1 Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan
2 Chengdu Institute of Biological Products, Chengdu 610063, Sichuan Province, PR China
Correspondence
Takaji Wakita
wakita{at}tmin.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences described in this study are given in Table 1.
Supplementary figures and tables are available in JGV Online.
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INTRODUCTION |
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Studies attempting to elucidate the molecular basis of JEV attenuation have analysed the genomes of both the virulent parental strain SA14 and the attenuated vaccine virus SA14-14-2 (Nitayaphan et al., 1990; Aihara et al., 1991
). Several JEV mutants have been obtained through
-ray irradiation (Chen et al., 1996
) or passage in cultured cells (Hasegawa et al., 1992
). These studies have indicated that some mutations in the E protein correlate with viral attenuation, including the amino acid mutation from glutamic acid (Glu) to lysine (Lys) at residue 138 of the E protein (E-138). Due to the multiple mutations present, the exact mutated residue changes in the E protein that are primarily responsible for JEV attenuation are not clearly understood. Since infectious Yellow fever virus RNA was transcribed successfully from a full-length cDNA template by using in vitro ligation of two cDNA fragments (Rice et al., 1989
), infectious flavivirus clones have been constructed in similar fashion for JEV (Sumiyoshi et al., 1992
), dengue virus type 2 (Polo et al., 1997
; Gualano et al., 1998
), dengue virus type 4 (Lai et al., 1991
) and other viruses (Khromykh & Westaway, 1994
; Gritsun & Gould, 1998
; Shi et al., 2002
; Hayasaka et al., 2004
). This method seems valuable in studying biological function, polyprotein processing and virulence of mutant viruses with specific mutations in various regions of the viral genome. However, irregular mutations occur frequently in full-length JEV cDNA clones during cloning into plasmid vectors, particularly in the 5' fragment, and this has seriously affected such studies (Sumiyoshi et al., 1995
; Yamshchikov et al., 2001
). Specific strategies are thus necessary to establish stable, full-length JEV infectious clones, such as insertion of short introns or cloning into bacterial artificial chromosomes (Yamshchikov et al., 2001
; Yun et al., 2003
).
The present report describes the construction of a stable, full-length cDNA clone of the wild-type JEV AT31 strain into the very low-copy-number plasmid pMW118 by using conventional molecular-cloning technology. Another plasmid, containing a Glu-to-Lys mutation at the E-138 residue, was also constructed. Recombinant JEVs comprising wild-type and mutant viruses were recovered after synthetic RNA transfection, and were characterized with regard to viral virulence, internalization and release.
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METHODS |
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The wild-type JEV strain AT31 was a gift from Dr Nakamura of the Nippon Institute for Biological Science, Japan. Attenuated JEV at222 strain was obtained after 222 passages through primary hamster kidney cells to obtain a vaccine strain, as described previously (Yasui, 2002). Recombinant wild-type rAT virus and mutant rAT-E138K were obtained from supernatants of Vero cells transfected with in vitro-transcribed RNAs. All cDNA sequences of viral RNAs were determined and deposited in GenBank/DDBJ/EMBL (Table 1
). The at222 strain contains several mutations, including the Glu-to-Lys mutation at residue E-138. All viruses were propagated once in Vero cells or C6/36 cells and supernatants were obtained and stored at 80 °C until used. Three-day-old BALB/c mice were inoculated intracerebrally with 104 p.f.u. JEV rAT-E138K. The inoculated mice were sacrificed at 8 days of age and their brains were harvested and homogenized. Reverted virus RE-138 was recovered from a single large plaque on Vero cells inoculated with the supernatant of homogenized brains. Rabbit anti-JEV serum was prepared as described previously (Kimura-Kuroda et al., 1993
).
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Construction of full-length cDNA and a point-mutant cDNA of JEV strain AT31.
