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1270 Differing Infection Patterns of Dengue and Yellow Fever Viruses in a Human Hepatoma Cell Line Philippe Marianneau, Anne-Marie Steffan, Cathy Royer, Marie-Thérèse Drouet, André Kirn, and Vincent Deubel Unité des Arbovirus et Virus des Fièvres Hémorragiques, Institut Pasteur, Paris, and Laboratoire de Virologie de La Faculté de Médecine, INSERM U74, Strasbourg, France Dengue (DEN) and yellow fever (YF) viruses are responsible for human diseases with symptoms ranging from mild fever to hepatitis and/or hemorrhages. Whereas DEN virus typically induces only limited foci of necrosis in the liver, YF virus infection is characterized by devastating lesions. In a human hepatoma cell line (HepG2), the kinetics of DEN and YF virus replication and release from the cells and the nature of host cell response to viral infection were compared. DEN virus infection was characterized by the early appearance of intracellular viral antigens, major ultrastructural cytopathic changes as early as 32 h after infection, extensive apoptotic cell death, and a low production of infectious particles. In contrast, YF virus grew exponentially to high titers and induced cytopathic changes only 72 h after infection. Differences between the infection processes of the two viruses observed in the hepatoma cell line may explain the different liver pathologies. Yellow fever (YF) and dengue (DEN) viruses are members of the flavivirus genus in the family Flaviviridae. They are positive-strand RNA viruses associated with a capsid protein and have an envelope assembled by budding through intracellular membranous structures [1]. These agents cause the most significant arthropodborne viral diseases in tropical countries and are responsible for infections with a large diversity of manifestations, from mild fever to hepatitis and/or hemorrhage. The pattern of symptoms depends on both viral and host factors [2]. In the first stages of infection, viruses may replicate in a variety of cells in different organs and are subsequently released into the bloodstream, from which they are taken up by hematophagous vectors. Although the majority of flaviviruses have the capacity to cross the blood-brain barrier in natural infections, the tropism of YF and DEN viruses may have changed during evolution, from nervous tissue to reticuloendothelial cells [3], with the liver as the ultimate target organ [4]. The hepatic pathology of these two viruses is different. In the course of YF infection, although the earliest cytopathologic changes and viral antigens appear in Kupffer cells, the hepatic parenchyma is the main target. The hepatocellular damage is marked by cell degeneration in the mesolobular and midzonal regions of the hepatic lobules, corresponding to coagulative necrosis of hepatocytes with microvacuolar fatty infiltrations Received 15 April 1998; revised 6 July 1998. Financial support: Direction des Recherches Etudes et Techniques (grant 95.1378). P.M. is supported by a grant from the Fondation Roux, Institut Pasteur, Paris. Reprints or correspondence: Dr. Vincent Deubel, Unité des Arbovirus et Virus des Fièvres Hémorragiques, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France ([email protected]). The Journal of Infectious Diseases 1998; 178:1270–8 q 1998 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/98/7805-0006$02.00 [5]. In DEN virus disease, the hepatic lesions resemble those of the early stage of YF virus infection but are less severe, with central and paracentral focal necrosis, hypertrophy of Kupffer cells, mild fatty changes, and patchy portal mononuclear cell infiltration [6]. Sinusoidal acidophilic Councilman bodies, which may correspond to apoptotic hepatocytes [7], are observed in both infections [2]. It is not known whether the differences in the extent of pathologic changes induced by these two viruses in the liver are due to different cytopathic events in the hepatocytes. In YF virus infection, damage to hepatocytes is presumably a direct result of viral infection, as these cells are the major site of viral replication [8, 9]. The target cells for DEN virus have not been clearly identified [8, 10], although the presence of virus-specific antigens and RNA inside hepatocytes suggests that these cells may also be susceptible to infection by DEN virus [11] (Couvelard A, Marianneau P, unpublished data). We previously demonstrated that a human hepatoma