Human enteroviruses (HEVs) are pathogens circulating commonly in the environment, with a seasonal peak during early fall. They belong to the Picornaviridae family, characterized by a single-stranded positive RNA genome (~7400 nucleotides) surrounded by an icosahedral capsid around 30 nm in size [1King AMQ, Ed. Picornaviridae In Van Regenmortel MHV Virus Taxonomy Seventh report of the international Comittee for the taxonomy of viruses. New York.: Academic Press 2000; pp. 657-73.]. Infection is normally asymptomatic or mild, but occasionally the virus spreads to secondary organs leading to more severe diseases such as aseptic meningitis or myocarditis [2Kandolf R, Sauter M, Aepinus C, Schnorr JJ, Selinka HC, Klingel K. Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system Virus Res 1999; 62: 149-58., 3Rotbart HA. Enteroviral infections of the central nervous system Clin Infect Dis 1995; 20: 971-81.]. Coxsackie B viruses (CVBs), belonging to the HEV-B group, have been associated to pancreatic diseases such as idiopathic pancreatitis [4Ursing B. Acute pancreatitis in coxsackie B infection Br Med J 1973; 3: 524-., 5Ozsvar Z, Deak J, Pap A. Possible role of Coxsackie-B virus infection in pancreatitis Int J Pancreatol 1992; 11: 105-8.] and type 1 diabetes (T1D) [6Frisk G, Fohlman J, Kobbah M. High frequency of Coxsackie-B-virus-specific IgM in children developing type I diabetes during a period of high diabetes morbidity J Med Virol 1985; 17: 219-7.-2Kandolf R, Sauter M, Aepinus C, Schnorr JJ, Selinka HC, Klingel K. Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system Virus Res 1999; 62: 149-58.]. T1D is considered to be an immune-mediated disorder leading to selective destruction of the insulin producing beta-cells in the endocrine islets of Langerhans [13Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus Diabetes 1965; 14: 619-33.]. Very little is known about the mechanism of infection in the pancreatic exocrine cells. Studies in mice have shown that infection with several CVB strains most often leads to necrosis of the mouse exocrine cells and infiltration of immune cells, reviewed in [14Ramsingh AI. Coxsackieviruses and pancreatitis Front Biosci 1997; 2: e53-62.]. Intriguingly the islets in these animal studies are usually left intact and uninfected, although virus proteins have been detected within endocrine cells in a few cases [15Bopegamage S, Kovacova J, Vargova A. Coxsackie B virus infection of mice: inoculation by the oral route protects the pancreas from damage but not from infection J Gen Virol 2005; 86: 3271-80., 16Drescher KM, Kono K, Bopegamage S, Carson SD, Tracy S. Coxsackievirus B3 infection and type 1 diabetes development in NOD mice insulitis determines susceptibility of pancreatic islets to virus infection Virology 2004; 329: 381-94.]. In two separate studies of CVB-4 inoculated mice, the endocrine beta-cells appeared damaged but visible virus particles could only be detected in the exocrine cells and immune-cells in the electron microscope [17Burch GE, Harb JM. Electron microscopic studies of viral pancreatitis in coxsackie B4 virus infected mice Exp Mol Pathol 1979; 31: 23-35., 18Harrison AK, Bauer SP, Murphy FA. Viral pancreatitis: ultrastructural pathological effects of Coxsackievirus B3 infection in newborn mouse pancreas Exp Mol Pathol 1972; 17: 206-19.]. The relevance of these studies in humans is however not clear. In human studies different serotypes of CVBs have been shown to replicate lytically in islet cells in vitro [11Roivainen M, Rasilainen S, Ylipaasto P. Mechanisms of Coxsackievirus-Induced Damage to Human Pancreatic {beta}-Cells J Clin Endocrinol Metab 2000; 85: 432-0., 19Elshebani A, Olsson A, Westman J, Tuvemo T, Korsgren O, Frisk G. Effects on isolated human pancreatic islet cells after infection with strains of enterovirus isolated at clinical presentation of type 1 diabetes Virus Res 2007; 124: 193-203., 20Frisk G, Diderholm H. Tissue culture of isolated human pancreatic islets infected with different strains of coxsackievirus B4: assessment of virus replication and effects on islet morphology and insulin release Int J Exp Diabetes Res 2000; 1: 165-75.]