RNA was extracted from the supernatant of C6/36 cells infected with AT31 by using Isogen-LS (Nippon Gene). Oligonucleotide primers were designed based on sequence data for JEV strain JaOArS982 (GenBank/EMBL/DDBJ accession no. M18370; Sumiyoshi et al., 1987) and the sequences of these primers are listed in Supplementary Table S1 (available in JGV Online). The RNA solution was subjected to reverse transcription (RT) using an antisense primer, JEV-10976R-30, and Moloney murine leukemia virus reverse transcriptase (Superscript II; Invitrogen) at 42 °C for 1 h. Both 5' and 3' fragments of JEV cDNA (nt 16125 and 538310976, respectively) were amplified by using two pairs of primers: SalT7GJE5P20S and JEV-6125R-30 for the 5' fragment or JEV-5383S-30 and ClaKpnJE3P30R for the 3' fragment, with TaKaRa LA Taq polymerase. PCR conditions comprised 30 cycles of denaturing at 95 °C for 10 s and annealing and extension at 68 °C for 6 min. PCR products containing 5' and 3' fragments of JEV cDNA were cloned into pBR322. Three cloned plasmids were selected for further reconstruction of a full-length JEV cDNA: pJEAT-5'-132 contained the 5' end to the SacI site (nt 2215) region; pJEAT-5'-258 contained the SacI site (nt 2215) to BamHI site (nt 5576) region; and pJEAT-3'-75 contained the BamHI site (nt 5576) to 3' end region (see Supplementary Fig. S1, available in JGV Online). For subcloning purposes, the second SacI site in JEV cDNA was abolished by using PCR-based mutagenesis (Kato et al., 2003a
), with an A-to-T mutation at nt 6713 of pJEAT-3'-75, producing pJEAT-3'-75dSac. Each insert from pJEAT-5'-132, pJEAT-5'-258 and pJEAT-3'-75dSac was cloned stably into a very low-copy-number plasmid vector, pMW118 (Nippon Gene), to construct the full-length JEV cDNA and the result was designated pMWJEAT (see Supplementary Fig. S1, available in JGV Online). A single G-to-A point mutation at nt 1389 was also introduced into pMWJEAT by PCR-based mutagenesis and designated pMWJEAT-E138K (Fig. 1a
).
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Plaque-reduction neutralization (PRNT) test.
The PRNT test was done as described previously (Zhao et al., 2003). PRNT titre was expressed as the maximum dilution of antibody yielding a 90 % reduction in viral infectivity (PRNT90).
Mouse experiments.
Groups of 3-week-old female BALB/c mice (n=5) were inoculated intracerebrally with 25 µl of a tenfold serially diluted virus solution (AT31, at222, rAT, rAT-E138K and RE-138). Similar groups of mice were inoculated intraperitoneally with 0·1 ml of a tenfold serially diluted virus solution. Mice were observed for 3 weeks after inoculation. End points of neurovirulence and neuroinvasiveness were identified as both mortality ratios and mean survival times. The LD50 was determined for each virus (Reed & Muench, 1938).
Groups of 3-week-old female BALB/c mice (n=30) were inoculated with 105 p.f.u. rAT in 25 µl or 107 p.f.u. rAT-E138K in 25 µl into the left footpad. At 2-day intervals after inoculation, two mice from each group were bled periorbitally before cardiac perfusion with HBSS solution (Invitrogen). Brains, livers, spleens and kidneys of mice were harvested. Tissue suspensions of 10 % (w/v) were prepared by using PBS, then homogenized immediately. After three cycles of freezing and thawing, these 10 % (w/v) tissue suspensions were centrifuged at 20 000 g at 4 °C for 1 h, then filtered through a 0·2 µm filter. Virus titres in filtered supernatant were determined by plaque assay on Vero cells.
Acid resistance of early viruscell interactions.
