1 Chang Gung Children's Hospital at Kaohsiung, Chang Gung University, 123 Ta-Pei Road, Niau-Sung, Kaohsiung 833, Taiwan
2 Department of Pathology, National Defense Medical Center, Hung Kuang Institute of Technology, Sha-Lu, Taichung 433, Taiwan
3 Department of Medical Research, Kuang Tien General Hospital, Hung Kuang Institute of Technology, Sha-Lu, Taichung 433, Taiwan
Correspondence
Kuender D. Yang
yangkd.yeh{at}msa.nihet.net
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ABSTRACT |
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INTRODUCTION |
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It is not clear why JEV causes overwhelming infections in humans yet is not transmissible from human beings. Studies of autopsies showed that JEV can only be isolated from brain tissues, but not tissue fluids or other organs (Iwasaki et al., 1986; Mukherji & Biswas, 1976
). This suggests that JEV tends to cause a neurotropic infection, attacking neural rather than non-neural tissues in humans. Experimental studies in rats have demonstrated that neuron cells, especially developing neurons, were the major infective source (Kimura-Kuroda et al., 1993
). Furthermore, animal studies have also shown that direct intracerebral inoculation of JEV induces extensive encephalitis in mice, while intraperitoneal inoculation does not (Hase et al., 1990a
). These studies indicate that JEV may not actively infect blood cells or non-neural cells after delivery into the subcutaneous tissues and blood through a mosquito bite. Although JEV does not replicate efficiently in blood cells, it may reside in certain leukocytes, enabling it to enter the central nervous system, resulting in encephalitis. Sharma et al. (1991)
showed that no JEV antigen or virus isolation could be found in blood or leukocytes from JE patients, although its antigen could be detected by indirect immunofluorescence in mouse embryonic fibroblasts after co-culturing with human mononuclear cells obtained from patients with JEV infections. We have attempted to establish a model suitable for identifying neurotropism and persistency of JEV infections in human cells. Employing human neuroblast-derived (NB) cell lines and human blood cells, including erythrocytes, lymphocytes, granulocytes and monocytes, we investigated whether JEV causes persistent infection in certain human leukocytes while causing neurocytotropic infections with cell apoptosis in human NB cells.
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METHODS |
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Determination of viable JEV titres by the p.f.u. assay.
The JEV titres were measured by the p.f.u. assay as modified from a previous description (Morens et al., 1985). In brief, baby hamster kidney (BHK-21) cells (1x105 cells per well) were cultured into monolayers on 24-well plates (Corning Laboratory Science). The BHK-21 cells were adsorbed with a series of 10-fold dilutions of virus-infected culture supernatants or virus-infected cell extracts for 60 min at 37 °C before they were overlaid with Eagle's minimal essential medium (EMEM) containing 2 % FCS and 1·5 % carboxymethylcellulose (Sigma Biochemicals) for 4 more days of incubation. The plaque-forming wells were fixed with 10 % formalin in phosphate-buffered saline (PBS) for 1 h, and stained with 0·5 % crystal violet for 30 min before counting (Shaio et al., 1994
; Yang, K. D. et al., 1995
). Results are presented with the standard error of the mean calculated from three triplicate experiments.
Determination of JEV titres by a real-time quantitative RT-PCR.
Viral RNA extracted from JEV-infected monocytes and their culture supernatants were subject to a fluorogenic quantitative RT-PCR detection of total JEV titres as modified from our previous description (Chen et al., 2001; Li et al., 2002
). In brief, 0·25 ml of culture supernatants or cultured cell pellets were added individually with 0·75 ml of Tri-reagent (Sigma) to separate RNA from DNA and protein fractions (Chen et al., 2001
). Fluorescent RT-PCR was carried out in a ABI 7700 quantitative PCR machine (Applied Biosystems) for 40 cycles using TaqMan technology, as described previously (Chen et al., 2001
). The forward primer used to amplify the JEV RNA was 5'-GGC TTA GCG CTC ACA TCC A-3'; the reverse primer was 5'-GCT GGC CAC CCT CTC TTC TT-3'; the nested fluorescent probe sequence was FAMCGC CCA CCA CTA TAG CTG CCG GATAMRA. Results were calculated from a standard curve made from a series of well-known virus titres (Chen et al., 2001
; Li et al., 2002
).
Preparation of human NB cells and myeloid leukaemic (HL60) cells.
