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Cell entry mechanisms of dengue virusSchaar, Hilde Margot van der

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General introduction

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Chapter 1

Dengue virus (DENV) currently causes the most common mosquito-borne viral infection in the world. According to the estimates of the World Health Organization, 50 to 100 million infections occur each year leading to 500,000 hospitalizations and 20,000 deaths (WHO, 2009). Around 2.5 billion people live in areas infested with the domesticated mosquito Aedes Aegypti, which transmits the virus, and thus are at risk for infection, see Figure 1. During the last few decades, the incidence of DENV infections has increased dramatically, which can be attributed to major societal and economical changes, including population growth, urbanization, lack of mosquito control, international trade, and modern transportation. These factors have promoted the geographical spread of all four DENV serotypes throughout the (sub)tropical regions of the world, which has resulted in larger and more severe outbreaks (Gubler, 2002; Mackenzie et al., 2004). DENV, an enveloped positive-sense RNA virus, is classified to the Flavivirus genus within the Flaviviridae family, which also includes yellow fever virus, West Nile virus, and tick-borne encephalitis virus. There are four closely related serotypes of DENV, called DENV-1 to DENV-4 (Lindenbach & Rice, 2001). Although many DENV infections are clinically inapparent,

Figure 1. Global distribution of the mosquito vector Aedes Aegypti and dengue epidemic activity in 2005. Adapted from the US Center for Disease Control and Prevention.

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General introduction

each serotype can cause disease in humans. Most individuals that do become ill experience uncomplicated dengue fever, an acute febrile illness accompanied by headache, often muscle and joint pain, nausea, vomiting, and rash. Yet, approximately 500,000 patients per year develop potentially life-threatening dengue hemorrhagic fever (DHF), which is characterized by high fever, plasma leakage, bleeding manifestations, and often hepatomegaly. In severe cases, plasma leakage can result in circulatory failure and hypovolemic shock, which is called dengue shock syndrome (DSS). Without proper treatment, such as effective replacement of the plasma loss, DHF/DSS can be fatal (Gibbons & Vaughn, 2002; Halstead, 2007; Rigau-Perez et al., 1998). Despite the high clinical impact of DENV, there are neither vaccines nor antiviral drugs available yet to prevent or treat the disease. The pathogenesis of DHF/DSS is not completely understood. Several clinical studies have established factors that correlate with disease severity. First of all, there is a considerably larger dengue-infected cell mass and higher viremia titers in the prelude of DHF (Libraty et al., 2002; Vaughn et al., 2000; Wang et al., 2003). Also, the serum levels of several pro- and anti-inflammatory cytokines and chemical mediators, such as TNF-a, IL-b, IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, and IL-18, are higher in patients with DHF (Bethell et al., 1998; Braga et al., 2001; Green et al., 1999b; Hober et al., 1993; Kurane et al., 1991; Mustafa et al., 2001; Pacsa et al., 2000; Raghupathy et al., 1998). Furthermore, lymphocytes isolated from DHF patients show markers of activation, such as expression of CD69 and secretion of soluble IL-2 receptor, soluble CD4, and soluble CD8 (Green et al., 1999a; Kurane et al., 1991). Other clinical studies have demonstrated that high serum levels of complement factor C3a and the SC5b-9 complement complex are associated with DHF as well (Avirutnan et al., 2006; Malasit, 1987). The elevated vascular permeability causing plasma leakage is presumably the consequence of a functional disturbance of the endothelial cells, most likely induced by the cytokine storm, as examination of the capillaries in skin biopsies of DHF patients has revealed that there is no severe damage to the endothelial cells (Basu & Chaturvedi, 2008; Sahaphong et al., 1980). What is the cause of the high viral load and the strong immune response in the onset of DHF? Intriguingly, many epidemiological studies have demonstrated that the development of DHF is strongly associated with secondary infections with a heterotypic serotype, i.e. a serotype different than the one(s) the individual has encountered before (Burke et al., 1988; Guzman et al., 1990; Halstead et al., 1969; Sangkawibha et al., 1984; Thein et al., 1997).

