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1 Université d'Auvergne, Laboratoire de Virologie-EA3843, UFR Médecine, 28 place Henri-Dunant, F-63001 Clermont-Ferrand, France
2 CHU Clermont-Ferrand, Laboratoire de Virologie, Centre de Biologie, F-63003 Clermont-Ferrand, France
Correspondence
Jean-Luc Bailly
j-luc.bailly{at}u-clermont1.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 133 sequences determined in this work are AM236601–AM236624, AM236921–AM236924, AM236927, AM236991–AM237042, AM237043–AM237081 and AM237316–AM237328.
Supplementary tables showing echovirus 30 strains analysed in this study and parameters determined for the rate matrix of the GTR model, and a supplementary figure showing a schematic representation of the enterovirus genome and primers used for RT-PCR amplification and sequencing, are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HEVs are the pathogen associated most commonly with acute meningitis worldwide and can cause sporadic cases, outbreaks and epidemics (Pallansch & Roos, 2001).
Echovirus 30 (E-30) was one of the most commonly isolated enterovirus serotypes in the multiple meningitis outbreaks that have been reported in the last 15 years (Bailly et al., 2002; Palacios et al., 2002; Wang et al., 2002; Chomel et al., 2003; Ozkaya et al., 2003; Antona et al., 2005; Zhao et al., 2005). In the USA, E-30 became a primary cause of meningitis outbreaks in 2003 and 2004 (CDC, 2003, 2006). The virus was recovered as the main serotype in some outbreaks (Yoshida et al., 1999; Henquell et al., 2001) but, in most instances, meningitis was associated with other serotypes (Chambon et al., 2001; Thoelen et al., 2003; Bernit et al., 2004). E-30 has also been associated with meningitis outbreaks caused by contaminated waters (Amvrosieva et al., 2001; Hauri et al., 2005), with nosocomial infections and with other diseases (Bailly et al., 2000a, b; Rabaud et al., 2002; Welch et al., 2003; Cabrera-Rode et al., 2005). Since the primary isolation of the virus in 1958, E-30 epidemiology has been characterized by sequential displacements among multiple genetic variants. Different variants have been detected and associated with local meningitis outbreaks, but a given variant can also be associated with several outbreaks in distant geographical areas (Oberste et al., 1999; Savolainen et al., 2001).
Molecular mechanisms of picornavirus variation and evolution result from point mutations and genomic rearrangements, in particular recombination (Agol, 1997; Agol et al., 1999). Although most attention has been directed towards mutation, recent studies have indicated that recombination might have an important role in enteroviruses (Santti et al., 1999; Guillot et al., 2000; Dahourou et al., 2002; Chan & AbuBakar, 2004; Arita et al., 2005). Putative recombinant genomes were detected through discordance of phylogenetic relationships for different parts of the genome in epidemiological studies of CA-9 and CB-4 (Mulders et al., 2000; Santti et al., 2000; Lindberg et al., 2003) and in investigations of epidemic or unrelated circulating strains from various geographical origins (Oprisan et al., 2002;Lukashev et al., 2003, 2004, 2005; Chevaliez et al., 2004). Despite the fact that close phylogenetic relationships have been reported between strains of different HEV-B serotypes, including E-30, the role of recombination in E-30 evolution was not investigated specifically in any of the above studies.
In a previous report, we detected different virus variants in patients during a local outbreak of E-30 meningitis in 2000 (Bailly et al., 2002). The virus sequences detected in patients were divided temporally into two main phylogenetic clusters that were not similar to those involved in a previous outbreak in 1997. The present study was carried out with a comprehensive set of E-30 strains isolated between 1991 and 2005 to investigate the genetic processes involved in the emergence of the different lineages. We showed that genetic recombination was involved in a time-correlated manner in their emergence and that drift occurred in all lineages, quasi-exclusively by synonymous nucleotide substitutions, indicating strong constraints against amino acid changes in both structural and non-structural genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Henquell et al., 2001; Peigue-Lafeuille et al., 2002; Archimbaud et al., 2004). Two E-30 strains (SE86788-98 and SE87177-98) isolated in patients admitted to the hospital of Saint-Etienne (France), some 150 km away, were included in the study. Three additional strains (CF1074-78, CF1260-78 and CF298-91) isolated between 1978 and 1981 were used as an outgroup of older viruses. Throughout the text, strain designation is given in the following format: CF or SE (for Clermont-Ferrand or Saint-Etienne), number of the strain, year of isolation.
All virus strains were isolated in MRC5 (human lung embryonic fibroblast; bioMérieux) cell cultures and were typed by seroneutralization tests with World Health Organization antiserum pools A–H according to standard procedures (Chambon et al., 2001).
Nucleic acid isolation, cDNA synthesis and gene amplification.
Virus RNA was extracted from 100 µl infected cell-culture supernatant by using a QIAamp Viral RNA kit (Qiagen) and synthesis of the cDNAs was carried out with the ThermoScript RT-PCR system (Life Technologies) as described earlier (Bailly et al., 2002). Three genome fragments (designated A, B and C) were obtained by RT-PCR [see Supplementary Fig. S1(a), available in JGV Online]. The locations and sizes of the corresponding PCR products and primers used in the amplification reactions are described in Supplementary Fig. S1. All PCRs were performed by using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) with 2–5 µl cDNA, as reported elsewhere (Bailly et al., 2002). Thermal cycling comprised 43–45 cycles as follows: one cycle of denaturation for 2 min at 94 °C, 41–43 cycles of denaturation for 15 s at 94 °C, annealing for 20 s at 50, 55 or 58 °C respectively for the A, B and C fragments and elongation for 50 s at 72 °C, and a last cycle of 10 min at 72 °C. The amplification of the 3CD segment in strains CF783-94, CF1845-94, CF51-96, CF746-98 and SE87177-98 was obtained with primers E30S3C2 and 3NCASC instead of primers E30S3C2 and E30R3D1. A long fragment [PCR product D in Supplementary Fig. S1(a); length, 4256 bp] was obtained with the primers E30SVP1 and 3NCASC for 13 representative strains. The following amplification conditions were used: one cycle of 2 min at 94 °C, 43 cycles consisting of 15 s at 94 °C, 20 s at 58.5 °C and 3.5 min at 68 °C, and a last cycle of 10 min at 72 °C.
