Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, 117597 Singapore
Correspondence
M. L. Ng
micngml{at}nus.edu.sg
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ABSTRACT |
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INTRODUCTION |
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In contrast, apoptosis is an energy-dependent process of cell death that displays distinct hallmarks, including cytoplasmic shrinkage, chromatin condensation and intranucleosomal cleavage, phosphatidylserine exposure, plasma membrane blebbing, activation of caspases and cell fragmentation into apoptotic bodies which are phagocytosed without provoking an inflammatory response (White, 1996; Vaux & Strasser, 1996
; O'Brien, 1998
; Hay & Kannourakis, 2002
). The execution of either necrosis or apoptosis in virus-infected cells reflects the pathogenicity of viruses (Hay & Kannourakis, 2002
).
West Nile (WN) virus is a single-stranded, plus-sense, mosquito-borne RNA virus. WN virus is a member of the family Flaviviridae and cross-neutralization studies showed that WN virus is antigenically related to the Japanese encephalitis virus serocomplex. WN virus can cause a spectrum of illnesses, which includes WN fever, chorioretinitis, acute flaccid paralysis syndrome and fatal meningoencephalitis (George et al., 1984; CDC, 2002
; Adelman et al., 2003
; Bains et al., 2003
). The clinical manifestations of WN virus infection are well defined but the mechanism of pathogenesis of WN virus has not been elucidated fully.
Previous studies have documented that WN virus could infect, and induce CPE in, a variety of cell cultures of human, primate, rodent and insect origin (Paul et al., 1969; Odelola & Fabiyi, 1977
; Jordan et al., 2000
). In several human cases, as well as in experimental animal studies, of fatal WN virus infection, the triggering of both necrosis and apoptosis in infected cells and tissue was observed (Sampson et al., 2000
; Senne et al., 2000
; Shieh et al., 2000
; Cantile et al., 2001
; Xiao et al., 2001
).
Using Mesocricetus auratus (golden hamster) as a model for WN virus encephalitis, Xiao et al. (2001) noted the activation of perivascular inflammation and microgliosis in response to neuronal cell death. The activation of an inflammatory response implied that there was a secondary response to WN virus-induced cell death, which may contribute to the fatal outcome. The intention of this study was to determine the circumstances that could influence the pathway of death in WN virus-infected cells.
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METHODS |
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To determine virus growth curves, Vero cells were infected with WN virus at an m.o.i. of 0·1, 1, 10 and 100 and productive virus was harvested at selected time periods for plaque assay. To determine cell viability, the trypan blue-exclusion method was used. Cell morphology was observed under an optical microscope (Olympus BX-60, Japan).
Reagents and antibodies.
Cytochalasin B was purchased from Sigma. Antibodies for poly(ADP-ribose) polymerase (PARP), HMGB1 protein and cytochrome c were purchased from BD Transduction Laboratories.
Indirect immunofluorescence microscopy.
For immunofluorescence microscopy, cell monolayers were grown on coverslips and infected with WN virus (strain Sarafend) at an m.o.i. of 10. The procedure was similar to that described by Chu & Ng (2002). In brief, at 12 h post-infection (p.i.), cells were fixed with cold absolute methanol (Merck) for 10 min, followed by a wash in cold PBS for 15 min. Cells were then washed further in cold PBS containing 0·1 % BSA to eliminate non-specific binding. Cells were incubated with the primary antibodies (at a 1 : 1000 dilution for anti-HMGB1 antibodies) in a humidity chamber for 1 h at 37 °C, washed and incubated with Texas red (TR)-conjugated secondary antibodies (Amersham Pharmacia). Before mounting onto ethanol-cleaned glass slides using Dabco, the processed coverslips were washed. Laser scanning confocal microscopy (Leica TCS SP2) was used to view the specimens at excitation wavelengths of 480 nm for FITC and 543 nm for TR using oil immersion objectives.
Ultrastructural studies.