. In children with systemic HEV infection, virus RNA [21Ylipaasto P, Klingel K, Lindberg AM, et al. Enterovirus infection in human pancreatic islet cells islet tropism in vivo and receptor involvement in cultured islet beta cells Diabetologia 2004; 47: 225-39., 22Foulis AK, McGill M, Farquharson MA, Hilton DA. A search for evidence of viral infection in pancreases of newly diagnosed patients with IDDM Diabetologia 1997; 40: 53-61.] and virus proteins [22Foulis AK, McGill M, Farquharson MA, Hilton DA. A search for evidence of viral infection in pancreases of newly diagnosed patients with IDDM Diabetologia 1997; 40: 53-61.] have been detected in the endocrine islets of Langerhans, but only occasionally in the exocrine cells [22Foulis AK, McGill M, Farquharson MA, Hilton DA. A search for evidence of viral infection in pancreases of newly diagnosed patients with IDDM Diabetologia 1997; 40: 53-61., 23Foulis AK, Farquharson MA, Cameron SO, McGill M, Schonke H, Kandolf R. A search for the presence of the enteroviral capsid protein VP1 in pancreases of patients with type 1 (insulin-dependent) diabetes and pancreases and hearts of infants who died of coxsackieviral myocarditis Diabetologia 1990; 33: 290-8.]. Moreover, a large-scale study by Richardson et al. showed that the human enterovirus capsid protein 1 (HEVP1) could be detected in the endocrine islets of about 61% of recent onset T1D subjects [10Richardson SJ, Willcox A, Bone AJ, Foulis AK, Morgan NG. The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes Diabetologia 2009; 52: 1143-51.]. HEV RNA was also detected in islet cells and in a few ductal cells from T1D patients [21Ylipaasto P, Klingel K, Lindberg AM, et al. Enterovirus infection in human pancreatic islet cells islet tropism in vivo and receptor involvement in cultured islet beta cells Diabetologia 2004; 47: 225-39.] and a child positive for islet related auto-antibodies [9Oikarinen M, Tauriainen S, Honkanen T. Analysis of pancreas tissue in a child positive for islet cell antibodies Diabetologia 2008; 51: 1796-802.]. In none of these cases was virus detected in the exocrine cells. Only rarely has HEVs been isolated from the pancreas at onset of T1D [12Yoon J-W, Austin M, Onodera T, Notkins AL. Virus-Induced Diabetes Mellitus N Engl J Med 1979; 300: 1173-9.].

One of the key determinants of a successful virus infection is the binding to a proper receptor. Two distinct receptors for CVBs have been characterized: The Coxsackie and Adenovirus receptor (CAR), located in tight junctions, and the decay and accelerating factor (DAF/CD55), which in some cells, is believed to act as a co-receptor, transporting the virus to the CAR [24Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5 Science 1997; 275: 1320-3.-26Shafren DR, Bates RC, Agrez MV, Herd RL, Burns GF, Barry RD. Coxsackieviruses B1, B3, and B5 use decay accelerating factor as a receptor for cell attachment J Virol 1995; 69: 3873-7.]. Some strains of CVB have been shown to infect cells lacking both of these receptors, which highlights the complexity of the virus tropism [27Frisk G, Elfstrom T, Diderholm H. The replication of certain Coxsackie B virus strains in CHO cells J Virol Methods 2001; 98: 161-5., 28Schmidtke M, Selinka HC, Heim A, et al. Attachment of coxsackievirus B3 variants to various cell lines: mapping of phenotypic differences to capsid protein VP1 Virology 2000; 275: 77-88.]. The reports on CAR expression on pancreatic exocrine and endocrine cells are diverging among various research groups, species and techniques [9Oikarinen M, Tauriainen S, Honkanen T. Analysis of pancreas tissue in a child positive for islet cell antibodies Diabetologia 2008; 51: 1796-802., 16Drescher KM, Kono K, Bopegamage S, Carson SD, Tracy S. Coxsackievirus B3 infection and type 1 diabetes development in NOD mice insulitis determines susceptibility of pancreatic islets to virus infection Virology 2004; 329: 381-94., 21Ylipaasto P, Klingel K, Lindberg AM, et al. Enterovirus infection in human pancreatic islet cells islet tropism in vivo and receptor involvement in cultured islet beta cells Diabetologia 2004; 47: 225-39., 29Mena I, Fischer C, Gebhard JR, Perry CM, Harkins S, Whitton JL. Coxsackievirus infection of the pancreas evaluation of receptor expression pathogenesis and immunopathology Virology 2000; 271: 276-88., 30Raschperger E, Thyberg J, Pettersson S, Philipson L, Fuxe J, Pettersson RF. The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis Exp Cell Res 2006; 312: 1566-80.]