key: cord-299786-wuve0tjz authors: Anderson, Robert title: Manipulation of cell surface macromolecules by flaviviruses date: 2004-02-27 journal: Adv Virus Res DOI: 10.1016/s0065-3527(03)59007-8 sha: doc_id: 299786 cord_uid: wuve0tjz Cell surface macromolecules play a crucial role in the biology and pathobiology of flaviviruses, both as receptors for virus entry and as signaling molecules for cell–cell interactions in the processes of vascular permeability and inflammation. This review examines the cell tropism and pathogenesis of flaviviruses from the standpoint of cell surface molecules, which have been implicated as receptors in both virus–cell as well as cell–cell interactions. The emerging picture is one that encompasses extensive regulation and interplay among the invading virus, viral immune complexes, Fc receptors, major histocompatibility complex antigens, and adhesion molecules. Flaviviruses comprise a rich and diverse family of agents that infect a variety of hosts and cause a wide spectrum of disease. Three disease types are recognized for flaviviruses, namely encephalitis, hemorrhagic fever, and fever-arthralgia-rash. Disease distinctions are not absolute, and overlapping pathologies among various flavivirus members are often observed. The ability of flaviviruses to cause such divergent clinical syndromes, associated with virus replication in a number of different organs, has profound implications for the types of cell surface molecules the virus recognizes as receptors. Mutational analyses of the flaviviral E protein have demonstrated a striking ability of flaviviruses to adapt to different cells and receptors. Given the considerable homologies among them, flaviviruses show a remarkable capacity to cause vastly different diseases with a minimum of alterations in the E protein. The cell surface molecules, which act as receptors for flaviviruses, are only starting to be identified. In addition to providing the molecules involved in virus attachment and penetration, the host cell erects a battery of surface structures that mediate communication with other cells and trigger host defense and pathological processes. Many of these are modulated by flavivirus infection and contribute to the overall picture of pathogenesis. The flavivirus E protein is a multifunctional protein involved in cell receptor binding (Anderson et al., 1992; Chen et al., 1996; He et al., 1995) and virus entry via fusion with a host cell membrane (Rice, 1996) . Some of the functional activities of the E protein, notably membrane fusion, are regulated by interaction with a second viral protein, prM. It is believed that the association of prM with E stabilizes certain pH-sensitive epitopes on the E protein, thereby preventing the conformational changes that normally occur at acidic pH and activate the fusogenic activity of the E protein Guirakhoo et al., 1992; Heinz et al., 1994) . In addition to its normal role in flavivirus assembly, the prM protein has also been included in novel recombinant formulations in which it is generally coexpressed with the E protein; the resultant E/prM complexes have been shown to be immunogenic and protective as vaccines against challenge with several flaviviruses, including Japanese encephalitis virus (Mason et al., 1991) , yellow fever virus (Pincus et al., 1992) , dengue virus (Fonseca et al., 1994) , and tick-borne encephalitis (TBE) virus . In TBE virus, the majority of extracellular virus is largely free of prM protein due to a late intracellular processing event that generates a carboxy-terminal fragment designated M and which together with the E and C proteins are believed to constitute the protein components of the mature virus particle (Heinz et al., 1994) . Cleavage of prM to M enhances low pH-dependent virus-cell fusion (Guirakhoo et al., 1991) and infectivity (Guirakhoo et al., 1992; Heinz et al., 1994; Randolph et al., 1990; Shapiro et al., 1972; Wengler, 1989) . Dengue virions containing prM are still infectious (Randolph et al., 1990) and bind to permissive cells in a manner that can be blocked using E-specific antibodies Wang et al., 1999) . Virus particles containing mainly E and prM also show antibody-enhanced binding to Fc receptor-bearing K562 cells as well as to platelets . Thus, in addition to being requisite precursors to mature virus particles, virus particles containing prM possess many properties associated with mature virus particles. Flaviviruses appear to gain entry to the cell by the endocytic pathway (Rice, 1996) . At low pH, the E protein undergoes a conformational change involving dissociation of the E dimer (Stiasny et al., 1996) , thereby exposing a hidden fusion peptide, followed by reorganization of E into a trimer , in which the fusion peptide is brought close to the membrane-anchoring carboxy terminus (Ferlenghi et al., 2001) . Remarkably similar structural features and conformational rearrangements have been noted between the flavivirus E protein and the alphavirus E1 Lescar et al., 2001; Pletnev et al., 2001; Strauss and Strauss, 2001) , suggesting a common evolutionary origin for these two virion surface proteins. Considerable homology exists among flaviviral E proteins, raising the possibility that different flaviviruses may have similar receptorbinding motifs. For example, many mosquito-borne flaviviruses contain an RGD sequence (e.g., residues 388-390 of the Murray Valley encephalitis virus E protein), which has been implicated in virulence (Lobigs et al., 1990) and receptor binding by analogy with integrin-binding motifs (Rey et al., 1995) . Mutagenesis studies of the yellow fever virus (Van der Most et al., 1999) and Murray Valley encephalitis virus (Hurrelbrink and McMinn, 2001 ) RGD motifs, however, have cast doubt on the role of integrins in flavivirus attachment or entry. Studies with TBE virus have identified important determinants for pathogenicity within the suspected receptor-binding site on the upperlateral surface of domain III (Mandl et al., 2000) . Acquisition of heparan sulfate-binding mutations by passaging TBE in cell culture has also implicated amino acids in this region in receptor binding (Mandl et al., 2001) . The selection of virus mutants on the basis of weak binding to brain membranes has been used with several neurotropic flaviviruses (Holbrook et al., 2001; Ni and Barrett, 1998; Ni et al., 2000) and has identified a variety of mutations within domain III as well as other regions of E. For dengue virus, blocking of virus cell binding correlates more closely to virus neutralization for mAb 3H5 than for mAb 1B7 . This may suggest that mAb 3H5 neutralizes dengue virus predominantly by blocking virus-cell attachment, whereas mAb 1B7 neutralizes dengue virus largely by a postattachment mechanism. The mAb 3H5-binding site on the dengue viral E protein has been partly characterized (Hiramatsu et al., 1996; Megret et al., 1992; Trirawatanapong et al., 1992) and probably encompasses, at a minimum, residues 383-385 (Hiramatsu et al., 1996) within domain III. More recent data involving a larger number of monoclonal antibodies indicate that mAbs that interact with domain III are in fact the most effective blockers of virus-cell attachment (Crill and Roehrig, 2001) . A putative heparan sulfate-binding site on the dengue-2 E protein is also located within this region , and comparative sequencing of dengue type 2 genomes has implicated amino acid 390 of the E protein as a major determinant of pathogenicity (Leitmeyer et al., 1999) . The pH-dependent conformational ''hinge'' region (between domains I and II) of the E protein has also been implicated in virulence, receptor interaction, and/or membrane fusion (Hurrelbrink and McMinn, 2001; Lee et al., 1997; Monath et al., 2002) . Further mutagenesis studies will undoubtedly help define the sites of the E protein involved in flavivirus-cell macromolecule recognition. Transmission of flaviviruses to humans generally occurs via the bite of an infected mosquito or tick. In the case of dengue, inoculated virus is thought to first replicate in skin Langerhans (dendritic) cells (Palucka, 2000; Taweechaisupapong et al., 1996a Taweechaisupapong et al., , 1996b Wu et al., 2000) . Dendritic cells have also been shown to be involved in the transport of intradermally inoculated West Nile virus to local draining lymph nodes, with a subsequent accumulation of leukocytes . It is likely that dendritic cells will prove to be efficient carriers of a wide number of flaviviruses from their cutaneous site of infection to lymphoid and possibly other tissues. Given the importance of dendritic cells in initiating immune responses (Banchereau et al., 2000) , they probably play a pivotal role in stimulating host defense against invading flaviviruses. Dengue virus infection of immature myeloid dendritic cells has been shown to induce their maturation accompanied by the expression of major histocompatibility complex (MHC) class I and II antigens; the costimulatory molecules CD40, CD80, and CD86; and the dendritic cell marker CD83 (Libraty et al., 2001) . Such changes were seen in both dengue-infected and bystander cells, indicating that upregulation of cell surface molecules could be a consequence of virus infection as well as virusinduced cytokine expression. Similarly, Langerhans cells infected with West Nile virus, as well as an alphavirus, Semliki Forest virus, express increased cell surface MHC class II and appear to undergo maturation to a cell type similar to lymphoid dendritic cells (Johnston et al., 1996) . The efficient presentation of both MHC class I-and II-associated viral peptides on the surface of dendritic cells permits the generation of potent cytotoxic and helper T cell responses (see also Section V,A). Monocytes and macrophages