At selected times p.i., Vero cells infected with WN virus at an m.o.i. of 0·1, 1, 10 and 100 were washed twice with cold PBS and fixed with 2·5 % glutaraldehyde and 2 % paraformaldehyde. Cells were then post-fixed for 90 min at 4 °C in 1 % osmium tetroxide. A series of ethanol dilutions of increasing concentration were used to dehydrate the samples before embedding in low-viscosity epoxy resin. Ultrathin sections (5070 nm) stained with 2 % uranyl acetate and post-stained with 2 % lead citrate were viewed under a Philips electron microscope (CM 120, BioTwin).
Lactate dehydrogenase (LDH) assay.
The release of LDH was detected using CytoTox 96 (Promega) in accordance with manufacturer's procedure (Miroslav, 1995). In brief, at different time points after virus infection with different m.o.i., supernatants collected were centrifuged to obtain cell-free supernatants. Of each sample, 50 µl per well was transferred to 96-well plates. LDH activity was detected by addition of freshly prepared reagents followed by incubation for 30 min in the dark at room temperature. The concentration of LDH was determined using an ELISA plate reader at an absorbance of 490 nm. The concentration of LDH was expressed as the fold increase in LDH activity from WN virus-infected cells with respect to that of mock-infected cells.
Cytochalasin B treatment of WN virus-infected cells.
Treatment of WN virus-infected cells with cytochalasin B was performed essentially as described by Chu et al. (2003). Briefly, Vero cell monolayers were infected with WN virus at an m.o.i. of 10. After adsorption for 1 h, 2 ml maintenance medium supplemented with cytochalasin B (10 µg ml-1) was added to the cultures. At different time intervals p.i., cell supernatants were harvested and the release of LDH was determined, as described above.
Fluorometric assay for caspase activity.
Vero cells were infected with WN virus at either an m.o.i. of 1 or an m.o.i. of 10. At selected time intervals p.i., WN virus-infected cells were harvested and processed in accordance with manufacturer's instructions (Clontech) to determine caspase-3, -8 or -9 activities. In brief, the total number of cells was first adjusted to 2x105 cells per well of the 96-well microtitre plate and lysed. The clarified cell lysate was incubated with the appropriate reactants for each caspase. Samples were analysed in a fluorescent plate reader with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The activity of each caspase was expressed as the fold increase in WN virus-infected cells with respect to that of mock-infected cells.
Chromosomal DNA fragmentation assay.
At appropriate time intervals after infection, mock- and WN virus-infected cells were scraped and sedimented by centrifugation at 1000 r.p.m. Ice-cold PBS was used to wash the cell pellets before lysis in TNT buffer (20 mM Tris-HCl, pH 7·5, 0·5 M NaCl and 1 % Triton X-100) supplemented with a commercial protease inhibitor mixture (Roche) at 4 °C for 30 min. The cell lysate was centrifuged for 10 min at 10 000 r.p.m. and the supernatant containing the fragmented DNA was incubated with 50 µg RNase ml-1 (Roche) for 2 h at 37 °C. The mixture was extracted using phenol/chloroform. DNA was precipitated in 70 % isopropanol and 20 µg glycogen ml-1 for 3 h at -70 °C. The DNA pellet was dissolved in TE buffer (10 mM Tris-HCl, pH 8, and 1 mM EDTA) and the DNA samples were analysed by 2 % agarose gel electrophoresis. The gel was stained with 1 µg ethidium bromide ml-1 and viewed under UV lighting.
Cytosolic cytochrome c assay.
Cytosolic cytochrome c from mock- and WN virus-infected cells was prepared as described by Carrascosa et al. (2002). In brief, cells were resuspended in extraction buffer (50 mM Tris-HCl, pH 7·6, 150 mM NaCl, 0·5 mM EDTA, 10 mM Na2HPO4 and 1 % Nonidet P-40) for 30 min at 4 °C. The cell lysate was then centrifuged at 20 000 g for 30 min and the supernatant was subjected to SDS-PAGE followed by Western blotting to detect cytosolic cytochrome c.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay.