Two human neuroblastoma cell lines, HTB-11 and IMR-32, and one human glioblastoma cell line, DBTRG-05MG, were obtained from the ATCC. Another human glioblastoma cell line, G5T/VGH, was obtained from the National Health Research Institute Culture Collection, Shin-Chu, Taiwan. These cells were cultured in EMEM supplemented with 10 % FCS, 1 mM glutamine, 0·1 mM non-essential amino acids, 1 mM sodium pyruvate, 50 µg gentamicin ml-1 and 100 µg penicillin ml-1 in a 5 % CO2 humidified incubator at 37 °C. Cells were regularly propagated at a density of 2x105 cells ml-1 every 7 days and harvested using 0·02 % EDTA in PBS (0·15 M, pH 7·37·4), as described previously (Yang, K. D. et al., 1993, 1995
). HL60 cells were cultured in RPMI 1640 medium with 10 % FCS as described previously (Yang, K. D. et al., 1994a
).
Preparations of human erythrocytes and different leukocytes.
Human erythrocytes and various leukocytes were isolated from peripheral whole blood donated by healthy volunteers after informed consent had been obtained. Heparinized peripheral blood was separated into plasma, leukocyte buffy-coat and erythrocyte layers by centrifugation at 1500 g for 15 min. After removing plasma, leukocytes were harvested from the buffy-coat layers; erythrocytes were harvested from the bottom layer and resuspended to the original volume in PBS for studies. Leukocytes in the buffy-coat layers were separated into polymorphonuclear (PMN) and mononuclear leukocytes (MNLs) by a gradient centrifugation in Ficoll-paque (Pharmacia Fine Chemicals) at 1500 g for 20 min (Yang, K. D. et al., 1988). PMNs were harvested from the cell pellet in the bottom. Residual erythrocytes were lysed twice with a hypotonic solution (0·2 % NaCl) for 30 s, followed by restoration of the osmolarity with 1·6 % NaCl. The PMNs were washed in PBS and suspended to 5x106 cells ml-1 in Hanks balanced salt solution (HBSS) containing 0·1 % human serum albumin (Hyland Baxter Healthcare). The purity and viability of the PMNs were greater than 95 and 98 %, respectively (Yang, K. D. et al., 1988
; Yang, M. Y. et al., 2002
). The MNL fraction was separated further by allowing monocytes to adhere onto a 6-well plate (Corning) for 90 min (Wang et al., 1996
; Yang, K. D. et al., 1994b
). Lymphocytes were harvested from the non-adherent cells, whereas monocytes were harvested from the adherent layer using a rubber policeman, as described previously (Yang, K. D. et al., 1995b
). All cells were resuspended at 5x105 cells ml-1 in RPMI 1640 medium (Gibco) containing 10 % FCS with penicillin G (100 µg ml-1) and streptomycin (100 µg ml-1) for studies.
JEV infectivity as determined by indirect immunofluorescence assay.
The JEV infectivity in leukocytes and NB cells was monitored by an indirect immunofluorescent analysis of NS3 antigen expression as modified from a previous description (Leake et al., 1986). Cells infected with JEV at an m.o.i. of 1 : 1 for 60 min were washed twice in PBS to remove extracellular virus before the cells were set for culture. The mAb (RP92-2) directed against the NS3 antigen of JEV (kindly provided by the Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan) (Ma et al., 1995
) was used as the specific antibody. Cells with and without JEV infection for the specified periods of time were spotted in triplicate onto a Teflon-coated chamber slide. These slides were fixed in acetone and stained with the anti-NS3 antibody at a dilution of 1 : 50 in PBS for 30 min, followed by an FITC-conjugated goat anti-mouse IgG at a dilution of 1 : 100 as the secondary antibody for another 30 min. After washing, these slides were observed under a Zeiss fluorescent microscope. Results are presented as the percentage of positive cells per 500 cells.
JEV replication in different human leukocytes and NB cell lines.
JEV replication in human monocytes and NB cells was assessed by standard p.f.u. assay and RT-PCR analysis as described above. In brief, monocytes or NB cells adsorbed with JEV at an m.o.i. of 1 : 1 for 60 min were washed twice in PBS to remove extracellular virus before the cells were cultured in medium for the indicated periods of time. In studying persistency of JEV infection in human monocytes, culture medium was changed every 7 days for 60 days. The culture supernatants and cell pellets from monocytes or neuroblastoma cells infected with JEV for the indicated periods of time were subjected to viral p.f.u. determination in BHK-21 cells as well as RT-PCR analysis of JEV titres. For those with positive RT-PCR but negative p.f.u. assays from JEV-infected monocytes, co-culture (0·1 ml) of the culture supernatants of JEV-infected monocytes with neuroblastoma (HTB-11) cells at 2 ml (5x105 cells ml-1) for 5 days was performed before a second round of assessment of p.f.u. ml-1 in the culture supernatants of neuroblastoma (HTB-11) cells. The virus titres determined by the p.f.u. assay were reciprocally compared with the host-cell viability as described below.