Vero cells (1x105) cultured in a six-well tissue-culture plate (Corning) were washed and pre-chilled at 4 °C for 2 h. After inoculation with 400 p.f.u. virus (m.o.i., 0·004 p.f.u. per cell) at 4 °C for 2 h, unbound virus was removed by washing three times with ice-cold MEM supplemented with 2 % FCS (MEM-2). Cells were treated with 2 ml glycine/HCl-buffered saline (8 g NaCl, 0·38 g KCl, 0·10 g MgCl2.6H2O, 0·10 g CaCl2.2H2O and 7·5 g glycine l1, pH adjusted to 3·0 with HCl) at room temperature for 5 min, as described by Hung et al. (1999), then overlaid with 1·25 % methyl cellulose/MEM for incubation at 37 °C for 5 days. Infected cells without acid treatment were used as controls. Penetration rates of acid-resistant intracellular viruses were calculated as 100x [no. plaques (acid-treated)/no. plaques (controls)].
Heparin assay and heparinase-treatment effect on Vero cells.
Inhibition of JEV binding to Vero cells by heparin (Sigma) was performed as described by Hung et al. (1999), with some modifications. The following two conditions were tested: (i) 400 p.f.u. virus plus heparin was pre-incubated at 37 °C for 1 h, then incubated in Vero cells cultured in a six-well plate at 37 °C for 2 h; and (ii) in total, 400 p.f.u. virus was reacted with heparin at 37 °C for 1 h, then inoculated into Vero cells at 4 °C for 2 h. Unbound virus was removed by three washes with MEM-2. Cells were overlaid with 1·25 % methyl cellulose/MEM to incubate at 37 °C for 5 days. Cells inoculated by using the same procedure without heparin were used as controls. Inhibition was described as: 100x [no. plaques (heparin treatment)/no. plaques (controls)].
The effectiveness of heparinase treatment was analysed on Vero cells that had been treated with different concentrations of heparinase I or III (Sigma Aldrich) at 37 °C for 1 h. Cells cultured in a six-well plate were pre-chilled at 4 °C for 1 h after washing and inoculated with 400 p.f.u. per well (m.o.i., 0·004 p.f.u. per cell) of JEV rAT or rAT-E138K at 4 °C for 2 h. After washing in MEM-2, cells were overlaid with methyl cellulose and incubated at 37 °C for 5 days. Cells not treated with heparinase were used as controls. Inhibition was calculated as described above.
Binding inhibition of recombinant JEV with lectins.
The ability of rAT and rAT-E138K to bind to Vero cells was examined by using concanavalin A (ConA; Sigma), wheatgerm agglutinin (WGA; Sigma) and phytohaemagglutinin P (PHA-P; Sigma) as described by Hung et al. (1999). A total of 400 p.f.u. virus was reacted with lectin at 37 °C for 1 h, then inoculated into Vero cells cultured in a six-well plate for 2 h. After washing in MEM-2, cells were overlaid with methyl cellulose and incubated at 37 °C for 5 days. Cells inoculated by using viral solutions without lectin were used as controls. Inhibition was calculated as: 100x [no. plaques (lectin treatment)/no. plaques (controls)].
Endocytosis inhibition.
Inhibition of rAT and rAT-E138K virus internalization into Vero cells was analysed by using different concentrations of chlorpromazine, cytochalasin D or nystatin (all from Sigma). Vero cells cultured in a six-well plate were treated with these reagents at 37 °C for 1·5 h. These cells were subsequently inoculated with 400 p.f.u. JEV at 37 °C for another 3 h in the presence of the reagents, followed by treatment with glycine/HCl-buffered saline at room temperature for 5 min to inactivate unpenetrated viruses on the cell surface. Inoculated cells without any inhibiting agent were used as controls. Inhibition was calculated as: 100x [no. plaques (agent treatment)/no. plaques (controls)].
Immunohistochemistry.
BALB/c mice (3 weeks old; n=5) received inoculation with 10 p.f.u. JEV rAT or 105 p.f.u. rAT-E138K into the right cerebral hemisphere. Virus titres were detected in the right hemispheres of sacrificed moribund mice. Left hemispheres were embedded immediately in Tissue-Tek embedding medium (Sakura Finetechnical) to examine the presence of JEV antigen with rabbit anti-JEV serum by using an immunofluorescence assay (IFA) as described previously (Zhao et al., 2003).
Statistical analysis.