WN virus-infected cells grown on coverslips were processed for TUNEL assays at the appropriate times p.i. Cells were washed with PBS once before fixation with 1 % paraformaldehyde for 10 min at room temperature. Cells were then permeabilized with 70 % ethanol at -20 °C. Apoptotic cells were stained using the ApoAlert DNA Fragmentation kit (Clontech), according to the manufacturer's instructions. Processed coverslips were mounted before viewing with a laser scanning confocal microscope (Leica TCS SP2) at an excitation wavelength of 480 nm for FITC using a 63x oil immersion objective.
PARP cleavage.
At appropriate time intervals after infection, mock- and WN virus-infected cells were scraped and sedimented by centrifugation at 1000 r.p.m. Cell pellets were washed twice in ice-cold PBS and lysed in TNT buffer containing protease inhibitors at 4 °C for 30 min. Cell nuclei were removed from the cell lysate with a centrifugation step of 800 g. The remaining supernatant was subjected to SDS-PAGE and cleavage of PARP into truncated forms was assessed by Western blot analysis with antibodies against PARP.
SDS-PAGE and Western blot analysis.
In brief, cell lysates were subjected to SDS-PAGE before transfer to nitrocellulose membranes (Bio-Rad) using the Phast system (Amersham Pharmacia). After transfer, blots were blocked in PBS containing 5 % skimmed milk for 1 h to saturate the non-specific protein-binding sites on the nitrocellulose membrane. The blot was washed three times with wash buffer (Genelabs Diagnostics) for 10 min each. Primary antibodies anti-HMGB1, anti-cytochrome c and anti-PARP at dilutions of 1 : 500, 1 : 500 and 1 : 200, respectively, were used to detect these proteins. The blot was incubated overnight at room temperature on an orbital shaker. Alkaline phosphatase-conjugated secondary antibodies were used to detect antibody binding with the addition of substrate (NBT).
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RESULTS |
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At 16 h p.i., there was approximately 95 % viable cells, which decreased to 50 % at 32 h p.i. (Fig. 1a, b, respectively). In relation to the slower virus-induced CPE, there were approximately 107 p.f.u. ml-1 of virus obtained at 32 h p.i. For cells that were infected with WN virus at high m.o.i. (10 or 100), by 14 h p.i., there was less than 35 % viable cells (Fig. 1c, d
, respectively). Approximately 109 p.f.u. ml-1 of WN virus was obtained from 14 h p.i. For mock-infected cells, the number of viable cells remained relatively constant throughout the experiment (data not shown).
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In contrast, there was a drastic reduction in LDH activity (approximately a 6-fold reduction) in WN virus-infected cells with cytochalasin B treatment (Fig. 3b) when compared to the untreated sample (Fig. 3a
). These results suggested that the loss of membrane integrity was due to the profuse budding of virus particles at the plasma membrane, eventually contributing to necrosis.
For WN virus-infected cells (m.o.i.1), there was only a slight increase in released LDH activity (1·8-fold) by 24 h p.i. (Fig. 3c
) and, subsequently, almost a 6-fold increase was observed after 32 h p.i. This level was still lower than infection with high m.o.i. and it took 20 h longer compared to data shown in Fig. 3(a)
.
Previous studies have suggested that HMGB1 protein is a specific marker for necrotic cells, as this protein leaks out rapidly into the extracellular space when membrane integrity is lost during necrosis (Falciola et al., 1997; Degryse et al., 2001
; Muller et al., 2001
; Scaffidi et al., 2002
). Fig. 4
(a) shows the release of HMGB1 proteins into the extracellular medium of WN virus-infected (m.o.i.=10) cells by 12 h p.i. Minimal HMGB1 protein was observed to associate with the nuclear fraction. Although some HMGB1 protein was observed in the extracellular medium of mock-infected cells, the protein was retained mostly within nuclear remnants (Fig. 4a
). This contrasted with the lack of nuclear retention of the HMGB1 protein in WN virus-infected cells.