Cell viability as determined by trypan-blue exclusion.
Cell viability was monitored by the trypan-blue-exclusion assay (Yang, K. D. et al., 1988; Yang, M. Y. et al., 2002
). Monocytes and NB cells with and without JEV infections for the indicated periods of time were incubated with 0·4 % trypan blue for 5 min before observation under a microscope (400x). Viable cells capable of excluding trypan blue showed no blue stain, whereas non-viable cells were incapable of excluding the dye and were stained blue by it. Results are presented as the percentage of viable cells per 500 cells.
Cell apoptosis as assessed by DNA fragmentation and flow cytometric analysis of apoptotic cells.
Cells undergoing apoptosis tend to express endonuclease resulting in DNA fragmentation in association with exposure of phosphatidylserine (PS) to the outer leaflet of the cell membrane. Employing flow cytometric analysis of PS expression and gel electrophoretic analysis of DNA fragmentation, we studied cell apoptosis in the NB cells and monocytes with and without JEV infection. Cells (2x105 per 0·1 ml) from monocytes and NB cells with and without JEV infection for 2 days were stained with FITC-labelled Annexin-V (50 µg ml-1; PharMingen) for 20 min at room temperature and analysed by flow cytometry (FACScan apparatus; Becton Dickinson), as described previously (Yang, M. Y. et al., 2002). DNA extracted from the culture supernatants of JEV-infected NB cells and monocytes was subjected to gel electrophoretic analysis of DNA fragmentation as modified from previous descriptions (Mabbott et al., 1995
; Yang, M. Y. et al., 2002
). In brief, culture supernatants (5 ml) were collected and subjected to DNA extraction by using an equal volume of chloroform/phenol solution. After this extraction, the DNA in the upper aqueous phase was further precipitated with 0·1 vol. of 5 M NaCl and 1 vol. of 2-propanol at -20 °C for 16 h. The DNA precipitates were suspended in 10 µl TE (10 mM Tris salt, 1 mM EDTA) buffer and incubated with 1 µg RNase A ml-1 at 65 °C for 10 min followed by another 60 min at 37 °C before electrophoresis in a 2 % agarose gel containing 1 µg ethidium bromide ml-1. The DNA fragmentation showing approximately 200 bp ladders was confirmed by a series of 100 bp reference markers (Sigma).
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RESULTS |
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DISCUSSION |
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We also found that the JEV titres in monocytes detected by RT-PCR were 1- to 3-log scale higher than those detected by p.f.u. assay, especially in the early initial infection period (Table 2). This indicates that non-viable JEV is present at much higher levels than viable JEV in human monocytes. In other words, this also suggests that certain mediators from JEV-infected monocytes may inactivate or, in part, kill the virus, resulting in poor transmission of JEV from the blood of human beings.
Pathogenesis of JE is still controversial. Two hypotheses are currently proposed. Studies with murine brain cell culture have shown that neuron cells, especially developing neurons, are the major infection sites (Kimura-Kuroda et al., 1993). JEV-infected mouse brain cells revealed impaired organelle functions before cell death (Hase et al., 1990b
), suggesting a direct virus-induced cell cytotoxicity. In contrast, some evidence demonstrates that damage of the CNS by JEV infection was indirectly mediated by immune reactions including antibody-mediated cytotoxicity or suppressor T cell activity (Burke et al., 1985
; Webb & Smith, 1966
). An understanding of the disease pathogenesis is crucial for prevention of neurologic sequelae mediated by JE in human beings. In support of direct cell cytotoxicity of JEV infections in humans, our study showed that JEV caused a massive infection in four human NB cell lines, followed by cell apoptosis in 2 days and almost 100 % cell death in 5 days. In addition, the fact that JEV can be only isolated in brain tissues during autopsies, but not cerebrospinal or tissue fluids (Iwasaki et al., 1986
; Mukherji & Biswas, 1976
), also suggests that active JEV replication is correlated to cerebral morbidity and mortality in JEV infections. In fact, evidence accumulated indicates that the titres of JEV neutralizing antibody were inversely correlated to JEV susceptibility in human beings (Burke et al., 1985
). Moreover, the case-fatality rate of JEV infections in dengue immunes is only one-third that of non-immunes (Grossman et al., 1974
; Hammon et al., 1958b
). These results suggest that JEV may specifically target NB cells and that specific or cross-reactive antibodies are protective but not offensive in human JEV infections. Further studies will investigate whether JEV-mediated programmed cell death of NB cells is preventable or ameliorable. An effective control of JEV-mediated programmed cell death may be useful in treating patients with JE.
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ACKNOWLEDGEMENTS |
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Received 16 June 2003;
accepted 22 October 2003.