have long been recognized as major targets of flavivirus replication in the human host (Halstead, 1989; Scott et al., 1980) . They are also important host cells for the antibody-enhanced replication of certain flaviviruses (see Section IV,C). Because of their presence in the circulation, blood monocytes may be particularly important to the pathogenesis of hemorrhagic viruses, such as dengue. Because most of the pathological changes associated with dengue virus are hemostatic in nature, it is suspected that blood cells, particularly virus-infected blood monocytes, orchestrate many of these effects. Dengue virus-infected human monocytes have been shown to be potent sources of vasoactive cytokines such as tumor necrosis factor (TNF)- and interleukin (IL)-1 (Chang and Shaio, 1994) . Monocytes are also known producers of several other vasoactive mediators, including IL-6, platelet-activating factor (PAF), prostaglandins, thromboxanes, leukotrienes, and nitric oxide (Bulger and Maier, 2000; Funk, 2001; Lefer, 1989; Maruo et al., 1992; Montrucchio et al., 2000; Szabo and Billiar, 1999) , any of which could have powerful effects on endothelial cell physiology. A crucial aspect in understanding dengue pathogenesis will be the identification of additional vasoactive mediators, which trigger the key dysfunctional events in vascular integrity. Various tissue macrophages are undoubtedly important in the pathogenesis of flaviviral diseases but have, to date, not received much attention. Skin mononuclear cells, pulmonary, splenic, and thymic macrophages and liver Kupffer cells have been recognized carriers of viral antigen (Halstead, 1989) . In the liver, virus or viral antigen has been found in Kupffer cells and hepatocytes in infections with yellow fever (Monath et al., 1989) and dengue (Bhamarapravati et al., 1967; Hall et al., 1991; Halstead, 1989; Rosen and Khin, 1989) . Destruction of Kupffer cells, possibly by apoptosis, has been reported in the liver of some patients with fatal dengue (Huerre et al., 2001) . Primary cultures of Kupffer cells apparently undergo an abortive infection with dengue virus in which viral antigen but no progeny virus is produced (Marianneau et al., 1999) . Many flaviviruses invade either visceral or central nervous system tissues following initial replication in dendritic cells, monocytes, or macrophages. Often this necessitates a transfer of virus across blood vessel endothelial layers. For neurotropic flaviviruses, endothelial cells of the cerebral microvasculature constitute a barrier that must be overcome in order to gain access to the central nervous system. How this occurs remains uncertain. Transendothelial passage of virus may direct infection of cerebral microvascular endothelial cells, may transport across the endothelial layer, or both (Dropulic and Masters, 1990) . Japanese encephalitis virus has been observed electron microscopically to traverse mouse cerebral endothelial cells by transcytosis (Liou and Hsu, 1998) . Alternatively, virus may spread from blood vessels to the olfactory neuroepithelium and from there to olfactory neurons (McMinn et al., 1996; Monath et al., 1983) . Even normally nonneurotropic flaviviruses may occasionally invade the central nervous system under certain conditions. Modulation of the blood-brain barrier by anesthetics (Ben-Nathan et al., 2000) or lipopolysaccharide (Lustig et al., 1992) has been reported to facilitate neuroinvasion by a normally noninvasive strain of West Nile virus. Flaviviruses may also trigger the production of soluble factors that perturb the integrity of the blood-brain barrier, leading to increased leakage of proteins and cells into the central nervous system (Chaturvedi et al., 1991) . These studies indicate that even nonneurotropic flaviviruses may infect tissues of the central nervous system or otherwise affect the integrity of the blood-brain barrier under special circumstances. Transendothelial migration of individual leukocytes (e.g., lymphocytes, monocytes, neutrophils, eosinophils) is regulated in a highly specific manner by the differential expression of selected adhesion molecules on endothelial cells (reviewed in Crockett, 1998; Lowell and Berton, 1999) . Flaviviruses, including dengue and West Nile (Shen et al., 1997) viruses, activate endothelial cell adhesion molecule expression by either direct (virus-mediated) or indirect (cytokine-mediated) mechanisms (see Section V,C). In the presence of leukocyte-attracting chemokines, such virus-triggered activation of the vascular endothelium may contribute toward the migration of leukocytes into extravascular tissues. In addition to being a mechanism for virus dissemination, this process may also be a factor in phenomena such as leukopenia and particularly neutropenia (loss of circulating leukocytes, neutrophils) often observed in flavivirus, particularly dengue, infection (reviewed in Halstead, 1989) . Due to the lack of suitable animal models for severe dengue disease, i.e., dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), there are difficulties in assessing the roles of such events, particularly the identification of adhesion molecules mediating the transendothelial migration of neutrophils using blocking antibodies against specific integrins, as has been performed for other disease states (Doerschuk et al., 1990; Gao et al., 1994; Issekutz and Issekutz, 1993; Laberge et al., 1995; Springer, 1995) . The hallmark feature of increased vascular permeability in hemorrhagic flavivirus (e.g., dengue) infection suggests that vascular endothelial cells may mediate the fluid leakage and hemorrhaging that occur in DHF/DSS. Endothelial cells line the inner surface of blood vessels and play essential roles in maintaining an antithrombogenic surface and regulating vascular permeability. Increased vascular permeability can arise from a variety of mediators associated with acute inflammation and shock (Bulger and Maier, 2000; Funk, 2001; Lefer, 1989; Michel, 1988; Montrucchio et al., 2000; Schnittler et al., 1990) . It is thought that vascular permeability is largely controlled by changes in endothelial cell-cell contact, which result in gap formation, thus allowing for fluid exchange between blood and interstitial tissue fluid (Michel, 1988 ). An electron microscopic study of endothelium from DHF biopsy samples revealed the occasional presence of gaps (Sahaphong et al., 1980) , thus providing evidence that endothelial cell features may indeed be perturbed during DHF/DSS. Although dengue virus infects endothelial cells in vitro (Andrews et al., 1978; Avirutnan et al., 1998; Killen and O'Sullivan, 1993) , there is no evidence that endothelial cell infection occurs clinically, as neither virus particles nor viral antigen has been detected in the endothelium of tissue specimens (Halstead, 1988 (Halstead, , 1989 Sahaphong et al., 1980) , in contrast to that seen in cases of ebola (Zaki et al., 1999) or hantaan hemorrhagic fever (Gavrilovskaya et al., 1999; Wang et al., 1997) . It is likely that dengue virus mediates endothelial cell activation via an indirect route, involving blood monocytes, which are a major cell target for dengue virus infection (Halstead et al., 1977b; Scott et al., 1980) . A major candidate event in such a route is the activation of endothelial cell adhesion molecules by a factor(s) (particularly TNF-) produced by dengue virus-infected blood monocytes . TNF is a key cytokine in a variety of normal and pathological immune responses, including immunoregulation, regulation of cell proliferation, cytotoxicity, and in the mediation of endotoxic shock (Fiers, 1991; Tartaglia and Goeddel, 1992; Tracey and Cerami, 1993; Vassalli, 1992) . Monocyte-derived TNF-appears to play a pivotal role in dengue-associated endothelial cell activation and may be an important effector in the manifestation of DHF/DSS. Support for the clinical significance of this observation comes from observations of elevated TNF levels in the sera of patients with severe dengue disease (Green et al., 1999b; Hober et al., 1993; Vitarana et al., 1991; Yadav et al., 1991) . Taken together, current evidence indicates that dengue virus represents a rather unique group of viruses that target monocytes, thereby triggering the production of factors such as TNF-, which in turn affect other cell targets, including endothelial cells. While the overall picture of endothelial cell dysfunction in DHF/DSS is obviously more complex than can be explained by any single factor, the role of TNF in dengue pathogenesis would seem to merit particular attention. Current knowledge of endothelial cell responses observed in endotoxic shock may be instructive for the understanding of vascular leakage in DHF/DSS. Plasma leakage induced by endotoxin (lipopolysaccharide, LPS) from gram-negative bacteria encompasses a complex cascade of processes, including activation and functional alteration of endothelial cells. Major mediators of endothelial cell perturbation in endotoxic shock are LPS itself, as well as cytokines such as TNF-and IL-1 (Bevilacqua, 1993) . These factors can modulate endothelial cell function to varying degrees by activating cytokine and vasoactive factor release (Rink and Kirchner, 1996; Shanley et al., 1995) , upregulating adhesion molecule expression (Bevilacqua, 1993; Luscinskas et al., 1991; Moser et al., 1989; Smith et al., 1989) , and mediating transendothelial migration of specific leukocytes Luscinskas et al., 1991; Morzycki et al., 1990; Moser et al., 1989; Smith et al., 1989) . Additional factors, particularly lipid mediators such as PAF, leukotrienes, thromboxanes, and prostaglandins, may contribute to further endothelial cell dysfunction, including vascular leakage (Bulger and Maier, 2000; Funk, 2001; Lefer, 