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Apoptosis observed in cells infected with WN virus at an m.o.i. of 1
In contrast, Vero cells infected with WN virus at an m.o.i. of 1 exhibited cytopathic changes similar to apoptosis, which were notably different from that of WN virus-infected cells at high infectious doses (m.o.i.10). Cytopathic changes occurred only by 24 h p.i. Membrane blebbing, cell shrinkage (Fig. 5
a, arrows) and detachment from substrum (cell rounding, arrowheads) were observed under optical microscopy. Using electron microscopy, further apoptotic features of chromatin condensation at the nuclear membrane (Fig. 5b
, arrows) and nuclear membrane blebbing were revealed. The formation of many membrane-bound apoptotic bodies (Fig. 5c
, arrows) was also observed at late times of infection (36 h p.i.).
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Release of holocytochrome c
The subsequent series of experiments was carried out to define the relevant apoptosis signal transduction pathway in WN virus-infected cells (m.o.i.=1). Release of cytochrome c from mitochondria is conserved in all mammalian cells undergoing apoptosis (Liu et al., 1996; Kluck et al., 1997
; Leist & Nicotera 1997
; Scorrano & Korsmeyer, 2003
). Cytochrome c is a small, 13 kDa protein which is attached loosely to the outer surface of the inner mitochondrial membrane.
Release of cytochrome c from the mitochondria into the cytosol of WN virus-infected cells was determined at different time periods after virus infection. Cytochrome c was detected as early as 14 h p.i. in the cytosolic fraction of WN virus-infected cells (Fig. 7a) but not in mock-infected cells (data not shown). By 18 h p.i., there was a remarkable increase in the presence of cytochrome c in the cytosolic fraction. Release of cytochrome c from the mitochondria is required for the formation of the apoptosome, which, in turn, is necessary for activation of pro-caspase-9.
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The cascade of caspase activation was investigated in WN virus-infected cells. Vero cells were infected with WN virus at an m.o.i. of 1 and cell lysates were processed for caspase-8, -9 and -3 activity determination assays. At selected time periods, cell lysates from both infected and mock-infected cells were subjected to fluorometric assays. There was a slight increase in caspase-8 activity (Fig. 7b) but activation of both caspase-9 and -3 was significantly induced by WN virus infection (Fig. 7c, d
, respectively). Activation of caspase-9 activity was also observed to occur much earlier and at higher levels (20 h p.i.) (Fig. 7c
) than caspase-3 (26 h p.i.) (Fig. 7d
). This time sequence of caspase-3 and -9 activation correlated well with the fact that caspase-9 is required for activation of downstream effector caspase-3. No such activation was observed when cells were infected with high infectious doses (m.o.i.
10).
Cleavage of PARP
Cleavage of PARP in WN virus-infected cells was determined using Western blotting. PARP is an abundant nuclear enzyme and is one of the earliest proteins targeted by caspase-3, -7 and -9 during apoptosis of mammalian cells (Casciola-Rosen et al., 1996). During apoptosis, caspase-3 cleaves the 116 kDa PARP into a stable 85 kDa fragment containing the c terminus. Intact, full-length PARP (116 kDa) was detected in the cell lysate harvested from mock-infected cells (Fig. 7e
). Cleavage of PARP into the 85 kDa fragment was observed by 28 h p.i. in WN virus-infected cells. As infection progressed from 32 to 36 h p.i., the 85 kDa cleavage product of PARP became the predominant band.
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DISCUSSION |
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Infections exhibiting necrosis often are accompanied by inflammatory responses. During WN virus infection in humans, inflammation was observed in several locations of the body (Senne et al., 2000; Deubel et al., 2001
; Sampson & Armbrustmacher, 2001
). The characteristic features observed in the necrotic process include swelling of cells (Fig. 2c
), vacuolation of cells (Fig. 2d
), a burst of LDH release (Fig. 3a
) and liberation of HMGB1 protein (Fig. 4
). Recent studies have demonstrated that HMGB1 protein is a critical factor that links necrotic cell death to inflammation (Degryse et al., 2001
; Muller et al., 2001
; Andersson et al., 2002
; Scaffidi et al., 2002
). This protein is a potent macrophage-activating factor and a pro-inflammatory mediator cytokine that is released exclusively by necrotic cells but not by apoptotic cells (Scaffidi et al., 2002
). The release of HMGB1 protein contributes to the pathogenesis of systemic inflammation (Andersson et al., 2002
).