Statistical analysis was performed by using Student's t-test. Survival rates of inoculated mice were analysed by using KaplanMeier methods. Values of P<0·05 were considered statistically significant.
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RESULTS |
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Generation of recombinant JEV on Vero and C6/36 cells
The cDNA-generated recombinant JEV strains rAT and rAT-E138K were obtained from the supernatant of Vero cells transfected with synthetic RNA using the full-length cDNA clone as described in Methods. Viral RNA was extracted from both rAT and rAT-E138K virus solutions and sequences were determined directly after RT-PCR. No mutations were found in the genomic sequences as compared with template plasmid DNA sequences. Parental wild-type AT31 and recombinant rAT viruses displayed similar plaque morphologies at 5 days after infection (diameter 3·1 mm), whereas rAT-E138K virus exhibited a smaller-plaque morphology (diameter
0·45 mm) on Vero cells (Fig. 1b
). Plaques similar to those of rAT-E138K were observed in all supernatant samples following seven passages on C6/36 cells. Reverted RE-138 virus was obtained as described in Methods. RE-138 virus also exhibited larger-size plaques (
3·1 mm) than those of AT31 and rAT viruses, as shown in Fig. 1(b)
.
Neurovirulence and neuroinvasiveness of recombinant JEV
Neurovirulence and neuroinvasiveness of JEV strains were determined by direct intracerebral inoculation or intraperitoneal infection using the tested viruses obtained from the supernatants of infected cells, as described in Methods. The JEV rAT strain displayed strong neurovirulence in 3-week-old BALB/c mice, as did the parent AT31 (2·5 and 2·2 p.f.u./LD50, respectively; Table 1). In contrast, the rAT-E138K strain displayed much lower neurovirulence in mice (15 200 p.f.u./LD50) and the attenuated JEV at222 strain displayed the lowest neurovirulence (148 000 p.f.u./LD50). However, RE-138 virus reverted from the rAT-E138K strain displayed strong neurovirulence (2·2 p.f.u./LD50; Table 1
), comparable to that seen with rAT and AT31 virus. After sequence analysis of RE-138 virus from the supernatant of infected Vero cells, only residue E-138 had been changed from Lys to Glu, compared with the rAT-E138K virus (data not shown). Furthermore, the JEV rAT strain demonstrated strong neuroinvasiveness, comparable to that of the parental AT31 strain (18 800 and 24 200 p.f.u./LD50, respectively). Both rAT-E138K and at222 strains exhibited weak neuroinvasiveness (>107 p.f.u./LD50 for both strains), as all mice inoculated with 107 p.f.u. of either rAT-E138K or at222 survived (Table 1
). These results demonstrate that the Glu-to-Lys mutation at residue E-138 of the JEV AT31 strain strongly attenuates viral neurovirulence and neuroinvasiveness in BALB/c mice.
Detection of JEV antigen in inoculated mouse brain
Positive signals were detected in both rAT- and rAT-E138K-infected mouse brains by IFA, and most positive cells displayed a neuronal morphology (Fig. 1c). The majority of positive signals were distributed in both the cerebral cortex and putaminal areas in brains of rAT-infected mice, whereas most positive cells were present in the frontal area of the cerebral cortex in rAT-E138K-infected mice. No positive signals were identified in the cerebella of either rAT- or rAT-E138K-infected mice (data not shown). These results indicate that the point mutation does not change viral neurotropism in BALB/c mice, although neurovirulence is attenuated.
JEV titre of infected mouse tissues
All mice inoculated with 107 p.f.u. rAT-E138K into the left footpad remained asymptomatic until 14 days post-inoculation, and no virus replication was detected in any tested tissue (Table 2). In contrast, viraemia (163 p.f.u. ml1) was detected in sera of mice inoculated with 105 p.f.u. of the rAT strain via the footpad on day 2 post-inoculation. Detectable levels of replicated rAT virus in brains of inoculated mice were identified at about 1·2x104 p.f.u. per tissue at 4 days post-inoculation, increasing to 8·6x107 p.f.u. per tissue by day 8. No virus was detected in any other tissue (Table 2
).