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Chapter 1

Furthermore, infants born to dengue-immune mothers have been shown to be at greater risk for developing DHF during a primary infection due to passively transferred maternal antibodies (Halstead et al., 2002; Kliks et al., 1988; Kliks et al., 1989; Nguyen et al., 2004; Simmons et al., 2007). Together these studies have led to two compatible hypotheses on enhancement of DENV infection that describe the role of antibodies and T cells in the development of severe disease (Fink et al., 2006; Halstead, 2007; Lei et al., 2001; Rothman & Ennis, 1999; Rothman, 2004). The first and widely accepted hypothesis concerns antibody-dependent enhancement (ADE) of DENV infection. ADE occurs when pre-existing antibodies direct the newly infecting virus particles towards the cells of the immune system, which are the natural host cells of DENV (Halstead, 2003; Kou et al., 2008; Pang et al., 2007; Rothman & Ennis, 1999; Sullivan, 2001; Wu et al., 2000). The antibodies facilitate more efficient cellular uptake of the virus via Fc receptor-mediated phagocytosis. Yet, the antibodies somehow fail to neutralize the virus intracellularly and thus increase the number of infected cells, which will result in a high viral load and induce the immunological cascade leading to plasma leakage. T cells are subsequently believed to play a pivotal role in this immunological cascade. The other hypothesis postulates that during a heterotypic secondary infection, there is a preferential activation of memory T cells with a higher avidity for primary serotype and thus a lower avidity for the newly infecting virus, a phenomenon called “original antigenic sin”, which results in altered T cell effector functions and increased cytokine production (Imrie et al., 2007; Mongkolsapaya et al., 2006; Zivny et al., 1999). Moreover, these cross-reactive memory T cells are expanded more rapidly in a secondary infection than the naïve T cell population (Mongkolsapaya et al., 2003), which could drive the immune response towards a more pathological outcome. Taken together, it appears that T cells primarily operate in the strong immune response in the prelude of DHF, while antibodies directly influence the infectious properties of the virus by altering the cell entry mechanisms. DENV enters its host cell via receptor-mediated endocytosis (Lindenbach & Rice, 2001), see Figure 2. The first step in this process is binding of the virus particle to a cellular receptor. Subsequently, the virus particle is internalized into the cell and transported to an intracellular vesicle called an endosome (Flint et al., 2004). Endosomes maintain an acidic lumen, which induces conformational changes in the major envelope protein E that promote fusion of the viral membrane with the endosomal membrane (Heinz et al., 2004; Kielian & Rey, 2006; Kuhn et al., 2002; Modis et al., 2004; Perera et al., 2008; Rey et al., 1995). Upon membrane fusion, the nucleocapsid enclosing the viral

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General introduction

genome is delivered into the cytosol to initiate viral replication. Following viral RNA replication and translation, immature virions are assembled by budding of newly formed nucleocapsids into the lumen of the ER, thereby acquiring a lipid bilayer envelope with the structural proteins prM and E (Lindenbach & Rice, 2001), see Figure 2. Subsequently, the immature virions mature during transport through the acidic trans-Golgi network, where the prM proteins are believed to stabilize the E proteins to prevent conformational changes that could lead to premature abortion of the productive cycle (Guirakhoo et al., 1992; Heinz et al., 1994; Li et al., 2008; Lorenz et al., 2002). Shortly before release of the virions from the host cell, the maturation process is completed when prM is cleaved by the cellular protease furin into a soluble pr peptide and virion-associated M. Upon secretion from the cell, the virus particles encounter a neutral pH, which promotes dissociation of the pr peptides from the virus particles and generates mature, infectious virions (Elshuber et al., 2003; Guirakhoo et al., 1991; Perera et al., 2008; Stadler et al., 1997).

Figure 2. Life cycle of dengue virus. See text for details.

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Chapter 1

Remarkably, several in vitro studies have demonstrated that, compared to other flaviviruses, cells infected with DENV release high numbers of immature particles containing unprocessed prM (Anderson et al., 1997; He et al., 1995; Henchal et al., 1985; Murray et al., 1993; Putnak et al., 1996; Randolph et al., 1990; Wang et al., 1999; Zybert et al., 2008). Multiple studies on other immature flavivirus particles have indicated that the maturation process is a prerequisite for viral infectivity (Elshuber et al., 2003; Heinz et al., 1994; Stadler et al., 1997). Indeed, immature DENV particles appear non-infectious in titration assays (Zybert et al., 2008), which might imply that these particles are an irrelevant byproduct of DENV infection. Interestingly, antibodies directed against prM are commonly found in sera of patients (Bray & Lai, 1991; Cardosa et al., 2002; Lai et al., 2008; Se-Thoe et al., 1999). Moreover, the antibody responses to prM are significantly elevated in patients after secondary dengue episodes (Lai et al., 2008), which might suggest a contribution of immature DENV to the pathogenesis of severe disease. To better understand the molecular mechanisms involved in enhancement of DENV infection by antibodies, it is essential to gain fundamental insights into the cell entry properties of the virus. Dissection of the cell entry pathway of DENV will enrich our knowledge on the critical determinants in DENV infection and may contribute to the rational design of safe antiviral drugs and vaccines. Additionally, in order to elucidate the potential role of immature particles in the disease pathogenesis, it is important to study the infectious properties of immature particles in the presence of anti-prM antibodies.