A previous study by Despres et al. (1998) also reported that dengue virus infection in Vero cells at a high infectious dose (m.o.i.
10) resulted in the activation of necrosis in these cells. The possible explanation provided for the activation of necrosis in these cells was due to the cytotoxic effect resulting from the accumulating dengue virus particles.
However, in WN virus infection, the loss of plasma membrane integrity was due largely to the profuse budding of the progeny virus particles at the cell surface (Ng et al., 2001), eventually resulting in cells undergoing necrosis. This was confirmed when Vero cells infected with WN virus at an m.o.i. of
10 were subjected to cytochalasin B treatment. The reason for this treatment was that in another study, actin filaments were established to have a prominent role in the mechanism of virus budding (Chu et al., 2003
). Disrupting actin filament formation with cytochalasin B would shut down the budding process leaving the plasma membrane intact (thus halting necrosis). The large reduction in released LDH activity (Fig. 3b
) was used as the marker to indicate that the process of necrosis was slowed down markedly. Thus, the proinflammatory necrotic cell death after high dose WN virus infection was induced by the subsequent extensive budding to release progeny virus particles at the plasma membrane.
Consistent with Yang et al. (2002), our study also showed that WN virus-induced apoptosis but only when a low titre inoculum was used (m.o.i.
1) (Figs 5 and 6
). During WN virus infection (at low infectious doses), cytochrome c was first detected in the cytosolic fraction by 14 h p.i. and increased substantially by 18 h p.i. (Fig. 7a
). Cytochrome c is one of the universal factors that is released from the mitochondria in response to the disruption of mitochondrial permeability. In the mitochondrial-mediated apoptotic pathway, the pro-apoptotic Bcl-2 family members Bak and Bax have direct effects on the endoplasmic reticular Ca2+ pool, with subsequent sensitization of mitochondria to calcium-mediated fluxes and cytochrome c release (Nutt et al., 2002
). In addition, Parquet et al. (2001)
have documented the upregulation of bax in WN virus-induced apoptosis. Upregulation of pro-apoptotic bax in WN virus-infected cells by acting on the upstream of the pathway would therefore activate the mitochondrial-mediated apoptosis pathway observed in this study.
The release of cytochrome c and other proteins then activates pro-caspase-9 in the apoptosome complex. Caspase-9 activity was substantially upregulated within 2 h after the release of cytochrome c in WN virus-infected cells (Fig. 7c). The activation of pro-caspase-9 led to activation of effector caspases, such as caspase-3 (Fig. 7d
) at 6 h later. This in turn results in the cleavage of a number of cellular proteins that contribute to the irreversible events in cell death (Kroemer et al., 1997
; Zamzami & Kroemer, 1999
). The cleavage of PARP was subsequently detected after the activation of caspase-3 (Fig. 7e
).
In conclusion, the mitochondrial-mediated apoptotic pathway was involved in cells infected with WN virus at low infectious doses. This differs from the necrotic process observed in Vero cells infected with WN virus at higher infectious doses (m.o.i.10).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersson, U., Erlandsson-Harris, H., Yang, H. & Tracey, K. J. (2002). HMGB1 as a DNA-binding cytokine. J Leukoc Biol 72, 10841091.
Bains, H. S., Jampol, L. M., Caughron, M. C. & Parnell, J. R. (2003). Vitritis and chorioretinitis in a patient with West Nile virus infection. Arch Ophthalmol 121, 205207.
Cantile, C., Del Piero, F., Di Guardo, G. & Arispici, M. (2001). Pathologic and immunohistochemical findings in naturally occurring West Nile virus infection in horses. Vet Pathol 38, 414421.
Carrascosa, A. L., Bustos, M. J., Nogal. , Gonzalez de Buitrago, G. & Revilla, Y. (2002). Apoptosis induced in an early step of African swine fever virus entry into Vero cells does not require virus replication. Virology 294, 372382.[CrossRef][Medline]
Casciola-Rosen, L., Nicholson, D. W., Chong, T., Rowan, K. R., Thornberry, N. A., Miller, D. K. & Rosen, A. (1996). Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J Exp Med 183, 19571964.[Abstract]
CDC (2002). Acute flaccid paralysis syndrome associated with West Nile virus infection in Mississippi and Louisiana. Morbid Mortal Wkly Rep 51, 825828.