1989; Montrucchio et al., 2000) . While the involvement of these vasoactive mediators is recognized in endotoxic shock, more needs to be learned of their role in the vascular dysfunction that occurs in severe dengue disease. Although lymphocytes are potently involved in the host response and immunopathology of flavivirus (especially dengue) diseases, their role as virus-permissive host cells is unclear. Dengue virus has been identified in circulating B cells from acutely ill dengue patients by immunocytochemistry and by recovery of infectious virus after passage in mosquitoes . In vitro studies showed that cells and cultured cell lines of both B and T cell derivation could be infected with dengue virus (Bielefeldt-Ohmann et al., 2001; Kurane et al., 1990; Marchette and Halstead, 1978; Mentor and Kurane, 1997; Sung et al., 1975; Takasaki et al., 2001; Theofilopoulos et al., 1976) . Continued passage of dengue virus in lymphoblastoid (Raji) cells can give rise to dengue virus variants capable of replication in human lymphocytes (Brandt et al., 1979) . Interestingly, lymphocytes do not appear to undergo antibody-enhanced dengue virus infection (Brandt et al., 1979; Kurane et al., 1990) , even though B cells do have Fc receptors (Dijstelbloem et al., 2001 ; see Section IV,C). The initial stages of pathogenesis for neurotropic flaviviruses appear to be common for flaviviruses in general in that the virus progresses from the subcutaneous site of inoculation to lymph nodes, followed by viremia and replication in extraneural tissues. Invasion into the central nervous system is marked by high virus titers in the brain and detectable virus or viral antigen in neurons (Albrecht, 1968) . Cell destruction in tick-borne encephalitis may be less extensive than that seen in herpes simplex type 1 encephalitis (Studahl et al., 2000) , although this is variable and may involve considerable inflammation (Chu et al., 1999; Matthews et al., 2000; Suzuki et al., 2000) . Susceptible cell types include both neurons and glial cells (Chu et al., 1999; Ramos et al., 1998; Steele et al., 2000) . As notorious producers of vasoactive mediators, mast cells have been a source of controversial speculation for years in dengue pathogenesis. Cells resembling degranulated mast cells have been reported in skin perivascular infiltrates from DHF/DSS cases (Bhamarapravati et al., 1967) . Dengue patients showed elevated levels of urinary histamine (a major granule product of mast cells), which correlated with disease severity (Tuchinda et al., 1977) , suggesting that mast cells may have a contributory role in the pathogenesis of dengue. Although antihistamine treatment does not resolve shock in severely dengue-diseased patients (Halstead, 1989) , histamine is only one of several potent vasoactive factors produced by mast cells (Benyon et al., 1991; Bradding et al., 1993; Galli et al., 1984; Grabbe et al., 1994; Marshall and Bienenstock, 1994; Moller et al., 1991 Moller et al., , 1993 Moller et al., , 1998 Nilsson et al., 1995; Schwartz and Austen, 1984) , some of which could cause vascular dysfunction in dengue infection. DHF/DSS patients have been reported to have elevated serum levels of IgE (Pavri et al., 1979) , which has been speculated to relate to IgE-triggered histamine release in the manifestation of shock (Pavri and Prasad, 1980) . Mast cells reside mainly in the tissues, often closely associated with blood vessels (Alving, 1991; Anton et al., 1998; Pesci et al., 1996; Pulimood et al., 1998; Selye, 1966; Selye et al., 1968) . They are present in large numbers in the skin (Marshall et al., 1987) , where transmission of insect-borne flaviviruses occurs. Basophils, however, comprise about 1% of total circulating cells and would be accessible to virus in the blood. Dengue virus infects basophil/mast cell-like KU812 cells in an antibody-enhanced manner, coupled with the release of vasoactive cytokines, IL-1 and IL-6 (King et al., , 2002 . This cell line, which can be differentiated easily toward either a basophil or mast cell phenotype (Saito et al., 1995) , may provide further insights into potential roles for basophils and mast cells in dengue disease. Dengue patients show increased serum levels of anaphylatoxins C3a and C5a (Malasit, 1987) , which can attract (Nilsson et al., 1996) and activate (Kownatzki, 1982) mast cells. Among the expected mast cell secretion products would be vasoactive factors, including histamine, which has been detected in elevated amounts in the urine of dengue patients (Tuchinda et al., 1977) . Evidence for platelet involvement in dengue pathogenesis comes from at least two (probably related) sources. First, thrombocytopenia (loss of circulating platelets) is one of the most consistent clinical features of severe dengue infection (Halstead, 1989) . Second, viral immune complexes have been detected on platelets from dengue patients Phanichyakarn et al., 1977a) . Functional studies on platelets in dengue-diseased individuals have been sparse, but include a markedly reduced half-life (Mitrakul et al., 1977) , deficient ADP release (Mitrakul et al., 1977) , increased adhesiveness (Doury et al., 1976) , increased tagging by complement fragments (Malasit, 1987) , and increased release of -thromboglobulin and platelet factor 4 (Srichaikul et al., 1989) . There is also evidence for platelet activation in dengue patients (Doury et al., 1976; Krishnamurti et al., 2001; Srichaikul et al., 1989) . Although these results relate to a variety of platelet functions, they do indicate a general alteration in platelet physiology, which is consistent with platelet involvement and triggering of thrombocytopenia in dengue disease. Dengue virus has been recovered from washed patient platelets (Scott et al., 1978) , and virus has been reported to bind to platelets in the absence of antibody as assayed using immunofluorescence and immunoperoxidase techniques (Funahara et al., 1987) . However, the levels of antibody-independent bound virus are very low compared to the levels of virus bound in the presence of dengue-specific antibodies . As noted earlier, dengue immune complexes have been demonstrated on platelets from dengue patients Phanichyakarn et al., 1977a) . Weiss and Halstead (1965) originally proposed the possibility that dengue virus interactions with platelets might be involved in the thrombocytopenia observed in severe dengue disease. The finding that dengue virus binding to platelets is dependent on a virus-specific antibody is consistent with epidemiological and experimental data linking preexisting host antibodies to an increased risk of DHF/DSS (reviewed in Halstead, 1990) . Several other viruses have been shown to bind directly to platelets (Bik et al., 1982; Danon et al., 1959; Forghani and Schmidt, 1983; Larke and Wheelock, 1970; Lee et al., 1993; Zucker-Franklin et al., 1990) . Platelet association may stabilize or protect blood-borne viruses (Larke and Wheelock, 1970) and may function as a mechanism of hematogenous dissemination (Forghani and Schmidt, 1983) . Virus binding to platelets has been suggested to be a contributing mechanism to thromobocytopenia arising from infections with vaccinia (Bik et al., 1982) , chikungunya (Larke and Wheelock, 1970) , and rubella (Bayer et al., 1965) . Thrombocytopenia in these virus infections is generally much milder than that observed in severe dengue disease. Levels of dengue virus in the blood can exceed 10 7 infectious units/ml (Gubler, 1988; Monath, 1994) . Such high viremic titers are likely necessary to ensure infection and transmission of the obligate mosquito intermediary host (Monath, 1994) . Assuming a reasonable particle:infectivity ratio of 100:1, virus particle titers in blood may rival normal platelet counts (3 Â 10 8 /ml). Such parity between numbers of virus particles and platelets suggests that antibodyenhanced binding of virus to platelets may have a profound effect on platelets. Circulating virus-immune complexes are detected in DHF/ DSS, and levels of immune complexes have been correlated with severity of disease (Ruangjirachuporn et al., 1979) and some of these are platelet associated Phanichyakarn et al., 1977a) . These observations suggest that sufficient binding of virus immune complexes to platelets may occur to tag the majority of circulating platelets. Such an event could lead to immune clearance by the reticuloendothelial system, thereby precipitating the thrombocytopenia frequently associated with severe dengue disease. It is likely that molecules other than Fc receptors on the platelet surface may mediate antibody-enhanced binding of dengue virus . Drug-induced thrombocytopenias provide interesting examples in this regard. It is known that given the appropriate accessory ligand (i.e., drug), IgG can bind to platelets through either the Fc receptor or other surface proteins. A variety of clinical thrombocytopenias are known that involve an immune component in pathogenesis. Many of these reflect activities of host antibodies, which react with proteins on the surface of platelets. These antibodies may be autoimmune in nature (i.e., antibodies that bind to platelet surface molecules) or dependent on a third party ligand (drug or protein), which then induces binding of the antibody-ligand complex to either the platelet Fc receptor or to another surface protein. For example, a number of individuals are susceptible to drug-dependent thrombocytopenia when administered drugs such as heparin or quinine/quinidine (Aster, 1989; Hackett et al., 1982) . While heparindependent antibodies bind to the platelet Fc receptor (Adelman et al., 1989; Chong et al., 1989a Chong et al., , 1989b Kelton et al., 1988) , quinine/quinidine-dependent antibodies bind to platelet protein heterodimers GPIIb/IIIa