. Lack of DAF expression has been shown in cultured islets [21Ylipaasto P, Klingel K, Lindberg AM, et al. Enterovirus infection in human pancreatic islet cells islet tropism in vivo and receptor involvement in cultured islet beta cells Diabetologia 2004; 47: 225-39.]. After entry into the host cell, the virus needs to replicate its genome and translate a number of structural and non-structural proteins in order to produce new virions. HEV replication complexes are associated to the surface of virus induced vesicles/chambers [31Belov GA, Nair V, Hansen BT, Hoyt FH, Fischer ER, Ehrenfeld E. Complex dynamic development of poliovirus membranous replication complexes J Virol 2012; 86: 302-12.-34Egger D, Bienz K. Intracellular location and translocation of silent and active poliovirus replication complexes J Gen Virol 2005; 86: 707-18.]. How these vesicle membranes are formed is not clear, but markers from several organelles, e.g. from the endoplasmatic reticulum (ER) and Golgi apparatus, have been associated to them, suggesting that the virus rearrange these structures for its own propagation [32Suhy DA, Giddings THJr, Kirkegaard K. Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins an autophagy-like origin for virus-induced vesicles J Virol 2000; 74: 8953-65., 35Wessels E, Duijsings D, Niu TK, et al. A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1 Dev Cell 2006; 11: 191-201.-37Rust RC, Landmann L, Gosert R, et al. Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex J Virol 2001; 75: 9808-18.].

Studies of the implication of HEV in T1D and pancreatitis in humans remain limited and inconclusive, partly due to limited access to primary human cells and tissues. Instead cell-lines and animal models, mostly mice, have been used to study the mechanisms behind the diseases, which might not reflect the human pathogenesis. Thus, the aim of this study was to investigate the tropism of two CVB-5 strains to primary human pancreatic endocrine and exocrine cell clusters in vitro. An additional aim was to study the subcellular localization of HEV particles and the ultrastructural changes induced by infection.

Immunolabeling of HEVP1, dsRNA and the electron micrographs showed that the two CVB-5 strains used in this study could not replicate in human exocrine cell clusters in vitro. This was also shown for a CVB-1 strain by immunohistochemical technique. These results diverge from what has been seen in numerous mouse studies in vivo [14Ramsingh AI. Coxsackieviruses and pancreatitis Front Biosci 1997; 2: e53-62., 17Burch GE, Harb JM. Electron microscopic studies of viral pancreatitis in coxsackie B4 virus infected mice Exp Mol Pathol 1979; 31: 23-35., 29Mena I, Fischer C, Gebhard JR, Perry CM, Harkins S, Whitton JL. Coxsackievirus infection of the pancreas evaluation of receptor expression pathogenesis and immunopathology Virology 2000; 271: 276-88., 41Vuorinen T, Kallajoki M, Hyypia T, Vainionpaa R. Coxsackievirus B3-induced acute pancreatitis: analysis of histopathological and viral parameters in a mouse model Br J Exp Pathol 1989; 70: 395-403.] and in vitro (present study). Our results are further supported by studies of children with systemic HEV infections, in which viral RNA was detected in the islets but not in the exocrine pancreas [21Ylipaasto P, Klingel K, Lindberg AM, et al. Enterovirus infection in human pancreatic islet cells islet tropism in vivo and receptor involvement in cultured islet beta cells Diabetologia 2004; 47: 225-39.]. The minor titer increase seen in a few donors is likely to be due to one or several cellular subpopulations, e.g. endocrine cells endothelial cells, ductal cells or blood cells which all have shown to be susceptible for HEV infection [11Roivainen M, Rasilainen S, Ylipaasto P. Mechanisms of Coxsackievirus-Induced Damage to Human Pancreatic {beta}-Cells J Clin Endocrinol Metab 2000; 85: 432-0., 20Frisk G, Diderholm H. Tissue culture of isolated human pancreatic islets infected with different strains of coxsackievirus B4: assessment of virus replication and effects on islet morphology and insulin release Int J Exp Diabetes Res 2000; 1: 165-75., 21Ylipaasto P, Klingel K, Lindberg AM, et al. Enterovirus infection in human pancreatic islet cells islet tropism in vivo and receptor involvement in cultured islet beta cells Diabetologia 2004; 47: 225-39., 42Zanone MM, Favaro E, Ferioli E, et al. Human pancreatic islet endothelial cells express coxsackievirus and adenovirus receptor and are activated by coxsackie B virus infection FASEB J 2007; 21: 3308-17., 43Smura T, Ylipaasto P, Klemola P, et al. Cellular tropism of human enterovirus D species serotypes EV-94.EV-70 and EV-68 in vitro implications for pathogenesis J Med Virol 2010; 82: 1940-9.]