cell line, HepG2, which conserved numerous characteristics of differentiated hepatocytes [12], is permissive for DEN virus infection and that infected cells died by an apoptotic mechanism as a direct consequence of the viral infection and not of a bystander effect [13]. To determine whether the differences in the extent of hepatic lesions associated with DEN and YF virus infections can be attributed to differences in the replication strategy of the viruses in the hepatocytes or to immunopathologic mechanisms [14], we compared infections of HepG2 cells with low-passaged clinical isolates of the two viruses. We investigated differences in the kinetics of appearance of viral antigen–containing cells, in the production of infectious virus particles and the propagation of the infection in cell culture, and in the strength and rapidity of both the cytopathic effect and the induction of apoptosis. Differences in virus morphogenesis and in subsequent cyto- JID 1998;178 (November) HepG2 Cell Infection by DEN and YF Viruses pathic effects were also sought in an ultrastructural study of HepG2 cells infected with the two viruses. Materials and Methods Cells and Viruses Human hepatoma cell line HepG2 (American Type Culture Collection, Rockville, MD) was cultured at 377C in Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 4 mM glutamine. Mosquito Aedes pseudoscutellaris AP61 cells were propagated in monolayers at 287C in Leibovitz L-15 medium (Gibco BRL) supplemented with 0.55% tryptose phosphate and 10% FCS. YF virus strain DakHD 78359 was recovered on AP61 cells from the serum of a child, collected 1 day before his death in North Cameroon in 1990 [15]. The virus isolate was used at its fourth passage on AP61 cells. DEN serotype 1 virus OSTER was recovered on Aedes albopictus C6/36 cells from a serum sample collected from a patient in French Guiana in 1989 and was used at its third passage [16, 17]. Virus stocks were prepared and titrated on AP61 cells as described previously [16, 17]. The titers of YF and DEN viruses were 4 3 10 6 and 5 3 10 7 focus-forming units (ffu)/mL. DEN virus was purified from supernatants by polyethylene glycol precipitation and sucrose gradient centrifugation for electron microscopy experiments requiring a high MOI [16, 17]. Virus Production and Indirect Immunofluorescence Assay (IFA) HepG2 cells were grown in an 8-microchamber Lab-Tek slide (Nunc, Roskilde, Denmark) for 24 h. Cells were infected with YF and DEN viruses at various MOIs from 1 to 50 ffu/cell. At various times after infection, supernatants were collected for virus titration, and cells were fixed with 3% paraformaldehyde in PBS. Viral antigens in infected cells were detected by IFA as previously described, using DEN- or YF-specific hyperimmune mouse ascitic fluids (1: 100) and fluorescein-conjugated anti-mouse IgG (1:100) [16, 17]. Transmission Electron Microscopy HepG2 cell monolayers were grown in Lab-Tek microchambers and infected with YF virus at an MOI of 20 ffu/cell or with DEN virus at MOIs of 20 and 100 ffu/cell. Cells were fixed with 2.5% glutaraldehyde in 75 mM sodium cacodylate (pH 7.3) containing 4% sucrose, 1 mM MgCl2, and 1 mM CaCl2, and postfixed in 1% OsO4. The cells were then dehydrated and embedded in LX112 (Ladd Research Industries, Burlington, VT) and observed under a microscope (EM410; Philips Electronic Instruments, Eindhoven, The Netherlands). Detection of Apoptosis Propidium iodide staining. HepG2 cells were grown in 8microchamber Lab-Tek slides for 24 h and infected with various MOIs of DEN or YF virus in medium containing 2% FCS. Cell monolayers were incubated for 1 h at 377C, washed six times with 1271 DMEM, and cultured in medium containing 2% FCS. At various times after infection, the cells were fixed in paraformaldehyde for 20 min, washed three times with PBS, treated with 50 mM NH4Cl in PBS at room temperature for 10 min, washed three times with PBS, and finally permeabilized with 0.5% Triton X-100 in PBS at room temperature for 4 min. Viral antigens in infected cells were detected by IFA as described above. Cells were then treated with 200 mg/mL RNase A in phosphate buffer (0.2 M, pH 7.0) at 377C for 1 h and washed three times with PBS, and nuclei were stained with 20 mg/mL propidium iodide in 0.1% sodium citrate, pH 7.0, at room temperature for 10 min. The slides were washed in PBS, mounted in 40% glycerol in PBS, and observed under a fluorescence microscope (Leitz, Rockleigh, NJ). Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) analysis. TUNEL analysis (Boehringer Mannheim, Indianapolis) with fluorescein-dUTP was used to label DNA strand breaks induced during apoptosis. Cells were treated as described for propidium iodide staining. Cells were permeabilized, incubated for 30 min at 377C with the TUNEL label, washed with PBS, and mounted with 40% glycerol in PBS. Fluorescent cells were counted under a fluorescence microscope. Detection of DNA fragmentation. Fragmentation of cellular DNA into characteristic apoptotic ladders was assessed as previously described [18]. HepG2 cells were infected with DEN or YF virus at an MOI of 20 ffu per cell in DMEM containing 2% FCS, incubated at 377C for 1 h, washed three times with DMEM, and cultured in medium containing 2% FCS. At 48 and 72 h after infection, 107 cells were washed in PBS and lysed in 0.5 mL of lysis buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA, pH 8.0; 0.2% Triton X-100) containing 100 mg/mL proteinase K, at room temperature for 1 h. The lysed samples were centrifuged at 15,000 g for 15 min to pellet the nuclei, and the cytoplasmic DNA was precipitated from the supernatant with 5 M NaCl in 1 vol of isopropanol at 2207C for 1 h. The DNA precipitates were recovered by centrifugation at 13,000 rpm at 47C for 15 min, air-dried at room temperature for 15 min, and resuspended in sterile water. DNA was then loaded onto a 1.8% agarose gel in Tris-acetate (40 mM)– EDTA (0.001 M) buffer. Results Kinetics of Viral Multiplication HepG2 cells were infected with YF and DEN virus at MOIs of 1, 20, and 50 ffu/cell. Viral antigen–containing cells, detected by immunofluorescence, were counted at 24, 48, and 72 h after infection. Twenty-four hours after infection, ∼17%–45% of the DEN virus–infected cells (figure 1A) displayed speckled fluorescent labeling throughout the cytoplasm, in some cases very intense in the perinuclear area (figure 2A). At later time points, the infected cell count never increased above 1.4 times that after 24 h, whatever the MOI used. The maximum infected cell count was reached 48 h after infection but did not exceed 65% of the cells present, even with the highest infectious dose of DEN virus. In the case of YF virus, the kinetics of appearance of viral antigen–containing cells was different. The number of YF virus antigen–containing cells was very low 24 h after infection 1272 Marianneau et al. JID 1998;178 (November) Figure 1. Analysis of sensitivity of HepG2 cells to infection by DEN and YF virus. HepG2 cells were infected with DEN (solid line) or YF virus (dashed line) at MOIs of 1 (v), 10 (m), and 20 (m). At various times after infection, % of viral antigen-positive cells was calculated by immunofluorescence assay (A), and virus particles produced in supernatants were titered on AP61 cells (B). Each point represents mean for 2 determinations in duplicate. Error bars are omitted for clarity. (∼10 times lower than DEN virus–infected cells), but the proportion of cells infected increased exponentially with time of infection to reach 100% 72 h after infection with the highest infectious dose (figure 1A). Fluorescently labeled YF virus proteins appeared homogeneously distributed in the cytoplasm (figure 2B), and this pattern was also different from that observed in DEN virus–infected cells. Kinetics of the Production of Infectious Virus Particles The release of infectious virus particles in the culture medium was followed by titration on AP61 cells (figure 1B). YF virus multiplication seemed to be very efficient: The number of in- fectious particles detected increased with time to a maximum titer of 9 3 10 5 ffu/mL 48 h after infection with an MOI of 20. In contrast, the number of infectious DEN virus particles produced was ∼100–1000 times lower. Detection of Apoptotic Cells DEN virus–infected HepG2 cells die ∼40 h after infection by an apoptotic mechanism, directly due to viral replication and not to a bystander effect [13]. We examined whether DEN and YF viruses differed in their capacity to induce apoptosis in hepatocytes. HepG2 cells were infected at various MOIs and tested by propidium iodide staining or TUNEL analysis to Figure 2. Immunofluorescent staining of viral antigens. HepG2 cells were infected with DEN virus (A) or YF virus (B) and fixed 24 h after infection. Viral antigens were detected by use of DEN- or YF-specific hyperimmune mouse ascitic