and GPIa/IX (Berndt et al., 1985; Chong et al., 1983; Christie et al., 1987; Devine and Rosse, 1995) . This latter category of immune-mediated thrombocytopenia may be relevant to the understanding of dengue-associated thrombocytopenia, as patient antibodies mediate dengue virus binding to platelets via a platelet surface protein other than the Fc receptor . Communication between platelets and endothelial cells is a frequent intermediate step in certain events such as platelet adhesion, aggregation, and regulation of vascular permeability. How this occurs in dengue infection and what the effects are on endothelial cell function are unknown. Binding of viruses to platelets can have potentially profound immunological effects [e.g., the stimulation of TGF-release by platelets bound by Epstein-Barr virus (Ahmad and Menezes, 1997) ]. In light of reports of altered platelet function in dengue patients, discussed earlier, there is a tantalizing need to determine the immunological consequences of antibody-enhanced dengue virus binding to platelets in terms of platelet as well as endothelial cell physiological responses. Many products of complement activation can also be deposited on platelets (Devine, 1992) . In view of evidence for complement activation in severe dengue disease (Halstead, 1989; Malasit, 1987) , binding of complement products might play a role in the immune destruction of platelets leading to thrombocytopenia. Platelets display surface receptors, e.g., C1q receptor Ghebrehiwet, 1987, 1998) , membrane cofactor protein (Seya et al., 1986) , and decay-accelerating factor (Devine et al., 1987) , for specific components of complement activation. In addition, the platelet surface can act as a substrate for the deposition of C3dg and C5b-9 (Devine, 1992) . Fragments of C3 have been detected on the platelets of DHF/DSS patients (Malasit, 1987) . In addition to immune complex deposition on platelets, thrombocytopenia associated with DHF/DSS might also arise by the immune destruction of platelets through antiplatelet autoantibodies. Antiplatelet autoantibodies have been reported in the sera of dengue patients (Lin et al., 2001) , although they have also been detected in patients recovering from a variety of viral infections (Imbach, 1994) . Antiplatelet antibodies are strongly linked to the pathogenesis of immunemediated thrombocytopenias, such as idiopathic thrombocytopenic purpura (Winkelstein and Kiss, 1997) . While this brief discussion of cell targets for flaviviruses is by no means complete, it highlights some of the major interactions as they relate to pathogenesis. Because pathogenesis is probably best understood for dengue, Fig. 1 illustrates the interactions of hemorrhagic flavivirus (e.g., dengue) with cell targets both within and outside the vascular system. Glycosaminoglycans and proteoglycans (i.e., proteins bearing glycosaminoglycans) are important cell surface molecules involved in a variety of ligand recognition and cell signaling processes (Gallo, 2000) . Because glycosaminoglycans are widely distributed on cells, they are attractive candidates as virus receptors. Some degree of specificity (i.e., virus tropism) may arise from the compositional heterogeneity of glycosaminoglycans, as well as quantitative differences in the degree of expression on various cell types. Flaviviruses seem to share, with a large number of virus families, the ability to bind glycosaminoglycans (Birkmann et al., 2001; Dechecchi et al., 2000 Dechecchi et al., , 2001 Duisit et al., 1999; Feldman et al., 1999 Feldman et al., , 2000 Giroglou et al., 2001; Goodfellow et al., 2001; Heil et al., 2001; Hsiao et al., 1999; Hulst et al., 2000 Hulst et al., , 2001 Lin et al., 2000; Liu and Thorp, 2002; Patel et al., 1993; Rue and Ryan, 2002; Shukla et al., 1999; Shukla and Spear, 2001) . Glycosaminoglycans such as heparin and its structural analogues have been investigated for their ability to bind dengue virus and thereby to gain insights as to the structural requirements for dengue receptors. Potential glycosaminoglycan-binding motifs have been identified on the dengue viral E protein at two sites, the best characterized of which appears to be composed of amino acids 188, 284-295, and 305-310 and which may also play a role in virus-cell attachment . Heparin (minimum of 10 carbohydrates) and an uncharacterized highly sulfated heparin sulfate isolated from bovine liver were found to show the best binding to dengue E protein . Attachment of dengue virus to human hepatoma cells has also been reported to be inhibited by heparin (Hilgard and Stockert, 2000) . A further study involving a panel of natural and synthetic polyanionic, sulfated compounds suggested that binding of the dengue E protein required a highly sulfated (and highly charged) oligosaccharide with a minimum size of 39Å and a high degree of structural flexibility (Marks et al., 2001) . The role of glycosaminoglycans in natural (i.e., nontissue cultureadapted) strains of flaviviruses needs to be studied further. It has long been recognized that dengue virus passaged in various host cell types can give rise to virus variants with altered cell specificity (Brandt et al., 1979; Halstead et al., 1984a Halstead et al., , 1984b Halstead et al., , 1984c . Passage-dependent mutations of the dengue virus E protein at a number of different amino acid residues have been documented (Lee et al., 1997) . Following passage of TBE virus in cultured BHK-21 cells, virus mutants were selected that contained more positively charged amino acids in the putative receptor-binding region of the E protein, resulting in dependence on cell surface heparan sulfate (Mandl et al., 2001) . Such mutants were diminished in their neurovirulence in mice as well as in their replication in primary chicken cells and plaque formation in porcine kidney cells (Mandl et al., 2001) . A large number of other viruses have also been shown to undergo loss of virulence upon adaptation to cell culture associated with heparan sulfate utilization (Bernard et al., 2000; Byrnes and Griffin, 2000; Klimstra et al., 1998 Klimstra et al., , 1999 Lee and Lobigs, 2000; Neff et al., 1998; Sa-Carvalho et al., 1997) . CD14 and the Toll-like receptor (TLR) pattern recognition receptors are involved in the innate response to lipopolysaccharide and other microbial products (Diamond et al., 2000; Imler and Hoffmann, 2000) . A role for CD14 and TLR4 has been found for respiratory syncytial virus (RSV) (Kurt-Jones et al., 2000) , suggesting that these receptors may have a broader involvement in host response than previously thought. A possible role for CD14 in dengue infection has been postulated on the basis of inhibition of dengue virus infection of human monocytes with bacterial lipopolysaccharide (Chen et al., 1999) . However, this has been disputed (Bielefeldt-Ohmann et al., 2001) and requires further investigation. As indicated earlier, flaviviruses are capable of initiating infection of appropriate host cells through as yet largely unidentified primary receptors. In addition, a number of flaviviruses are capable of using subneutralizing levels of virus-specific antibodies to attach to and gain entry to cells bearing Fc and/or complement receptors (Cardosa et al., 1983; Halstead, 1982; Halstead and O'Rourke, 1977a; Schlesinger and Brandriss, 1981a ) by a process known as antibody-dependent enhancement (ADE) of infection (Table I) . ADE has been documented for dengue , West Nile (Peiris and Porterfield, 1979) , yellow fever (Schlesinger and Brandriss, 1981b) , tick-borne encephalitis (Phillpotts et al., 1985) and Japanese encephalitis (Cecilia and Ghosh, 1988) viruses. Early work with dengue virus and monocytes differentiated between trypsin-sensitive and trypsinresistant cell surface molecules as the putative receptors for antibody-independent and antibody-dependent infection, respectively (Daughaday et al., 1981) . To date, dengue virus appears to be the only flavivirus in which strong evidence exists for antibody-dependent enhancement as a major contributing factor to severe disease (Halstead, 1980; Thein et al., 1997) . Severe dengue disease, encompasing conditions known as dengue hemorrhagic fever/dengue shock syndrome, involves several well-defined hemostatic abnormalities, including the leakage of plasma into interstitial spaces, as well as thrombocytopenia and bleeding (Halstead, 1990; Kurane et al., 1994) . The potential to cause severe hemorrhagic disease is a general property of dengue viruses and is not limited to any one viral serotype (Gubler, 1998; Rigau-Perez et al., 1998) . Although different strains of dengue may influence the severity of hemorrhagic symptoms (Leitmeyer et al., 1999; Rico-Hesse et al., 1997) , it is also generally accepted that pathogenesis depends on immunopathological processes (Rothman and Ennis, 1999) . Thus the roles of prior immunity, antibody-enhanced virus infection, and immune-mediated pathologic effects on the vascular system are key points in understanding the pathogenesis of dengue hemorrhagic disease. While the pathogenesis of severe dengue disease is not completely understood, it is clear from laboratory and epidemiological studies that a considerable risk factor is prior immunity. Severe dengue disease, DHF/DSS, rarely occurs in seronegative individuals suffering their first dengue infection, but instead occurs in individuals who have preexisting dengue viral antibodies, either from a previous infection or from passive antibody transfer, e.g., following maternal transmission of antibodies to the fetus (Kliks et al., 1988 (Kliks et al., , 1989 . Estimates suggest that 99% of children suffering from DHF/DSS have preexisting immunity from a prior dengue virus infection (Halstead, 1988) . Consequently, from