. This is further supported by the rapidly declining virus titers after the initial increase in exocrine cell clusters from organ donors. The chromogranin A and dithizone staining clearly shows that endocrine cells are present in various amounts even in handpicked exocrine cell clusters, suggesting that this is the main replication source. In addition to potentially different expression of virus receptors, variations in antiviral response between virus-inoculated cells, could contribute to the divergence in virus tropism. IFN response is an important tropism determinant which has been shown in various HEV infected tissues [44Ida-Hosonuma M, Iwasaki T, Yoshikawa T, et al. The alpha/beta interferon response controls tissue tropism and pathogenicity of poliovirus J Virol 2005; 79: 4460-9., 45Yoshikawa T, Iwasaki T, Ida-Hosonuma M, et al. Role of the alpha/beta interferon response in the acquisition of susceptibility to poliovirus by kidney cells in culture J Virol 2006; 80: 4313-25.] including human and mouse islets [46Flodstrom M, Maday A, Balakrishna D, Cleary MM, Yoshimura A, Sarvetnick N. Target cell defense prevents the development of diabetes after viral infection Nat Immunol 2002; 3: 373-82., 47Hultcrantz M, Huhn MH, Wolf M, et al. Interferons induce an antiviral state in human pancreatic islet cells Virology 2007; 367: 92-101.]. However, if the difference in antiviral defence can explain the tropism patterns seen between the exocrine and endocrine cells and between the species in this study remains to be studied.

Our findings show for the first time that CVB infections might not be a major cause of pancreatitis, although it cannot be excluded that there exists susceptible individuals or specific virus serotypes/strains that have tropism for the human exocrine pancreas.

The second aim in our study was to determine the subcellular location of HEV in virus-inoculated pancreatic cells. It was shown that virus particles were observed restrictively in endocrine cells, almost exclusively in beta-cells. The virus particles were often assembled in crystalline structures and the infected cells displayed chromatin condensation and increased number of vesicle and vacuoles, which are hallmarks of HEV infection [11Roivainen M, Rasilainen S, Ylipaasto P. Mechanisms of Coxsackievirus-Induced Damage to Human Pancreatic {beta}-Cells J Clin Endocrinol Metab 2000; 85: 432-0.]. Interestingly the virus particles were mainly found in close association to insulin granules, both the membranes and the core. The insulin granules in infected cells were often enlarged and contained more than one insulin crystal or barely recognizable insulin crystals. The close association of virus particles to insulin granules suggests that the virus can utilize these as replication scaffolds and/or affect the secretion of insulin. A normal beta-cell contains over 10.000 insulin granules (reviewed in [48Rorsman P, Renstrom E. Insulin granule dynamics in pancreatic beta cells Diabetologia 2003; 46: 1029-45.]), which might enable efficient virus propagation. This is consistent with previous studies, in which it has been shown that HEV replication complexes often contain markers from the endoplasmatic reticulum and Golgi apparatus [32Suhy DA, Giddings THJr, Kirkegaard K. Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins an autophagy-like origin for virus-induced vesicles J Virol 2000; 74: 8953-65., 36Schlegel A, Giddings TH Jr, Ladinsky MS, Kirkegaard K. Cellular origin and ultrastructure of membranes induced during poliovirus infection J Virol 1996; 70: 6576-88.] as well as proteins from the secretory pathway [33Bienz K, Egger D, Pfister T, Troxler M. Structural and functional characterization of the poliovirus replication complex J Virol 1992; 66: 2740-7., 37Rust RC, Landmann L, Gosert R, et al. Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex J Virol 2001; 75: 9808-18.].

In conclusion the tropism of two CVB-5 strains is in vitro restricted to human primary pancreatic endocrine cells. This is the opposite from what has been shown in mice studies in which the exocrine cell clusters are infected while the islets usually are spared.