fluids. JID 1998;178 (November) HepG2 Cell Infection by DEN and YF Viruses Figure 3. Kinetics of apoptotic cell death of infected HepG2 cells. HepG2 cells were infected with DEN (solid line) or YF virus (dashed line) at MOIs of 1 (v), 10 (m), and 20 (m). At various times after infection, number of apoptotic nuclei was determined by immunofluorescent terminal deoxynucleotidyl transferase–mediated dUTP nickend labeling. Each point represents mean for 2 determinations in duplicate. Error bars are omitted for clarity. apoptotic nuclei. These two different techniques gave identical results. For both viruses, no apoptotic cells were detected 24 h after infection (figure 3). Forty-eight hours after infection with the highest MOI, the number of apoptotic cells in DEN virus–infected cultures was higher than that in YF virus–infected cultures. The percentage of apoptotic cells increased with the time of infection with both viruses but remained higher whatever the MOI used with DEN virus. Isolation of DNA from infected cultures at 72 h after infection confirmed the occurrence of an apoptotic process in cells infected with both viruses and the greater extent of this phenomenon in DEN virus–infected than YF virus–infected HepG2 cells (figure 4). 1273 tures of DEN virus particles sequestered by the invaginating plasma membrane (figure 5G). HepG2 infection with DEN virus. One of the most prominent features in DEN virus–infected HepG2 cells was a very rapid (within 32 h) cytopathic effect. The effect consisted first in the proliferation and dilatation of the endoplasmic reticulum (figure 6A, 6B) and second in the formation of huge contiguous vacuoles invading the whole cytoplasm and forming a network around the nucleus (figure 6A, 6E). The ultrastructural evidence for the infection of these cells was the presence of intracellular virus particles. The frequent continuity between these vacuoles and the perinuclear cisternae suggested that they were of endoplasmic reticulum origin (figure 6A). A small number of virus particles were found inside intracytoplasmic vesicles (figure 6C). Extracellular DEN virus particles were rarely observed. The ultrastructure of the virus particles associated with cellular debris (figure 6D) seems to have been altered (cf. inset in figure 6C). Apoptotic cells with chromatin condensation and nuclear fragmentation were visible as early as 48 h after infection and more numerous 72 h after infection (figure 6F). HepG2 infection with YF virus. The ultrastructural features of HepG2 cells infected with YF virus were different from those observed with DEN virus. At a low magnification, YF virus–infected cells or virus-producing cells were easily recognizable: Numerous electron-dense inclusions, not observed in DEN virus–infected cells, were scattered throughout the cytoplasm, especially in the perinuclear region (figure 7A). At a higher magnification, other features typical of flavivirus infection were observed, with proliferation of intracellular membranous structures (figure 7B) and dilated rough endoplasmic reticulum (figure 7C) into which virus particles were budding. Numerous nascent YF virus particles were found inside the rough endoplasmic reticulum (figure 7C, 7E), and sometimes Ultrastructural Study of Infected HepG2 Cells Penetration of DEN virus particles. HepG2 cells were infected with highly purified and concentrated virus at an MOI of 100 for 1 h at 377C and were then fixed for electron microscopy. Numerous DEN virus particles were found attached to the plasma membrane (figure 5A). They entered the cells via several different mechanisms. Many single particles seemed to be internalized via clathrin-coated pits (figure 5B, 5C) or small uncoated vesicles (figure 5D). Several particles forming aggregates at the cell surface could be engulfed simultaneously by a mechanism of phagocytosis or macropinocytosis via typical cytoplasmic processes (figure 5E). These particles were found enclosed inside phagosomes (figure 5F). We obtained several pic- Figure 4. Effect of DEN and YF virus infection on DNA fragmentation. Soluble DNA was extracted from HepG2 cell lysates 72 h after mock infection (M.I.) or infection at MOI of 20 with indicated virus. Sizes of DNA markers are indicated. 