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General introduction

Scope of this thesisThe studies described in this thesis focus on the mechanisms involved in cell entry that control the infectious properties of DENV. Chapter 2 provides a general overview of the potential mechanisms involved in cell entry of enveloped viruses. Subsequently, the internalization route of flaviviruses, including DENV, is outlined as well as the intracellular trafficking behavior prior to membrane fusion. Since this chapter is published as a review article, results described in Chapters 3 and 4 are also discussed here. Finally, the current hypotheses on how antibodies can either neutralize or enhance viral infection through alteration of the cell entry process of DENV are addressed. In Chapter 3, the infectious properties of DENV are investigated by determination of the physical particle to infectious unit ratio using several biochemical and titration assays. In order to explain the observed ratio, a method based on real-time fluorescence microscopy is used to visualize the cell entry process of individual DENV particles in living cells. The cellular binding properties of DENV, its intracellular transport behavior and the time point of membrane fusion are examined to determine the critical steps in cell entry that define the infectivity of DENV. Chapter 4 presents an elaborate study on the cell entry pathway of DENV to elucidate the internalization route, trafficking through the endocytic network, and the organelle where membrane fusion occurs. Single virus particles are simultaneously tracked with endocytic structures illuminated by fluorescent marker proteins in living cells, which enables us to gather unique information on the mechanisms and kinetics in DENV entry. In Chapter 5, the effect of an anti-prM antibody on the infectious pro-perties of immature DENV particles is investigated in Fc receptor-expressing cells. To this end, the different stages in the cell entry process of antibody-opsonized immature particles are examined. The binding efficiency of immature particles to Fc receptor-bearing cells is determined as well as the involvement of furin in the entry process. Chapter 6 gives a summarizing discussion of the results and conclusions presented in this thesis.

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ReferencesAnderson R, Wang S, Osiowy C, and Issekutz AC. 1997. Activation of endothelial cells via

antibody-enhanced dengue virus infection of peripheral blood monocytes. J. Virol. 71: 4226-4232.

Avirutnan P, Punyadee N, Noisakran S, Komoltri C, Thiemmeca S, Auethavornanan K, Jairungsri A, Kanlaya R, Tangthawornchaikul N, Puttikhunt C, Pattanakitsakul SN, Yenchitsomanus PT, Mongkolsapaya J, Kasinrerk W, Sittisombut N, Husmann M, Blettner M, Vasanawathana S, Bhakdi S, and Malasit P. 2006. Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. J. Infect. Dis. 193: 1078-1088.

Basu A and Chaturvedi UC. 2008. Vascular endothelium: the battlefield of dengue viruses. FEMS Immunol. Med. Microbiol. 53: 287-299.

Bethell DB, Flobbe K, Cao XT, Day NP, Pham TP, Buurman WA, Cardosa MJ, White NJ, and Kwiatkowski D. 1998. Pathophysiologic and prognostic role of cytokines in dengue hemorrhagic fever. J. Infect. Dis. 177: 778-782.

Braga EL, Moura P, Pinto LM, Ignacio SR, Oliveira MJ, Cordeiro MT, and Kubelka CF. 2001. Detection of circulant tumor necrosis factor-alpha, soluble tumor necrosis factor p75 and interferon-gamma in Brazilian patients with dengue fever and dengue hemorrhagic fever. Mem. Inst. Oswaldo Cruz 96: 229-232.

Bray M and Lai CJ. 1991. Dengue virus premembrane and membrane proteins elicit a protective immune response. Virology 185: 505-508.

Burke DS, Nisalak A, Johnson DE, and Scott RM. 1988. A prospective study of dengue infections in Bangkok. Am. J. Trop. Med. Hyg. 38: 172-180.