this and other studies, it has been calculated that prior exposure to dengue increases the risk for hemorrhagic disease in a Okayama et al. (2000) , Anselmino et al. (1989) , and Tuijnman et al. (1993) . b From . c From Wu et al. (2000) and Libraty et al. (2001) . d From King et al. (2000) . e Abortive infection, but expressing viral antigen (Marianneau et al., 1999) . f From Littaua et al. (1990) and Kontny et al. (1988). second dengue infection by at least 15-fold (Halstead, 1980; Thein et al., 1997) . Preexisting serum antibodies can potentiate virus infection by the mechanism of antibody-dependent enhancement, giving rise to amplified virus replication and to an increased potential for the development of hemorrhagic symptoms (Halstead, 1989) . Viremic titers are higher in secondary dengue infections in both humans (Gubler et al., 1979) and experimental monkeys (Halstead et al., 1973) . Antibody-enhanced dengue virus infection of human blood monocytes is necessary for the production of endothelial cell activators , thereby providing a link between antibody-dependent enhancement and alteration of endothelial cell properties, which might contribute to vascular permeability in dengue infection. For certain other viruses, e.g., influenza (Tamura et al., 1993) and HIV (Takeda et al., 1990 (Takeda et al., , 1992 , distinct ''neutralizing'' and ''antibodyenhancing'' epitopes have been identified on the respective viral attachment proteins. Surprisingly, no systematic approach has yet been undertaken to identify regions on the E protein that are essential for ADE, even though this issue was raised as a challenge to research on dengue many years ago (Halstead, 1988) . Human Fc receptors are currently categorized into three classes: FcRI (CD64), FcRII (CD32), and FcRIII (CD16). While FcRI shows high affinity for monomeric IgG, FcRII and FcRIII bind monomeric IgG poorly and are more likely involved in binding immune complexes (Dijstelbloem et al., 2001) . FcRII is the most widely distributed, being expressed on most circulating leukocytes (van de Winkel and Anderson, 1991) . Monocytes express all three FcRs to varying degrees (van de Winkel and Anderson, 1991) , although FcRI and FcRII predominate, whereas FcRIII appears to be limited to a subpopulation ($10%) of monocytes (Anderson et al., 1990; Passlick et al., 1989) . FcRIII constitutes the major FcR on macrophages (Fanger et al., 1989) , although FcRI and FcRII are also present (Tuijnman et al., 1993; van de Winkel and Anderson, 1991) . It is also important to recognize that FcR expression on cells, including macrophages, can vary depending on the microenvironment (Tomita et al., 1994) . Although strong evidence exists for FcR involvement in ADE of dengue virus, the participating FcRs in vivo have not yet been identified rigorously. In cultured cell lines (monocytic U937 or erythroleukemic K562 cells), FcRI (Kontny et al., 1988) and FcRII (Littaua et al., 1990) have been shown to mediate ADE of dengue virus infection. That FcRI has the ability to mediate ADE of dengue has been demonstrated using COS cells transfected with FcRI (Schlesinger and Chapman, 1999) . Dengue and DHF patients show elevated serum levels of interferon (IFN)- (Kurane et al., 1991) . Because IFN-can upregulate both MHC class I and II molecules as well as Fc R (particularly Fc RI) expression in monocytes (Erbe et al., 1990; Perussia et al., 1983) , the chances for ADE may be increased, thereby creating a vicious cycle involving positive cytokine feedback and virus amplification (Kurane and Ennis, 1992) . IFN-has been shown to enhance ADE of dengue virus infection of human monocytic U937 cells (Kontny et al., 1988) , although any enhancing effect on dengue infection of peripheral blood monocytes may be negated by the antiviral properties of IFN- (Sittisombut et al., 1995) . Mast cells and basophils express mainly Fc RII (Anselmino et al., 1989; Okayama et al., 2001a; Wedi et al., 1996) and some (IFN--inducible) Fc RI (Okayama et al., 2000 (Okayama et al., , 2001b as well as the highaffinity Fc eRI for IgE (Guo et al., 1992; Sperr et al., 1994) . As noted previously, the basophil/mast cell KU812 cell line exhibits antibodyenhanced dengue virus infection and produces vasoactive cytokines . Although Fc R-mediated ADE of flaviviruses has been examined extensively as a mechanism for virus amplification, the biological consequences for the participating host cell are not well understood. Because Fc R-mediated cell signaling is complex, the functional effects of virus-antibody interactions with cell surface Fc Rs need to be investigated. Monocytes infected with dengue virus in the presence of antibody release cytokines such as TNF- . Induction of TNF-requires infectious virus , suggesting that virus replication (or perhaps expression of one or more crucial viral genes) is responsible for the stimulation of TNF-release. Therefore, in this case, the Fc R is likely facilitating antibody-enhanced virus replication rather than providing a signal triggered by virus binding to the Fc R. Similarly, antibody-enhanced dengue virus infection of KU812 basophil/mast cells produces IL-1, IL-6 (King et al., , 2002 , and selected chemokines (King et al., 2002) . Suppressive effects of antibody-enhanced flavivirus or alphavirus infection on monocyte cytokine secretion have also been reported (Lidbury and Mahalingam, 2000; Yang et al., 2001) . Both activating (FcRI, FcRIIa, and FcRIIIa) and inhibitory (FcRIIb) forms of FcRs exist, which mediate signal transduction via a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) or inhibitory (ITIM) motif, respectively (Dijstelbloem et al., 2001) . The ITAM and associated molecules are necessary for the endocytosis of FcR-bound immune complexes (Amigorena and Bonnerot, 1999 ) and therefore play a likely role in the initiating events of antibody-enhanced flavivirus infection. Although not necessary for FcRII, an accessory subunit (homo-or heterodimeric or chains) is required for signaling through FcRI and FcRIIIa (Ravetch, 1994) . A further FcR (FcRIIIb) lacks transmembrane and cytoplasmic domains and is instead anchored to the cell surface membrane via a glycosylphosphatidylinositol (GPI) linkage (Selvaraj et al., 1988; Simmons and Seed, 1988) . It apparently does not participate in signal transduction and has been speculated to sequester and accumulate immune complexes at specific sites on the cell surface (Huizinga et al., 1988; Selvaraj et al., 1988) . The roles of activating and inhibitory FcRs in viral ADE have not yet been ascertained. Activating FcRs are expressed on monocytes, macrophages, granulocytes, natural killer (NK) cells, and platelets but not on most lymphocytes (Dijstelbloem et al., 2001) . Inhibitory FcRs, however, are found on B cells, dendritic cells, and macrophages (Dijstelbloem et al., 2001) . Interestingly, ADE of dengue virus is best documented for monocytes/macrophages and related cell lines (Halstead, 1989) . In contrast, lymphocytic cells (Brandt et al., 1979; Kurane et al., 1990) and dendritic cells (Wu et al., 2000) do not appear to support antibody-enhanced dengue virus infection. Whether this is due to differential expression of activating versus inhibitory FcRs remains to be investigated. FcRs for IgE (primarily the high-affinity FceRI) are expressed on cells such as monocytes, macrophages, mast cells, basophils, and dendritic cells and are structurally related to FcRs (Ravetch, 1994) . Their role in binding IgE and/or immune-complexed flaviviruses, such as dengue, remains unexplored. Similarly unexplored is the potential role of the neonatal Fc IgG receptor (FcRn), structurally related to MHC class I and involved in IgG transport across cells (Ghetie and Ward, 2000) . In addition to being expressed on certain epithelial and endothelial cells, FcRn is also expressed functionally on monocytes, macrophages, and dendritic cells (Zhu et al., 2001) . In addition to the FcR, the antibody-complexed flavivirus has been shown to be taken up by a macrophage cell line using the complement receptor-3 (Cardosa et al., 1983) . In the case studied-West Nile virus infection of mouse P388D1 macrophages-ADE was mediated by the presence of antiviral IgM and was inhibited with a CR3-blocking antibody. This mode of ADE was, however, found to be quantitatively less productive than the more commonly studied route of ADE, i.e., involving FcR-mediated uptake route of IgG-virus complexes (Cardosa et al., 1983) . The recent demonstration of DC-SIGN as a functional dengue virus receptor on human dendritic cells represents an important advance in the definitive identification of flavivirus receptors (Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003) . Several studies have identified cell surface proteins that bind flaviviruses, generally assayed by virus overlay blots of SDS-PAGE-resolved cell proteins (Table II) . Further work is required to confirm the involvement of these and other proteins as receptors in flavivirus infection. A number of flaviviruses are able to stimulate the expression of cell surface molecules. Notable among these are adhesion molecules and major histocompatibility antigens. Multiple mechanisms appear to be involved, including virus-and cytokine-dependent pathways. Flavivirus infection of a number of cell types causes an increase in cell surface MHC class I expression (King and Kesson, 1988; King et al., 1989; Libraty et al., 2001; Liu et al., 1989; Lobigs et al., 1996; Shen et al., 1995a Shen et al., , 1997 . Evidence for both virus-dependent (Lobigs et al., 1996) and cytokine-dependent (Libraty et al., 2001; Shen et al., 1997) mechanisms has been reported. One process appears to be driven by the amount of flaviviral peptides generated by proteolysis and