Chu, J. J. H. & Ng, M. L. (2002). Infection of polarized epithelial cells with flavivirus West Nile: polarized entry and egress of virus occur through apical surface. J Gen Virol 83, 24272435.
Chu, J. J. H., Choo, B. G. H., Lee, J. W. M. & Ng, M. L. (2003). Actin filaments participate in West Nile (Sarafend) virus maturation process. J Med Virol 71, 463471.[CrossRef][Medline]
Degryse, B., Bonaldi, T., Scaffidi, P., Muller, S., Resnati, M., Sanvito, F., Arrigoni, G. & Bianchi, M. E. (2001). The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. J Cell Biol 152, 11971206.
Despres, P., Frenkiel, M. P., Ceccaldi, P. E., Duarte Dos Santos, C. & Deubel, V. (1998). Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses. J Virol 72, 823829.
Deubel, V., Fiette, L., Gounon, P., Drouet, M. T., Khun, H., Huerre, M., Banet, C., Malkinson, M. & Despres, P. (2001). Variations in biological features of West Nile viruses. Ann N Y Acad Sci 951, 195206.
Falciola, L., Spada, F., Calogero, S., Langst, G., Voit, R., Grummt, I. & Bianchi, M. E. (1997). High mobility group 1 protein is not stably associated with the chromosomes of somatic cells. J Cell Biol 137, 1926.
Fernandes-Alnemri, T., Litwack, G. & Alnemri, E. S. (1994). CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 -converting enzyme. J Biol Chem 269, 3076130764.
George, S., Gourie-Devi, M., Rao, J. A., Prasad, S. R. & Pavri, K. M. (1984). Isolation of West Nile virus from the brains of children who had died of encephalitis. Bull World Health Organ 62, 879882.[Medline]
Goudie, R. B. (1985). Molecular and cellular pathology of tissue damage. In Muir's Textbook of Pathology, 12th edn. Edited by J. R. Anderson. Baltimore, MD: Edward Arnold.
Hay, S. & Kannourakis, G. (2002). A time to kill: viral manipulation of the cell death program. J Gen Virol 83, 15471564.
Jordan, I., Briese, T., Fischer, N., Lau, J. Y. & Lipkin, W. I. (2000). Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells. J Infect Dis 182, 12141217.[CrossRef][Medline]
Kluck, R. M., Bossy-Wetzel, E., Green, D. R. & Newmeyer, D. D. (1997). The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 11321136.
Koyama, A. H., Irie, H., Ueno, F., Ogawa, M., Nomoto, A. & Adachi, A. (2001). Suppression of apoptotic and necrotic cell death by poliovirus. J Gen Virol 82, 29652972.
Kroemer, G., Zamzami, N. & Susin, S. A. (1997). Mitochondrial control of apoptosis. Immunol Today 18, 4451.[CrossRef][Medline]
Leist, M. & Nicotera, P. (1997). The shape of cell death. Biochem Biophys Res Commun 236, 19.[CrossRef][Medline]
Liu, X., Kim, C. N., Yang, J., Jemmerson, R. & Wang, X. (1996). Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147157.[Medline]
Miroslav, C. (1995). Using Promega's CytoTox 96 Non-Radioactive Cytotoxicity Assay to measure cell death mediated by NMDA receptor subunits. In Promega Notes, chapter 51, p. 21. Madison, WI: Promega.
Muller, S., Scaffidi, P., Degryse, B., Bonaldi, T., Ronfani, L., Agresti, A., Beltrame, M. & Bianchi, M. E. (2001). The double life of HMGB1 chromatin protein: architectural factor and extracellular signal. EMBO J 20, 43374340.