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Inhibition of viruscell interactions with lectins
Regulation of viral entry by carbohydrates on dengue virus type 2 glycoproteins has been reported (Hung et al., 1999). To explore the effect of the point mutation in JEV glycoproteins on this regulation, various lectins were used to inhibit viruscell interactions. No significant differences in plaque formation were seen following ConA treatment between JEV rAT and rAT-E138K strain infection on Vero cells (Fig. 4
a). However, significant differences in plaque formation were found between rAT and rAT-E138K strains when virus was treated with >5 µg WGA ml1 pre-inoculation (P<0·01 or P<0·001; Fig. 4b
). Finally, PHA-P did not reduce plaque formation in either JEV rAT or rAT-E138K strains at any tested concentration (Fig. 4c
). These results indicate that the point mutation affects inhibition of viruscell interactions with WGA, but exerts less effect with ConA and PHA-P. The higher efficiency of WGA inhibition against the rAT strain than against the rAT-E138K strain suggests that residues of
-N-acetylmuramic acid or
-N-acetylneuraminic acid on rAT virus are more involved in viruscell interactions (Fig. 4b
).
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DISCUSSION |
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By analogy with three-dimensional structure models of TBE virus (Rey et al., 1995; Mandl et al., 2001
) and Dengue virus (Zhang et al., 2003b
), the E glycoprotein of JEV is predicted to contain an extended structure with seven
-sheets, two
-helices and three domains (Kolaskar & Kulkarni-Kale, 1999
). The E glycoprotein is the viral haemagglutinin and induces protective immune responses and mediates receptor-specific viral attachment to cell surfaces. Previous reports have indicated that mutations in the JEV E protein affect viral neurovirulence or neuroinvasiveness in vivo (Yu et al., 1981
; Ni & Barrett, 1998
) or viral binding and entry into cultured cells in vitro (Hasegawa et al., 1992
; Lee & Lobigs, 2002
). The molecular basis of viral attenuation in flaviviruses has been analysed by using site-directed mutagenesis of infectious cDNA clones (Kinney et al., 1997
; Hurrelbrink et al., 1999
), production of chimeric viruses (Arroyo et al., 2001
) and deletions in the 3' untranslated regions of genomes (Whitehead et al., 2003
). Residue 138 of the E protein has been considered to be located on the E0
-strand in domain I and to be exposed on the surface of the E protein (Lee et al., 2004
). The data described in the present manuscript demonstrate that mutation of the E-138 residue in JEV from Glu to Lys inhibits viral spread from cell to cell, explaining the small-plaque morphology. Previous studies have indicated that mutations in domain III of the flavivirus E protein modulate virus binding and entry into host cells (Hung et al., 2004
; Liu et al., 2004
; Modis et al., 2004
). The dimer-to-trimer structural transition of the E protein induced by low-pH conditions is considered crucial for viruscell binding and entry processes (Stiasny et al., 1996
; Chen et al., 1997
; Arroyo et al., 2001
) and this binding may be affected by mutations in the E protein. The E-138 mutation is not located in domain III, but enhances viruscell attachment greatly under acidic conditions (Fig. 2
).