Cardosa MJ, Wang SM, Sum MS, and Tio PH. 2002. Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses. BMC. Microbiol. 2: 9.

Elshuber S, Allison SL, Heinz FX, and Mandl CW. 2003. Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. J. Gen. Virol. 84: 183-191.

Fink J, Gu F, and Vasudevan SG. 2006. Role of T cells, cytokines and antibody in dengue fever and dengue haemorrhagic fever. Rev. Med. Virol. 16: 263-275.

Flint,S.J., L.W.Enquist, V.R.Racaniello, and A.M.Skalka. 2004. Attachment and entry. In: Principles of virology. ASM Press, Washington DC, pp. 126-180.

Gibbons RV and Vaughn DW. 2002. Dengue: an escalating problem. BMJ. 324: 1563-1566.Green S, Pichyangkul S, Vaughn DW, Kalayanarooj S, Nimmannitya S, Nisalak A, Kurane

I, Rothman AL, and Ennis FA. 1999a. Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever. J. Infect. Dis. 180: 1429-1435.

Green S, Vaughn DW, Kalayanarooj S, Nimmannitya S, Suntayakorn S, Nisalak A, Rothman AL, and Ennis FA. 1999b. Elevated plasma interleukin-10 levels in acute dengue correlate with disease severity. J. Med. Virol. 59: 329-334.

Gubler DJ. 2002. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol. 10: 100-103.

Guirakhoo F, Bolin RA, and Roehrig JT. 1992. The Murray Valley encephalitis virus prM

17

General introduction

protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology 191: 921-931.

Guirakhoo F, Heinz FX, Mandl CW, Holzmann H, and Kunz C. 1991. Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions. J. Gen. Virol. 72 ( Pt 6): 1323-1329.

Guzman MG, Kouri GP, Bravo J, Soler M, Vazquez S, and Morier L. 1990. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic study. Am. J. Trop. Med. Hyg. 42: 179-184.

Halstead SB. 2003. Neutralization and antibody-dependent enhancement of dengue viruses. Adv. Virus Res. 60: 421-467.

Halstead SB. 2007. Dengue. Lancet 370: 1644-1652.Halstead SB, Lan NT, Myint TT, Shwe TN, Nisalak A, Kalyanarooj S, Nimmannitya S,

Soegijanto S, Vaughn DW, and Endy TP. 2002. Dengue hemorrhagic fever in infants: research opportunities ignored. Emerg. Infect. Dis. 8: 1474-1479.

Halstead SB, Scanlon JE, Umpaivit P, and Udomsakdi S. 1969. Dengue and chikungunya virus infection in man in Thailand, 1962-1964. IV. Epidemiologic studies in the Bangkok metropolitan area. Am. J. Trop. Med. Hyg. 18: 997-1021.

He RT, Innis BL, Nisalak A, Usawattanakul W, Wang S, Kalayanarooj S, and Anderson R. 1995. Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2. J. Med. Virol. 45: 451-461.

Heinz FX, Stiasny K, and Allison SL. 2004. The entry machinery of flaviviruses. Arch. Virol. Suppl 133-137.

Heinz FX, Stiasny K, Puschner-Auer G, Holzmann H, Allison SL, Mandl CW, and Kunz C. 1994. Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198: 109-117.

Henchal EA, McCown JM, Burke DS, Seguin MC, and Brandt WE. 1985. Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies. Am. J. Trop. Med. Hyg. 34: 162-169.

Hober D, Poli L, Roblin B, Gestas P, Chungue E, Granic G, Imbert P, Pecarere JL, Vergez-Pascal R, Wattre P, and . 1993. Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta) in dengue-infected patients. Am. J. Trop. Med. Hyg. 48: 324-331.

Imrie A, Meeks J, Gurary A, Sukhbataar M, Kitsutani P, Effler P, and Zhao Z. 2007. Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes. J. Virol. 81: 10081-10091.

Kielian M and Rey FA. 2006. Virus membrane-fusion proteins: more than one way to make a hairpin. Nat. Rev. Microbiol. 4: 67-76.

Kliks SC, Nimmanitya S, Nisalak A, and Burke DS. 1988. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am. J. Trop. Med. Hyg. 38: 411-419.

Kliks SC, Nisalak A, Brandt WE, Wahl L, and Burke DS. 1989. Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am. J. Trop. Med. Hyg. 40: 444-451.

