM, Favaro E, Conaldi PG, Greening J, Bottelli A, Perin PC, Klein NJ, Peakman M, Camussi G (2003) Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules. J Immunol 171(1):438–446

  14. 14.

    Ju Y, Wang T, Li Y, Xin W, Wang S, Li J (2007) Coxsackievirus B3 affects endothelial tight junctions: possible relationship to ZO-1 and F-actin, as well as p38 MAPK activity. Cell Biol Int 31(10):1207–1213

  15. 15.

    Henke A, Mohr C, Sprenger H, Graebner C, Stelzner A, Nain M, Gemsa D (1992) Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes. J Immunol 148(7):2270–2277

  16. 16.

    Jarasch N, Martin U, Zell R, Wutzler P, Henke A (2007) Influence of pan-caspase inhibitors on coxsackievirus B3-infected CD19 + B lymphocytes. Apoptosis 12:1633–1643

  17. 17.

    Heller R, Unbehaun A, Schellenberg B, Mayer B, Werner-Felmayer G, Werner ER (2001) L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem 276(1):40–47

  18. 18.

    Stahmann N, Woods A, Carling D, Heller R (2006) Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2 +/calmodulin-dependent protein kinase beta. Mol Cell Biol 26(16):5933–5945

  19. 19.

    Knowlton KU, Jeon ES, Berkley N, Wessely R, Huber S (1996) A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3. J Virol 70(11):7811–7818

  20. 20.

    Slifka MK, Pagarigan R, Mena I, Feuer R, Whitton JL (2001) Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8 + T cells during picornavirus infection. J Virol 75(5):2377–2387

  21. 21.

    Verstrepen WA, Kuhn S, Kockx MM, Van De Vyvere ME, Mertens AH (2001) Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single-tube real-time reverse transcription-PCR assay. J Clin Microbiol 39(11):4093–4096

  22. 22.

    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45

  23. 23.

    Caserta TM, Smith AN, Gultice AD, Reedy MA, Brown TL (2003) Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties. Apoptosis 8(4):345–352

  24. 24.

    Melnikov VY, Faubel S, Siegmund B, Lucia MS, Ljubanovic D, Edelstein CL (2002) Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice. J Clin Invest 110(8):1083–1091

  25. 25.

    Martin U, Jarasch N, Nestler M, Rassmann A, Munder T, Seitz S, Zell R, Wutzler P, Henke A (2007) Antiviral effects of pan-caspase inhibitors on the replication of coxsackievirus B3. Apoptosis 12(3):525–533

  26. 26.

    Wilsky S, Sobotta K, Wiesener N, Pilas J, Althof N, Munder T, Wutzler P, Henke A (2012) Inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus B3 replication. Arch Virol 157(2):259–269

  27. 27.

    Feuer R, Mena I, Pagarigan R, Slifka MK, Whitton JL (2002) Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro. J Virol 76(9):4430–4440

  28. 28.

    Venkatesan A, Sharma R, Dasgupta A (2003) Cell cycle regulation of hepatitis C and encephalomyocarditis virus internal ribosome entry site-mediated translation in human embryonic kidney 293 cells. Virus Res 94(2):85–95

  29. 29.

    Feuer R, Mena I, Pagarigan RR, Hassett DE, Whitton JL (2004) Coxsackievirus replication and the cell cycle: a potential regulatory mechanism for viral persistence/latency. Med Microbiol Immunol 193(2–3):83–90

  30. 30.

    Henke A, Huber S, Stelzner A, Whitton JL (1995) The role of CD8 + T lymphocytes in coxsackievirus B3-induced myocarditis. J Virol 69(11):6720–6728

  31. 31.

    Olivetti G, Abbi R, Quaini F, Kajstura J, Cheng W, Nitahara JA, Quaini E, Di Loreto C, Beltrami CA, Krajewski S, Reed JC, Anversa P (1997) Apoptosis in the failing human heart. N Engl J Med 336(16):1131–1141

  32. 32.

    Martin U, Nestler M, Munder T, Zell R, Sigusch HH, Henke A (2004) Characterization of coxsackievirus B3-caused apoptosis under in vitro conditions. Med Microbiol Immunol 193(2–3):133–139

  33. 33.

    Sumikoshi M, Hashimoto K, Kawasaki Y, Sakuma H, Suzutani T, Suzuki H, Hosoya M (2008) Human influenza virus infection and apoptosis induction in human vascular endothelial cells. J Med Virol 80(6):1072–1078

  34. 34.

    Lagunoff M, Bechtel J, Venetsanakos E, Roy AM, Abbey N, Herndier B, McMahon M, Ganem D (2002) De novo infection and serial transmission of Kaposi’s sarcoma-associated herpesvirus in cultured endothelial cells. J Virol 76(5):2440–2448

  35. 35.

    Saijets S, Ylipaasto P, Vaarala O, Hovi T, Roivainen M (2003) Enterovirus infection and activation of human umbilical vein endothelial cells. J Med Virol 70(3):430–439

  36. 36.

    Viemann D, Schmolke M, Lueken A, Boergeling Y, Friesenhagen J, Wittkowski H, Ludwig S, Roth J (2011) H5N1 virus activates signaling pathways in human endothelial cells resulting in a specific imbalanced inflammatory response. J Immunol 186(1):164–173

  37. 37.

    Wang L, Damania B (2008) Kaposi’s sarcoma-associated herpesvirus confers a survival advantage to endothelial cells. Cancer Res 68(12):4640–4648

  38. 38.

    Bozym RA, Morosky SA, Kim KS, Cherry S, Coyne CB (2010) Release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells. PLoS Pathog 6(10):e1001135

  39. 39.