Flaviviruses are considered to be internalized after binding to glycosaminoglycan (GAG) residues on cells, and other molecules are also involved in virus entry for some viruses (Mandl et al., 2001; Lee et al., 2004
; Liu et al., 2004
). The binding reaction can be inhibited in vitro by using heparin (Chen et al., 1997
; Hurrelbrink & McMinn, 2001
; Lee & Lobigs, 2002
). However, the molecular effects of heparin on virus-binding activity with JEV attenuation are not well understood. Previous studies have indicated that mutations of residues E-49, E-138, E-306 or E-389 on the JEV E protein reduce the efficiency of viral binding to heparan sulphate residues on target cells (Lee et al., 2004
; Liu et al., 2004
). Furthermore, cell-adapted TBE virus infection of BHK-21 cells is inhibited by GAGs, whereas this is not the case for the parental virus (Mandl et al., 2001
). Mutation at residue E-138 in both JEV and West Nile virus shows clearly that this residue is a strong determinant of viral binding to GAG residues on target cells (see Supplementary Table S2, available in JGV Online). In our study, binding by rAT-E138K was inhibited significantly by heparin, whereas the rAT strain was unaffected. Cells digested with heparinase III displayed specifically reduced binding activity to rAT-E138K (Fig. 3
). This indicates that rAT-E138K virus binding to cell surfaces is more dependent on heparan sulphate B residues than rAT virus. The better interaction of heparin with rAT-E138K virus in comparison with rAT virus is explained by the fact that heparin is negatively charged and the substitution of negatively charged Glu for positively charged Lys provides a more highly charge-mediated interaction, based on the consideration that residue 138 of the E protein is exposed on the surface (Lee et al., 2004
); alternatively, this mutation may expose other positively charged residues on the surface of the virion. Residue E-138 is negatively charged and conserved among the JEV serocomplex, but not among other flaviviruses (See Supplementary Fig. S2, available in JGV Online). Furthermore, a Glu-to-Lys mutation was found not only in JEV, but also in West Nile virus. Taken together, the E-138 residue is suggested to be an important determinant of GAG binding and virulence of the JEV serocomplex.
Flaviviruses generally bud into the lumen of the endoplasmic reticulum in infected cells and are subsequently secreted through the vesicle-transport pathway. A recent study has indicated that a single Pro-to-Ser point mutation at position 63 in the prM protein of TBE virus may induce a reduction in virus-particle secretion from RNA-transfected BHK-21 cells (Yoshii et al., 2004). Interestingly, the Glu-to-Lys mutation at E-138 also affects the efficiency of viral release from infected cells. Changes in not only infection efficiency, but also viral secretion efficiency, may play important roles in virus attenuation. Some studies have reported that about 180 glycoprotein E monomers are present on the surface of a mature flavivirus particle with the (pre)ME heterodimer (Lescar et al., 2001
; Kuhn et al., 2002
; Zhang et al., 2003a
, b
). The Glu-to-Lys mutation changes the charge of the side chain from negative to positive. About 180 positively charged side chains at E-138 residues would change to 180 negative charges in each rAT-E138K virus particle. Clearly, this would affect both the binding of rAT-E138K virus to target cells and intracellular transport of viral particles. Furthermore, the E-138 residue exists near the hinge region between domains I and II, and the three-dimensional structure of the E protein might therefore change due to mutation of this residue. Taken together, these mechanisms may attenuate JEV virulence cooperatively in vivo, but further study is needed to clarify this point.
Neuroinvasiveness of both at222 and rAT-E138K viruses was attenuated (Table 1). The bloodbrain barrier (BBB) can inhibit viral invasion into mouse brains, and both rAT-E138K and at222 viruses proved unable to enter the brain. A recent publication demonstrated that West Nile virus replication in peripheral tissues triggers a Toll-like receptor inflammatory response that alters the permeability of the BBB (Wang et al., 2004
). Increased BBB permeability allows West Nile virus to cross the BBB. West Nile virus is a flavivirus that is genetically related very closely to JEV. Similar mechanisms might thus be important for the neuroinvasiveness of JEV. The data in Table 2
demonstrate clearly that rAT-E138K virus does not cause viraemia and, thus, inflammatory responses might be insufficient to replace BBB permeability. This issue warrants further analysis in future.
Previous investigators of the JEV SA14-14-2 strain have demonstrated that change of residue E-279 from methionine to lysine in domain II affects viral neurovirulence significantly (Monath et al., 2002). Our studies indicate that residue E-138, which is located at the link region between domains I and II, is an important determinant of neurovirulence and neuroinvasivenes in JEV. However, the at222 virus contains additional mutations besides E-138 and demonstrated characteristics of further attenuation, compared with rAT-E138K with the single mutation at E-138. The function of point mutations at other residues in the JEV E protein should thus be examined by using full-length cDNA clones in future studies.
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ACKNOWLEDGEMENTS |
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Received 25 September 2004;
accepted 21 April 2005.
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