1274 Marianneau et al. JID 1998;178 (November) Figure 5. Ultrastructural study of penetration of DEN virus into HepG2 cells 1 h after infection at MOI of 100. A, Attachment of numerous DEN virus particles to the membrane (original magnification, 3130,000). B and C, Uptake of single particles via clathrin-coated pits (3130,000). D, Uptake of DEN virus particle by uncoated vesicle (3170,000). E, Engulfment of several particles by cytoplasmic process (342,500). F, Numerous DEN virus particles enclosed in intracytoplasmic vacuole (386,000). G, Capture of virus particles by invaginating plasma membrane (arrows) (368,000). they formed small crystalline arrays in the cytoplasm (figure 7F). No significant alterations in intensively virus-producing cells were observed 48 h after infection, although after 72 h, some infected cells displayed an intense vacuolization and vesiculization of the cytoplasm (figure 7D). Only a few cells in the apoptotic state were observed (figure 7G). Discussion Postmortem analysis reveals that the degree of hepatic lesions associated with DEN and YF diseases is different. The whole liver seems to be damaged by an invasive process during YF virus infection, whereas in DEN infection the lesions are mostly well-limited foci of necrosis. To evaluate the possibility that the type and degree of hepatocyte cell damage caused by YF and DEN viruses could be different, we comparatively analyzed the viral replication and the response to viral infection in a human hepatoma cell line, HepG2. Two low-passaged isolates repre- sentative of YF and DEN serotype 1 virus were used in these experiments. Cellular receptors specific for flavivirus envelope glycoprotein have not been identified on mammalian cells [17, 19]. Although electron microscopy studies have revealed that receptor-mediated endocytosis of YF virus in Vero cells occurred through coated vesicles [20], the mechanism by which DEN virus penetrates target cells remains unclear. We showed in this study that numerous DEN virus particles attached to the plasma membrane before apparently entering cells by diverse mechanisms. The first mechanism appeared to be receptor-mediated endocytosis in clathrin-coated vesicles, consistent with the mode of entry of other flavivirus, including YF, Kunjin, and West Nile viruses [20–22]. The second mechanism involved macropinocytosis or phagocytosis, with the engulfment of the viruses by cytoplasmic processes, as already described for DEN virus in blood monocytes [23]. This clathrin-independent route may explain the broad spectrum of cells of different origins sensitive JID 1998;178 (November) HepG2 Cell Infection by DEN and YF Viruses 1275 Figure 6. Ultrastructural features of HepG2 cells infected with DEN virus at MOI of 20. A, Cytopathic effect observed 48 h after infection, with extensive proliferation of endoplasmic reticulum (arrows), and enlargement of vacuoles to form network, which are sometimes in continuity with (*) perinuclear cisternae (original magnification, 360,000). B, Dilatation of endoplasmic reticulum. C, Presence of DEN virus particles inside intracytoplasmic vesicles 48 h after infection (329,000). Inset: 2 virus particles inside vesicle (arrow) (3135,000). D, Extracellular clumps of DEN virus particles with altered ultrastructure associated with cellular debris, 48 h after infection (380,000). E, Typical cytopathic effect with huge vacuoles forming network around nucleus 48 h after infection (34300). F, Characteristic aspect of apoptotic cell 48 h after infection, with densification and fragmentation of nucleus (39400). 1276 Marianneau et al. JID 1998;178 (November) Figure 7. Ultrastructural features of YF virus–infected HepG2 cells 72 h after infection at MOI of 20. A, Typical electron-dense areas corresponding to sites of accumulation of nucleocapsids (original magnification, 313,000). B, Proliferation of membranous structures inside cytoplasm (arrows) (368,000). C, Enlargement of rough endoplasmic reticulum containing several YF virus particles (357,600). D, Cytopathic effect in HepG2 cell with intense proliferation of intracytoplasmic vesicles (318,200). E, Higher magnification of YF viruses in cytoplasmic vesicle shown in figure 4C (3108,000). F, Intracytoplasmic virus particles forming crystalloid area (3108,000). G, Apoptotic cell displaying nuclear fragmentation and shrinkage of cytoplasm (34300). JID 1998;178 (November) HepG2 Cell Infection by DEN and YF Viruses to DEN and other flavivirus infections in vitro. Direct penetration of the virus particle by fusion with the plasma membrane, as shown for DEN virus in mosquito cells, was not observed [23]. The different modes of penetration of DEN virus into HepG2 cells are thus very similar to those described for other flaviviruses. The pathway of YF virus penetration into HepG2 cells was not studied because the titer of purified and concentrated YF virus was not sufficiently high. The number of infected cells (viral antigen–containing cells) was much higher 24 h after DEN than YF infection, suggesting that DEN virus uses a more efficient pathway of infection, leading to earlier viral protein synthesis. The patterns of immunofluorescent labeling in infected HepG2 cells were different and consisted of large perinuclear inclusions and small cytoplasmic foci of DEN virus antigens and of homogeneous YF virus antigens distributed throughout the cytoplasm. These differences in the cellular localization of the viral antigens have already been described for DEN virus–infected Vero cells [22] and YF virus–infected hamster kidney cells [24]. Presumably, therefore, the different cytoplasmic distributions of the viral proteins are a consequence of the virus rather than the host cells. DEN virus infection of HepG2 cells led to only a small amount of infectious particles released and a slight increase in the number of viral antigen–containing cells with the time of infection. In contrast, YF virus replicated to high titers and infected all exposed cells. These results are in accordance with the ultrastructural study, which evidenced that YF virus particles were more frequently observed than DEN virus particles in the cytoplasm, forming crystalloid areas, and in vesicles. In contrast, DEN virus–infected Vero cells produced 2–4 log more virus [17] and showed numerous virus particles inside intracytoplasmic vacuoles or clustered in the cytoplasm in crystalloid arrays [25]. Furthermore, neither the huge electron-dense intracytoplasmic areas corresponding to the accumulation of nucleocapsids [20] nor the extensive proliferation of membranous structures observed in YF virus–infected HepG2 cells were obvious with DEN virus. Whereas numerous rough endoplasmic reticulum–derived vacuoles typical of flavivirus infection in mammalian and mosquito cells [25] were present in HepG2 cells infected with both viruses, very early striking cytopathologic changes corresponding to an exaggerated dilatation of endoplasmic reticulum cisternae with a rarification of the cytoplasm were found only in DEN virus–infected HepG2 cells. Although this virus-induced extended vacuolization does not correspond to cytologic changes generally occurring during apoptosis, it is probable that these cells subsequently die by apoptosis, as they decrease in number between 48 and 72 h after infection concomitantly with an increase in the number of apoptotic cells. We observed that DEN virus–infected cells died rapidly by apoptosis. The highly productive YF virus–infected cells, in contrast, did not show early cytopathic changes. It was unclear 1277 whether the defect in the production of mature infectious DEN virus particles is the cause or the consequence of the early cytopathic effect and the apoptotic process. However, we have previously reported that the apoptotic process in DEN virus–infected HepG2 cells could be generated by the accumulation of viral proteins in the endoplasmic reticulum [13]. Any defect in DEN morphogenesis in HepG2 cells may thus have two consequences: first, a very low production of infectious virus particles, and second, the induction of an early apoptotic process. We are now investigating the cause of the very low production of DEN virus particles in HepG2 cells. Preliminary results showing the presence of large amounts of intracellular viral RNA and proteins reinforce the idea that a defect in viral morphogenesis is a significant factor. The results of our studies, showing the release of only small amounts of infectious DEN virus from infected HepG2 cells contrasting with the large amounts of YF virus particles produced, allow us to hypothesize on the events in hepatocytes during the course of natural infection. Our observations may explain the differences between the pathologic changes caused in the liver. YF infection is characterized by devastating liver lesions following extensive growth of the virus in liver cells, whereas in DEN infection, the low production of viral progeny by infected hepatocytes could lead to limited foci of infection. 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