Nagata, S., Nagase, H., Kawane, K., Mukae, N. & Fukuyama, H. (2003). Degradation of chromosomal DNA during apoptosis. Cell Death Differ 10, 108116.[CrossRef][Medline]
Ng, M. L., Tan, S. H. & Chu, J. J. H. (2001). Transport and budding at two distinct sites of visible nucleocapsids of West Nile (Sarafend) virus. J Med Virol 65, 758764.[CrossRef][Medline]
Nicholson, D. W. & Thornberry, N. A. (1997). Caspases: killer proteases. Trends Biochem Sci 22, 299306.[CrossRef][Medline]
Nutt, L. K., Pataer, A., Pahler, J., Fang, B., Roth, J., McConkey, D. J. & Swisher, S. G. (2002). Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores. J Biol Chem 277, 92199225.
O'Brien, V. (1998). Viruses and apoptosis. J Gen Virol 79, 18331845.
Odelola, H. A. & Fabiyi, A. (1977). Biological characteristic of Nigerian strains of West Nile virus in mice and cell cultures. Acta Virol 21, 161164.[Medline]
Parquet, M. C., Kumatori, A., Hasebe, F., Morita, K. & Igarashi, A. (2001). West Nile virus-induced Bax-dependent apoptosis. FEBS Lett 500, 1724.[CrossRef][Medline]
Paul, S. D., Singh, K. R. & Bhat, U. K. (1969). A study on the cytopathic effect of arboviruses on cultures from Aedes albopictus cell line. Indian J Med Res 57, 339348.[Medline]
Porter, A. G. & Janicke, R. U. (1999). Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6, 99104.[CrossRef][Medline]
Sampson, B. A. & Armbrustmacher, V. (2001). West Nile encephalitis: the neuropathology of four fatalities. Ann N Y Acad Sci 951, 172178.
Sampson, B. A., Ambrosi, C., Charlot, A., Reiber, K., Veress, J. F. & Armbrustmacher, V. (2000). The pathology of human West Nile Virus infection. Human Pathol 31, 527531.[Medline]
Scaffidi, P., Misteli, T. & Bianchi, M. E. (2002). Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191195.[CrossRef][Medline]
Scorrano, L. & Korsmeyer, S. J. (2003). Mechanisms of cytochrome c release by proapoptotic Bcl-2 family members. Biochem Biophys Res Commun 304, 437444.[CrossRef][Medline]
Senne, D. A., Pedersen, J. C., Hutto, D. L., Taylor, W. D., Schmitt, B. J. & Panigrahy, B. (2000). Pathogenicity of West Nile virus in chickens. Avian Dis 44, 642649.[Medline]
Shieh, W. J., Guarner, J., Layton, M. & 7 other authors (2000). The role of pathology in an investigation of an outbreak of West Nile encephalitis in New York, 1999. Emerg Infect Dis 6, 370372.[Medline]
Solomon, T. & Vaughn, D. W. (2002). Pathogenesis and clinical features of Japanese encephalitis and West Nile virus infections. Curr Top Microbiol Immunol 267, 171194.[Medline]
Vaux, D. L. & Strasser, A. (1996). The molecular biology of apoptosis. Proc Natl Acad Sci U S A 93, 22392244.
White, E. (1996). Life, death, and the pursuit of apoptosis. Genes Dev 10, 115.[CrossRef][Medline]
Xiao, S. Y., Guzman, H., Zhang, H., Travassos da Rosa, A. P. & Tesh, R. B. (2001). West Nile virus infection in the golden hamster (Mesocricetus auratus): a model for West Nile encephalitis. Emerg Infect Dis 7, 714721.[Medline]
Yang, J. S., Ramanathan, M. P., Muthumani, K. & 10 other authors (2002). Induction of inflammation by West Nile virus capsid through the caspase-9 apoptotic pathway. Emerg Infect Dis 8, 13791384; erratum 9, 406.
Zamzami, N. & Kroemer, G. (1999). Condensed matter in cell death. Nature 401, 127128.[CrossRef][Medline]
Zou, H., Li, Y., Liu, X. & Wang, X. (1999). An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274, 1154911556.
Received 20 June 2003;
accepted 20 August 2003.