imported into the transporter associated with antigen processing (TAP), which results in increased cell surface expression of peptideloaded MHC class I (Momburg et al., 2001) . The upregulation of MHC class I molecules by flaviviruses is perhaps reminiscent of that observed in infections by coronaviruses (Suzumura et al., 1986) but stands in contrast to the virus-manipulated downregulation of MHC class I by viruses such as herpesviruses (Jennings et al., 1985; Ploegh, 1998) , adenoviruses (Sparer and Gooding, 1998) , poxviruses (Boshkov et al., 1992) , and HIV (Scheppler et al., 1989) . Although enhanced Chu and Ng (2003) MHC class I expression would be expected to lead to greater cytotoxic T (Tc) cell-mediated cytolysis, it would render cells less susceptible to recognition by NK cells. Evidence has been presented that flavivirusinfected cells in fact show reduced susceptibility to NK cells at the cost of enhanced Tc cell-mediated lysis (Lobigs et al., 1996) . It has been suggested that such a response may permit flaviviruses to evade an early NK cell response and thereby allow for substantial amplification of virus during the viremic phase of infection (Momburg et al., 2001 ). Nevertheless, evidence shows that NK cells are activated during dengue infection (Green et al., 1999a) , and NK cell-mediated cytotoxicity has been reported to correlate with the severity of disease (Homchampa et al., 1988) . Dendritic cells also undergo upregulation of MHC class I molecules following infection with dengue virus (Libraty et al., 2001) . Compared to other antigen-presenting cells, dendritic cells have superior T cellstimulating activities (McKinney and Streilein, 1989; Timares et al., 1998) . Because antigen presentation via dendritic cell MHC class I can provoke exceptionally strong proliferation in CD8-bearing T cells (Bhardwaj et al., 1994; Elbe et al., 1994; McKinney and Streilein, 1989) , much of the overall cytotoxic T cell response arising in flavivirus infection may be dictated at the level of the dendritic cell. West Nile virus infection induces MHC class II expression in mouse macrophages (Shen et al., 1995a) , mouse astrocytes (Liu et al., 1989) , rat Schwann cells (Argall et al., 1991) , and human myoblasts (Bao et al., 1992) . Upregulation of dendritic cell MHC class II occurs in response to dengue (Libraty et al., 2001) and West Nile (Johnston et al., 1996) virus infection. Given the potent ability of dendritic cells to activate T cells (Banchereau et al., 2000) , the communication between dendritic cell MHC class II-peptide complexes and recognition molecules on CD4-expressing T cells should provide insights into some of the molecular processes underlying T cell activation. Adhesion molecules are expressed on a variety of cells and mediate a spectrum of processes (Ley, 2001; Roebuck and Finnegan, 1999; Springer, 1995) . From the standpoint of flaviviruses, the most significant processes likely concern adhesion molecules on vascular endothelial cells, as these cells regulate permeability as well as transendothelial migration of leukocytes (Springer, 1995) . Of particular importance are intercellular adhesion molecule 1 (ICAM-1; CD54), vascular cell adhesion molecule-1 (VCAM-1; CD106), and E-selectin (CD 62E), which are upregulated on the surface of the endothelium by inflammatory cytokines, cellular stress, and virus infection (Roebuck and Finnegan, 1999) . In the case of dengue, activation of endothelial cells occurs in vitro via TNF-released from antibody-enhanced dengue virus infection of monocytes . Such activation involves upregulation of adhesion molecules E-selectin, ICAM-1, and VCAM-1. Evidence that similar activation processes occur in vivo comes from clinical studies showing elevated serum levels of TNF- (Green et al., 1999b; Hober et al., 1993; Vitarana et al., 1991; Yadav et al., 1991) and soluble VCAM-1 (Murgue et al., 2001) in dengue-and DHF/DSS-infected patients. Surprisingly, serum levels of soluble ICAM-1 were actually found to be lower than those of control subjects, although this may reflect plasma protein loss through leakage (Bethell et al., 1998) . Moreover, the function of soluble forms of ICAM-1 remains unclear, and their expression appeazrs to be regulated differently from that of membranebound ICAM-1 (Komatsu et al., 1997; van Den Engel et al., 2000) . Two phases of ICAM-1 upregulation have been noted in West Nile and Kunjin virus infection of human embryonic fibroblasts, namely an early ($2 h postinfection) virus-dependent process and a later ($24 h postinfection) event that is mediated by type 1 interferon (Shen et al., 1995b) . For neurotropic flaviviruses, such as West Nile virus in the mouse, the development of encephalitis has been correlated with viremia (Weiner et al., 1970) , suggesting virus penetration of the blood-brain barrier. The endothelium of the brain microvasculature normally represents a block between circulating virus and the central nervous system. Expression of endothelial cell adhesion molecules, thereby facilitating leukocyte adherence and diapedesis through the endothelium, may be an important mode of dissemination of virus-infected monocytes or other leukocytes into the brain. West Nile virus infection of human endothelial cells causes the upregulation of E-selectin, ICAM-1, and VCAM-1 (Shen et al., 1997) , which could mediate the transendothelial migration of leukocytes. Upregulation of these adhesion molecules was observed to occur early (2-4 h) in infection and appeared to be triggered by the virus rather than by cytokines (Shen et al., 1997) . Further studies are required to clarify the role of endothelial cell adhesion molecule expression in the neuroinvasion of certain flaviviruses. Assuming such a role is confirmed, it will be incumbent to identify the mechanisms by which either free or cell-borne flaviviruses are stimulated to cross the vascular endothelial layer. For virusinfected leukocytes, such stimulation likely arises, at least in part, from chemokines produced by cells of the central nervous system. Astrocytes infected with JE virus have been reported to release chemokines (RANTES and MCP-1), which may play a role in the transendothelial migration of leukocytes (including those possibly carrying virus) across the blood-brain barrier (Chen et al., 2000) . Thus, once neural infection is initiated, the process could be amplified by the production of leukocyte-attracting chemokines at the site of infection. Complement activation is well documented in dengue disease (Nishioka, 1974; Phanichyakarn et al., 1977b; Russell et al., 1969) , with peak activation and the production of C3a and C5a occurring at the time of vascular leakage and/or shock (Malasit, 1987) . Complement activation is likely to be largely mediated by immune complexes consisting of IgG and virus (Bokisch et al., 1973a (Bokisch et al., , 1973b Shaio et al., 1992; Sobel et al., 1975) , although the low levels of circulating immune complexes detected in patients have stimulated thought as to other possible mechanisms (Malasit, 1987) . Receptors for C3a and C5a are found on a wide variety of cells, including many human peripheral blood leukocytes (Chenoweth and Hugli, 1978; Fureder et al., 1995; Kretzschmar et al., 1993; Nilsson et al., 1996; van Epps and Chenoweth, 1984) . C5a receptors have been reported on endothelial cells, although at lower levels than myeloid cells (Zwirner et al., 1999) . Although endothelial cells do not appear to be major targets for dengue virus in vivo (Halstead, 1988 (Halstead, , 1989 Sahaphong et al., 1980) , endothelial cells infected with dengue virus in vitro can become a substrate for deposition of C3dg and C5b-9, provided the dengue antibody is present (Avirutnan et al., 1998) . The presence of complement activation products on the endothelial cell surface could be a contributing factor to vascular permeability . Furthermore, anaphylotoxins and/or deposition of sublytic C5b-9 on the endothelial cell surface has the potential to activate the expression of adhesion molecules (Foreman et al., 1994) , cytokines (Saadi et al., 2000) , chemokines (Selvan et al., 1998) , cyclooxygenase-2 (Bustos et al., 1997) , tissue factor , heparan sulfate proteoglycan proteinases (Ihrcke and Platt, 1996) , and even functional or morphological changes such as permeability loss and gap formation . Thus, in addition to being activated by leukocyte-derived cytokines , endothelial cells may also be coaxed toward a more permeability-enhancing state by virus infection and virusmediated complement deposition. At present, the lack of evidence for in vivo infection of endothelial cells by dengue virus would suggest that the cytokine-mediated pathway is dominant. Figure 2 shows a model illustrating the potential role of endothelial cell perturbation by monocyte-derived cytokines and complement activation products in initiating vascular permeability and leukocyte extravasation in severe hemorrhagic flavivirus disease. or by deposition of C5b-9 and other products of complement activation (Avirutnan et al., 1998) . C5b-9 is represented as a membrane attack complex pore structure, although the deposition of C5b-9 on dengueinfected cells appears associated with sublytic, rather than lytic, responses (Avirutnan et al., 1998) . Increased adhesion molecule expression, along with uncharacterized vasoactive factors, can lead to endothelial leakage and can mediate rolling, adhesion, and transendothelial migration of leukocytes into extravascular tissues. Similar processes may also contribute to the invasion of cell-borne neurotropic flaviviruses through the endothelial blood-brain barrier. Much remains to be learned about the primary receptors for flaviviruses, though much knowledge has been gained about the initial interactions of flaviviruses with cell surface structures. The ability of flaviviruses to affect cell entry through heparan sulfate-type proteoglycans, as well as their dexterity to adjust mutationally to different receptors, depending on host cell type, illustrates the plasticity of the viral E protein to adapt to changing conditions and to ensure successful virus replication. Beyond this, certain flaviviruses, notably dengue virus, are masters at exploiting host antibody and Fc receptor-bearing cells to dramatically amplify viral replication. Flavivirus replication is coupled to altered cellular expression of cytokines, chemokines, and cell surface molecules, which shape the host response and immunopathogenesis associated with flavivirus infections. Ongoing and future characterization of the cell surface structures that mediate these events will be helpful in understanding the mechanisms of flavivirus-induced disease and in developing therapeutic and/or preventive strategies. 