18

Chapter 1

Kou Z, Quinn M, Chen H, Rodrigo WW, Rose RC, Schlesinger JJ, and Jin X. 2008. Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells. J. Med. Virol. 80: 134-146.

Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones CT, Mukhopadhyay S, Chipman PR, Strauss EG, Baker TS, and Strauss JH. 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108: 717-725.

Kurane I, Innis BL, Nimmannitya S, Nisalak A, Meager A, Janus J, and Ennis FA. 1991. Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. J. Clin. Invest 88: 1473-1480.

Lai CY, Tsai WY, Lin SR, Kao CL, Hu HP, King CC, Wu HC, Chang GJ, and Wang WK. 2008. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J. Virol. 82: 6631-6643.

Lei HY, Yeh TM, Liu HS, Lin YS, Chen SH, and Liu CC. 2001. Immunopathogenesis of dengue virus infection. J. Biomed. Sci. 8: 377-388.

Li L, Lok SM, Yu IM, Zhang Y, Kuhn RJ, Chen J, and Rossmann MG. 2008. The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319: 1830-1834.

Libraty DH, Endy TP, Houng HS, Green S, Kalayanarooj S, Suntayakorn S, Chansiriwongs W, Vaughn DW, Nisalak A, Ennis FA, and Rothman AL. 2002. Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections. J. Infect. Dis. 185: 1213-1221.

Lindenbach D, and Rice CM. 2001. Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Malcolm AM, Roizman B, and Straus SE (eds). Fields Virology. Lippincott Williams & Wilkins, Philadelphia, pp. 991-1041.

Lorenz IC, Allison SL, Heinz FX, and Helenius A. 2002. Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J. Virol. 76: 5480-5491.

Mackenzie JS, Gubler DJ, and Petersen LR. 2004. Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat. Med. 10: S98-109.

Malasit P. 1987. Complement and dengue haemorrhagic fever/shock syndrome. Southeast Asian J. Trop. Med. Public Health 18: 316-320.

Modis Y, Ogata S, Clements D, and Harrison SC. 2004. Structure of the dengue virus envelope protein after membrane fusion. Nature 427: 313-319.

Mongkolsapaya J, Dejnirattisai W, Xu XN, Vasanawathana S, Tangthawornchaikul N, Chairunsri A, Sawasdivorn S, Duangchinda T, Dong T, Rowland-Jones S, Yenchitsomanus PT, McMichael A, Malasit P, and Screaton G. 2003. Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat. Med. 9: 921-927.

Mongkolsapaya J, Duangchinda T, Dejnirattisai W, Vasanawathana S, Avirutnan P, Jairungsri A, Khemnu N, Tangthawornchaikul N, Chotiyarnwong P, Sae-Jang K, Koch M, Jones Y, McMichael A, Xu X, Malasit P, and Screaton G. 2006. T cell responses in dengue hemorrhagic fever: are cross-reactive T cells suboptimal? J. Immunol. 176: 3821-3829.

Murray JM, Aaskov JG, and Wright PJ. 1993. Processing of the dengue virus type 2 proteins

19

General introduction

prM and C-prM. J. Gen. Virol. 74 ( Pt 2): 175-182.Mustafa AS, Elbishbishi EA, Agarwal R, and Chaturvedi UC. 2001. Elevated levels of

interleukin-13 and IL-18 in patients with dengue hemorrhagic fever. FEMS Immunol. Med. Microbiol. 30: 229-233.

Nguyen TH, Lei HY, Nguyen TL, Lin YS, Huang KJ, Le BL, Lin CF, Yeh TM, Do QH, Vu TQ, Chen LC, Huang JH, Lam TM, Liu CC, and Halstead SB. 2004. Dengue hemorrhagic fever in infants: a study of clinical and cytokine profiles. J. Infect. Dis. 189: 221-232.

Pacsa AS, Agarwal R, Elbishbishi EA, Chaturvedi UC, Nagar R, and Mustafa AS. 2000. Role of interleukin-12 in patients with dengue hemorrhagic fever. FEMS Immunol. Med. Microbiol. 28: 151-155.

Pang T, Cardosa MJ, and Guzman MG. 2007. Of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (DHF/DSS). Immunol. Cell Biol. 85: 43-45.

Perera R, Khaliq M, and Kuhn RJ. 2008. Closing the door on flaviviruses: entry as a target for antiviral drug design. Antiviral Res. 80: 11-22.