    Coyne CB, Bozym R, Morosky SA, Hanna SL, Mukherjee A, Tudor M, Kim KS, Cherry S (2011) Comparative RNAi screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers. Cell Host Microbe 9(1):70–82

  40. 40.

    Xie Y, Liao J, Li M, Wang X, Yang Y, Ge J, Chen R, Chen H (2012) Impaired cardiac microvascular endothelial cells function induced by Coxsackievirus B3 infection and its potential role in cardiac fibrosis. Virus Res 169(1):188–194

  41. 41.

    Bergelson JM, Mohanty JG, Crowell RL, St John NF, Lublin DM, Finberg RW (1995) Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55). J Virol 69(3):1903–1906

  42. 42.

    Agrez MV, Shafren DR, Gu X, Cox K, Sheppard D, Barry RD (1997) Integrin alpha v beta 6 enhances coxsackievirus B1 lytic infection of human colon cancer cells. Virology 239(1):71–77

  43. 43.

    Roivainen M, Piirainen L, Hovi T, Virtanen I, Riikonen T, Heino J, Hyypiä T (1994) Entry of coxsackievirus A9 into host cells: specific interactions with alpha v beta 3 integrin, the vitronectin receptor. Virology 203(2):357–365

  44. 44.

    Zautner AE, Korner U, Henke A, Badorff C, Schmidtke M (2003) Heparan sulfates and coxsackievirus-adenovirus receptor: each one mediates coxsackievirus B3 PD infection. J Virol 77(18):10071–10077

  45. 45.

    Luo H, Zhang J, Dastvan F, Yanagawa B, Reidy MA, Zhang HM, Yang D, Wilson JE, McManus BM (2003) Ubiquitin-dependent proteolysis of cyclin D1 is associated with coxsackievirus-induced cell growth arrest. J Virol 77(1):1–9

  46. 46.

    Esfandiarei M, McManus BM (2008) Molecular biology and pathogenesis of viral myocarditis. Annu Rev Pathol 3:127–155

  47. 47.

    Yajima T, Knowlton KU (2009) Viral myocarditis: from the perspective of the virus. Circulation 119(19):2615–2624

  48. 48.

    Rehren F, Ritter B, Dittrich-Breiholz O, Henke A, Lam E, Kati S, Kracht M, Heim A (2013) Induction of a broad spectrum of inflammation-related genes by coxsackievirus B3 requires interleukin-1 signaling. Med Microbiol Immunol 202(1):11–23

  49. 49.

    Feuer R, Mena I, Pagarigan RR, Harkins S, Hassett DE, Whitton JL (2003) Coxsackievirus B3 and the neonatal CNS: the roles of stem cells, developing neurons, and apoptosis in infection, viral dissemination, and disease. Am J Pathol 163(4):1379–1393

  50. 50.

    Saraste A, Arola A, Vuorinen T, Kyto V, Kallajoki M, Pulkki K, Voipoi-Pulkki LM, Hyypia T (2003) Cardiomyocyte apoptosis in experimental coxsackievirus B3 myocarditis. Cardiovasc Pathol 12(5):255–262

  51. 51.

    Yuan JP, Zhao W, Wang HT, Wu KY, Li T, Guo XK, Tong SQ (2003) Coxsackievirus B3-induced apoptosis and caspase-3. Cell Res 13(3):203–209

  52. 52.

    Huber SA, Budd RC, Rossner K, Newell MK (1999) Apoptosis in coxsackievirus B3-induced myocarditis and dilated cardiomyopathy. Ann N Y Acad Sci 887:181–190

  53. 53.

    Carthy CM, Granville DJ, Watson KA, Anderson DR, Wilson JE, Yang D, Hunt DW, McManus BM (1998) Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. J Virol 72(9):7669–7675

  54. 54.

    Colston JT, Chandrasekar B, Freeman GL (1998) Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis. Cardiovasc Res 38(1):158–168

  55. 55.

    Carthy CM, Yanagawa B, Luo H, Granville DJ, Yang D, Cheung P, Cheung C, Esfandiarei M, Rudin CM, Thompson CB, Hunt DW (2003) McManus BM (2003) Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release, activation of multiple caspases, and virus release following coxsackievirus B3 infection. Virology 313(1):147–157

  56. 56.

    Henke A, Launhardt H, Klement K, Stelzner A, Zell R, Munder T (2000) Apoptosis in coxsackievirus B3-caused diseases: interaction between the capsid protein VP2 and the proapoptotic protein siva. J Virol 74(9):4284–4290

  57. 57.

    Henke A, Nestler M, Strunze S, Saluz HP, Hortschansky P, Menzel B, Martin U, Zell R, Stelzner A, Munder T (2001) The apoptotic capability of coxsackievirus B3 is influenced by the efficient interaction between the capsid protein VP2 and the proapoptotic host protein Siva. Virology 289(1):15–22

  58. 58.

    Si X, Luo H, Morgan A, Zhang J, Wong J, Yuan J, Esfandiarei M, Gao G, Cheng C, McManus BM (2005) Stress-activated protein kinases are involved in coxsackievirus B3 viral progeny release. J Virol 79(22):13875–13881

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We thank Elke Teuscher for their excellent technical assistance.

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Correspondence to A. Henke.

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A. Kühnl and C. Rien have contributed equally to this manuscript.

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Kühnl, A., Rien, C., Spengler, K. et al. Characterization of coxsackievirus B3 replication in human umbilical vein endothelial cells. Med Microbiol Immunol 203, 217–229 (2014).

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  • Coxsackievirus B3
  • Human umbilical vein endothelial cells
  • Replication
  • Coxsackievirus/adenovirus receptor
  • Serum deprivation
  • Apoptosis