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Clinical laboratory responses to heterologous infection Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2 An amino acid substitution in the coding region of the E2 glycoprotein adapts Ross River virus to utilize heparan sulfate as an attachment moiety The machinery for flavivirus fusion with host cell membranes Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM Heparan sulfate proteoglycans initiate dengue virus infection of hepatocytes Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: Monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) in dengue-infected patients Amino acid substitution(s) in the stem-anchor region of langat virus envelope protein attenuates mouse neurovirulence Natural killer cells in dengue hemorrhagic fever/dengue shock syndrome. Asian Pac Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children The PI-linked receptor FcRIII is released on stimulation of neutrophils Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein E(rns) Interaction of classical swine fever virus with membrane-associated heparan sulfate: Role for virus replication in vivo and virulence Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: Evidence for proteolysis of cell-surface molecules Harmful and beneficial antibodies in immune thrombocytopenic purpura Toll and Toll-like proteins: An ancient family of receptors signaling infection CD11/CD18-independent transendothelial migration of human polymorphonuclear leokocytes and monocytes: Involvement of distinct and unique mechanisms A major portion of polymorphonuclear leukocyte and T lymphocyte migration to arthritis in the rat is via LFA-1/MAC-1 independent mechanisms Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells Phenotypic changes in Langerhans' cells after infection with arboviruses: A role in the immune response to epidermally acquired viral infection? Langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovirus Heparin-induced thrombocytopenia: Laboratory studies Detection of dengue virus by in situ hybridization B cells are the principal circulating mononuclear cells infected by dengue virus Dengue virus selectively induces human mast cell chemokine production Release of vasoactive cytokines by antibody-enhanced dengue virus infection of a human mast cell/basophil line Interferon-independent increases in class I major histocompatibility complex antigen expression follow flavivirus infection Induction of class I major histocompatibility complex antigen expression by West Nile virus on gamma interferonrefractory early murine trophoblast cells Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants Antibodydependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever Infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor Differential up-regulation of circulating soluble and endothelial cell intercellular adhesion molecule-1 in mice Gamma interferon augments Fc receptor-mediated dengue virus infection of human monocytic cells A putative host cell receptor for tick-borne encephalitis virus identified by anti-idiotypic antibodies and virus affinoblotting Triggering of mast cells Chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin C3a Mechanisms of hemorrhage in dengue without circulatory collapse Immunity and immunopathology in dengue virus infections Activation of T lymphocytes in dengue virus infections: High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferongamma in sera of children with dengue Dengue-2 virus infection of human mononuclear cell lines and establishment of persistent infections Immunopathologic mechanisms of dengue hemorrhagic fever and dengue shock syndrome Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus Role of VLA-4 and LFA-1 in allergen-induced airway inflammation in the rat Stabilization of chikungunya virus infectivity by human blood platelets Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry Changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the protein Effect of plateletassociated virus on assays of HIV-1 in plasma Trends in shock research: Significance of lipid mediators in shock states Dengue virus structural differences that correlate with pathogenesis The fusion glycoprotein shell of Semliki Forest virus: An icosahedral assembly primed for fusogenic activation at endosomal pH Pathways and bottlenecks in the web of inflammatory adhesion molecules and chemoattractants Human dendritic cells are activated by dengue virus infection: Enhancement by gamma interferon and implications for disease pathogenesis Specific ablation of antiviral gene expression in macrophages by antibody-dependent enhancement of Ross River virus infection Generation of IgM anti-platelet autoantibody in dengue patients Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo Japanese encephalitis virus is transported across the cerebral blood vessels by endocytosis in mouse brain Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection Cell surface heparan sulfate and its roles in assisting viral infections Flavivirus infection up-regulates the expression of class I and class II major histocompatibility antigens on and enhances T cell recognition of astrocytes in vitro Flavivirus-induced up-regulation of MHC class I antigens: Implications for the induction of CD8+ T-cell-mediated autoimmunity Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence Integrin signal transduction in myeloid leukocytes Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1 Viral neuroinvasion and encephalitis induced by lipopolysaccharide and its mediators Complement and dengue hemorrhagic fever/shock syndrome Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo Phytohemagglutinin enhancement of dengue-2 virus replication in nonimmune rhesus monkey peripheral blood leukocytes Infection of primary cultures of human Kupffer cells by Dengue virus: No viral progeny synthesis, but cytokine production is evident Probing the interaction of dengue virus envelope protein with heparin: Assessment of glycosaminoglycan-derived inhibitors The role of mast cells in inflammatory reactions of the airways, skin and intestine Formalin sensitivity and differential staining of mast cells in human dermis Identification of a putative coreceptor on vero cells that participates in dengue 4 virus infection IL-6 increases endothelial permeability in vitro Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection Morphological features of Murray Valley encephalitis virus infection in the central nervous system of Swiss mice On the extraordinary capacity of allogeneic epidermal Langerhans cells to prime cytotoxic T cells in vivo A comparison of the spread of Murray Valley encephalitis viruses of high or low neuroinvasiveness in the tissues of Swiss mice after peripheral inoculation Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein Dengue virus infection of human T lymphocytes Capillary permeability and how it may change Hemostatic and platelet kinetic studies in dengue hemorrhagic fever Mast cells and their mediators in immediate and delayed immune reactions Comparative cytokine gene expression: Regulation and release by human mast cells Human mast cells produce IL-8 Modulation of transporter associated with antigen processing (TAP)-mediated peptide import into the endoplasmic reticulum by flavivirus infection Dengue: The risk to developed and developing countries Single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: Relevance to development and safety testing of live, attenuated vaccines Detection of yellow fever viral RNA by nucleic acid hybridization and viral antigen by immunocytochemistry in fixed human liver Mode of entry of a neurotropic arbovirus into the central nervous system: Reinvestigation of an old controversy Role of platelet-activating factor in cardiovascular pathophysiology Interleukin-1 and tumour necrosis factor alpha induced polymorphonuclear leukocyte-endothelial cell adhesion and transendothelial migration in vitro: The effect of apical versus basal monolayer stimulation Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage Putative dengue virus receptors from mosquito cells Plasma concentrations of sVCAM-1 and severity of dengue infections Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infecton of human dendritic cells by mosquito-cell-derived dengue viruses Foot-and-mouth disease virus virulent for cattle utilizes the integrin alpha(v)beta3 as its receptor Attenuation of Japanese encephalitis virus by selection of its mouse brain membrane receptor preparation escape variants Interaction of yellow fever virus French neurotropic vaccine strain with monkey brain: Characterization of monkey brain membrane receptor escape variants C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway Constitutive and inducible cytokine mRNA expression in the human mast cell line HMC-1 Serum complement level in dengue hemorrhagic fever A comparison of mediators released or generated by IFN-gamma-treated human mast cells following aggregation of Fc gamma RI or Fc epsilon RI Further characterization of FcgammaRII and FcgammaRII expression by cultured human mast cells Expression of a functional high-affinity IgG receptor, Fc gamma RI, on human mast cells: Up-regulation by IFN-gamma Dengue virus and dendritic cells Identification and characterization of a novel monocyte subpopulation in human peripheral blood Cell-surface heparan sulfate proteoglycan mediates HIV-1 infection of T-cell lines Immunoglobulin E in dengue haemorrhagic fever (DHF) cases T suppressor cells: Role in dengue hemorrhagic fever and dengue shock syndrome Human blood platelets possess specific binding sites for C1q Platelet receptors for the complement component C1q: Implications for hemostasis and thrombosis Antibody-mediated enhancement of Flavivirus replication in macrophage-like cell lines Immune interferon induces the receptor for monomeric IgG1 on human monocytic and myeloid cells Mast cells in bronchiolitis obliterans organizing pneumonia: Mast cell hyperplasia and evidence for extracellular release of tryptase Studies on dengue hemorrhagic fever: IV. Fluorescence staining of the immune complexes on platelets Studies on Dengue hemorrhagic fever: III. Serum complement (C3) and platelet studies Antibody-dependent enhancement of tick-borne encephalitis virus infectivity Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold Viral strategies of immune evasion Quantitative and ultrastructural analysis of rectal mucosal mast cells in acute infectious diarrhea Dengue virus in the brain of a fatal case of hemorrhagic dengue fever A 65-kDa trypsin-sensible membrane cell protein as a possible receptor for dengue virus in cultured neuroblastoma cells Acidotropic amines inhibit proteolytic processing of flavivirus prM protein Fc receptors: Rubor redux The envelope glycoprotein from tick-borne encephalitis virus at 2A resolution ''Fields Virology Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas Dengue and dengue haemorrhagic fever Recent progress in the tumor necrosis factor-alpha field Regulation of intercellular adhesion molecule-1 (CD54) gene expression Recovery of virus from the liver of children with fatal dengue: Reflections on the pathogenesis of the disease and its possible analogy with that of yellow fever Immunopathogenesis of dengue hemorrhagic fever Dengue-2 virus infection of human bone marrow: Characterization of dengue-2 antigen-positive stromal cells Circulating immune complexes in serum from patients with dengue haemorrhagic fever Characterization of pseudorabies virus glycoprotein C attachment to heparan sulfate proteoglycans Anti-dengue immunoglobulins and serum beta 1 c/a globulin levels in dengue shock syndrome Endothelial cell activation by pore-forming structures: Pivotal role for interleukin-1alpha Complementmediated regulation of tissue factor activity in endothelium Transient perturbation of endothelial integrity induced by natural antibodies and complement Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle Electron microscopic study of the vascular endothelial cell in dengue haemorrhagic fever Development of tryptase-positive KU812 cells cultured in the presence of Steel factor Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus Down-modulation of MHC-I in a CD4+ T cell line, CEM-E5, after HIV-1 infection Antibody-mediated infection of macrophages and macrophage-like cell lines with 17D-yellow fever virus Growth of 17D yellow fever virus in a macrophage-like cell line, U937: Role of Fc and viral receptors in antibody-mediated infection Influence of the human high-affinity IgG receptor FcgammaRI (CD64) on residual infectivity of neutralized dengue virus Role of actin and myosin in the control of paracellular permeability in pig, cat and human vascular endothelium Structure and function of the chemical mediators of mast cells A preliminary report on the isolation of viruses from the platelets and leukocytes of dengue patients Isolation of dengue viruses from peripheral blood leukocytes of patients with hemorrhagic fever Complement-induced expression of chemokine genes in endothelium: Regulation by IL-1-dependent and -independent mechanisms The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal nocturnal haemoglobinuria Mast cells and necrosis Influence of mast cells and vasoconstrictors upon various acute connective-tissue reactions Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b Complement pathway activity in serum from patients with classical dengue fever The role of cytokines and adhesion molecules in the development of inflammatory injury Change involving a viral membrane glycoprotein during morphogenesis of group B arboviruses Adherence status regulates the primary cellular activation responses to the flavivirus West Nile Early induction of interferon-independent virus-specific ICAM-1 (CD54) expression by flavivirus in quiescent but not proliferating fibroblasts: Implications for virus-host interactions Early E-selectin, VCAM-1, ICAM-1, and late major histocompatibility complex antigen induction on human endothelial cells by flavivirus and comodulation of adhesion molecule expression by immune cytokines A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry Herpesviruses and heparan sulfate: An intimate relationship in aid of viral entry The Fc receptor of natural killer cells is a phospholipid-linked membrane protein Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro C1q deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum Suppression of MHC class I antigen presentation by human adenoviruses The human cardiac mast cell: Localization, isolation, phenotype, and functional characterization Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration Platelet function during the acute phase of dengue hemorrhagic fever Pathology of fatal West Nile virus infections in native and exotic birds during the Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus Virus evolution: How does an enveloped virus make a regular structure Difference in pathogenesis between herpes simplex virus type 1 encephalitis and tick-borne encephalitis demonstrated by means of cerebrospinal fluid markers of glial and neuronal destruction Dengue carrier culture and antigen production in human lymphoblastoid lines Japanese encephalitis virus up-regulates expression of macrophage migration inhibitory factor (MIF) mRNA in the mouse brain Coronavirus infection induces H-2 antigen expression on oligodendrocytes and astrocytes Novel roles of nitric oxide in hemorrhagic shock Electron microscopic study of persistent dengue virus infection: analysis using a cell line persistently infected with dengue-2 virus Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120 Two receptors are required for antibody-dependent enhancement of human immunodeficiency virus type 1 infection: CD4 and Fc gamma R Neutralization and infectionenhancement epitopes of influenza A virus hemagglutinin Two TNF receptors DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells In vivo and in vitro studies on the morphological change in the monkey epidermal Langerhans cells following exposure to dengue 2 (16681) virus Langerhans cell density and serological changes following intradermal immunisation of mice with dengue 2 virus Risk factors in dengue shock syndrome Replication of dengue-2 virus in cultured human lymphoblastoid cells and subpopulations of human peripheral leukocytes Quantitative analysis of the immunopotency of genetically transfected dendritic cells Immunohistochemical phenotyping of liver macrophages in normal and diseased human liver Tumor necrosis factor, other cytokines and disease Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody Histamine content in 24-hour urine in patients with dengue haemorrhagic fever Tissue distribution of human IgG Fc receptors CD16, CD32 and CD64: an immunohistochemical study Circulating forms of intercellular adhesion molecule (ICAM)-1 in mice lacking membranous ICAM-1 Mutagenesis of the RGD motif in the yellow fever virus 17D envelope protein Biology of human immunoglobulin G Fc receptors Analysis of the binding of fluorescent C5a and C3a to human peripheral blood leukocytes The pathophysiology of tumor necrosis factors Elevated tumour necrosis factor in dengue fever and dengue haemorrhagic fever PrM-and cell-binding domains of the dengue virus E protein Antibodyenhanced binding of dengue-2 virus to human platelets Transmission electron microscopic study of the hemorrhagic spots in patients with epidemic hemorrhagic fever in the early stage Human HMC-1 mast cells exclusively express the Fc gamma RII subtype of IgG receptor Experimental encephalitis following peripheral inoculation of West Nile virus in mice of different ages Studies of hemostasis in Thai hemorrhagic fever Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release Immunohematologic disorders Human skin Langerhans cells are targets of dengue virus infection Dengue haemorrhagic fever and dengue shock syndrome: Are they tumour necrosis factor-mediated disorders? Antibodydependent enhancement of heterotypic dengue infections involved in suppression of IFN-gamma production A novel immunohistochemical assay for the detection of Ebola virus in skin: Implications for diagnosis, spread, and surveillance of Ebola hemorrhagic fever MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells Internalization of human immunodeficiency virus type 1 and other retroviruses by megakaryocytes and platelets Expression of the anaphylatoxin C5a receptor in non-myeloid cells