Putnak R, Barvir DA, Burrous JM, Dubois DR, D’Andrea VM, Hoke CH, Sadoff JC, and Eckels KH. 1996. Development of a purified, inactivated, dengue-2 virus vaccine prototype in Vero cells: immunogenicity and protection in mice and rhesus monkeys. J. Infect. Dis. 174: 1176-1184.

Raghupathy R, Chaturvedi UC, Al-Sayer H, Elbishbishi EA, Agarwal R, Nagar R, Kapoor S, Misra A, Mathur A, Nusrat H, Azizieh F, Khan MA, and Mustafa AS. 1998. Elevated levels of IL-8 in dengue hemorrhagic fever. J. Med. Virol. 56: 280-285.

Randolph VB, Winkler G, and Stollar V. 1990. Acidotropic amines inhibit proteolytic processing of flavivirus prM protein. Virology 174: 450-458.

Rey FA, Heinz FX, Mandl C, Kunz C, and Harrison SC. 1995. The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375: 291-298.

Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P, Sanders EJ, and Vorndam AV. 1998. Dengue and dengue haemorrhagic fever. Lancet 352: 971-977.

Rothman AL. 2004. Dengue: defining protective versus pathologic immunity. J. Clin. Invest 113: 946-951.

Rothman AL and Ennis FA. 1999. Immunopathogenesis of Dengue hemorrhagic fever. Virology 257: 1-6.

Sahaphong S, Riengrojpitak S, Bhamarapravati N, and Chirachariyavej T. 1980. Electron microscopic study of the vascular endothelial cell in dengue hemorrhagic fever. Southeast Asian J. Trop. Med. Public Health 11: 194-204.

Sangkawibha N, Rojanasuphot S, Ahandrik S, Viriyapongse S, Jatanasen S, Salitul V, Phanthumachinda B, and Halstead SB. 1984. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am. J. Epidemiol. 120: 653-669.

Se-Thoe SY, Ng MM, and Ling AE. 1999. Retrospective study of Western blot profiles in immune sera of natural dengue virus infections. J. Med. Virol. 57: 322-330.

Simmons CP, Chau TN, Thuy TT, Tuan NM, Hoang DM, Thien NT, Lien lB, Quy NT, Hieu NT, Hien TT, McElnea C, Young P, Whitehead S, Hung NT, and Farrar J. 2007. Maternal antibody and viral factors in the pathogenesis of dengue virus in infants. J. Infect. Dis. 196: 416-424.

20

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Stadler K, Allison SL, Schalich J, and Heinz FX. 1997. Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol. 71: 8475-8481.

Sullivan NJ. 2001. Antibody-mediated enhancement of viral disease. Curr. Top. Microbiol. Immunol. 260: 145-169.

Thein S, Aung MM, Shwe TN, Aye M, Zaw A, Aye K, Aye KM, and Aaskov J. 1997. Risk factors in dengue shock syndrome. Am. J. Trop. Med. Hyg. 56: 566-572.

Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, Endy TP, Raengsakulrach B, Rothman AL, Ennis FA, and Nisalak A. 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J. Infect. Dis. 181: 2-9.

Wang S, He R, and Anderson R. 1999. PrM- and cell-binding domains of the dengue virus E protein. J. Virol. 73: 2547-2551.

Wang WK, Chao DY, Kao CL, Wu HC, Liu YC, Li CM, Lin SC, Ho ST, Huang JH, and King CC. 2003. High levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: implications for pathogenesis. Virology 305: 330-338.

WHO, 2009: http://www.who.int/mediacentre/factsheets/fs117/enWu SJ, Grouard-Vogel G, Sun W, Mascola JR, Brachtel E, Putvatana R, Louder MK, Filgueira

L, Marovich MA, Wong HK, Blauvelt A, Murphy GS, Robb ML, Innes BL, Birx DL, Hayes CG, and Frankel SS. 2000. Human skin Langerhans cells are targets of dengue virus infection. Nat. Med. 6: 816-820.

Zivny J, DeFronzo M, Jarry W, Jameson J, Cruz J, Ennis FA, and Rothman AL. 1999. Partial agonist effect influences the CTL response to a heterologous dengue virus serotype. J. Immunol. 163: 2754-2760.

Zybert IA, van der Ende-Metselaar H, Wilschut J, and Smit JM. 2008. Functional importance of dengue virus maturation: infectious properties of immature virions. J. Gen. Virol. 89: 3047-3051.

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