Nucleotide sequencing of PCR products.
The PCR products obtained were purified with a MinElute gel extraction kit (Qiagen). Forward and reverse sequencing reactions were carried out with the oligonucleotides used in gene-amplification reactions, using a Big Dye Terminator Cycle Sequencing kit, version 1.1 (Applied Biosystems). The 4256 bp long PCR products were sequenced with oligonucleotides SEQE30SVP1, 3NCASC and with specific primers along the genome. After a purification step with a DyeEx 2.0 kit (Qiagen), the sequencing products were analysed on an ABI 310 Genetic Analyzer (Applied Biosystems).
Phylogenetic clustering of E-30 nucleotide sequences.
Multiple sequence alignments corresponding to partial 5' untranslated region (UTR), the 5' end of the VP0 sequence, the complete VP1 and partial 3CD sequences were constructed to determine the phylogenetic clustering among the isolates. Sequences retrieved from international databases were also used to compare the E-30 isolates with other enteroviruses. The datasets were constructed and edited by two computer programs, CLUSTAL_W (Thompson et al., 1994) and GENEDOC (available at http://www.psc.edu/biomed/genedoc/). Two different methods were employed (Bailly et al., 2000b, 2004) for phylogenetic inference: the neighbour-joining and maximum-likelihood (ML) methods implemented in the MEGA (http://www.megasoftware.net) and TREE-PUZZLE (http://www.tree-puzzle.de/) programs, respectively. Reliability of the phylogenetic topologies was determined by the bootstrap-resampling test with 1000 replicates (Felsenstein, 1985). The phylogenetic trees were rooted by the outgroup method and the sequences included in the outgroup for the different datasets were selected on the basis of their genetic relatedness with the ingroup (E-30 sequences).
Analysis of subgenomic segments in 13 representative isolates.
Subgenomic segments 4878 nt in length, covering the eight genes that encode the VP1, 2Apro, 2B, 2C, 3A, 3B, 3Cpro and 3Dpol virus proteins, were assembled from the B and D PCR products [see Supplementary Fig. S1(a), available in JGV Online] obtained for 13 virus isolates that were representative of phylogenetic clusters. Similarity and bootscanning analyses along the subgenomic segments were performed with SimPlot software (3.2 beta version), using a sliding window of 400 nt moving in steps of 20 nt (Salminen et al., 1995).
Potential recombination breakpoints determined by the scanning analysis were investigated further by phylogenetic analysis. The 13 subgenomic sequences were aligned with outgroup sequences and the resulting alignment was split into five datasets that corresponded to virus genes 1D, 2A, 2BC, 3AB and 3CD. Each dataset was investigated to determine the parameter values for the best-fitting nucleotide-substitution model (see Supplementary Table S2, available in JGV Online), chosen according to the Akaike information criterion (AIC) among 21 models by the FINDMODEL web application at http://hiv-web.lanl.gov/ (Posada & Buckley, 2004). For all datasets, the AIC-selected model was the general time-reversible (GTR) model with four categories in the gamma distribution of rate variation among nucleotide sites (GTR 
4). Each dataset was then subjected to phylogenetic analysis with the ML method implemented in the TREE-PUZZLE program, using the parameters determined for the rate matrix of GTR 4 (base composition, parameter alpha of gamma distribution of among-site rate variation, rate parameters for the substitution matrix). Topologies in the different trees obtained were evaluated by using 1000 bootstrap replicates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The E-30 5' UTR sequences formed a monophyletic cluster that excluded any other EV sequence and that was divided further into two groups supported by high bootstrap values (PB, 99 %). One group comprised sequences of the most recent viruses isolated during the second part of the 2000 meningitis outbreak, the 2005 outbreak and in sporadic cases (2001, 2002). This group was designated C4 according to the nomenclature adopted earlier (Bailly et al., 2002). The other group included the sequences obtained in isolates recovered between 1991 and 2001 and comprised four major clusters (C0–C3). The genetic groupings investigated with the partial VP0 sequences resulted in a similar tree topology (data not shown).
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, b, 2002). Phylogenetic analysis showed five distinct clusters similar to those observed with the sequences located in the 5' end of the genome (Fig. 1b
). Nucleotide sequences differed by 0.5–2.9 % within a given cluster and by 4.7–9.8 % between clusters, and all of the isolates shared similar amino acid sequences (overall amino acid variation, 1.4 %). The C1 cluster comprised sequences of viruses isolated between 1994 and 2001 and was related directly to the C2 cluster (PB, 98 %), which included sequences corresponding to the 1997 outbreak. The C3 cluster grouped viruses of the 1996–2000 period but, in contrast to the 5' UTR tree topology, it was related directly to the C4 cluster (PB, 94 %), which included 17 sequences of recent viruses. The viruses isolated in the early 1990s were clearly differentiated from all viruses recovered later and the cluster was designated C0. Within this cluster, the CF670-91 sequence fell close to the node linking the five lineages, with little nucleotide variation from the putative ancestor sequence of the five lineages. High genetic diversity was observed within the C1 cluster and the branching orders between the sequences were poorly supported. Some sequences (CF51-96, CF746-98 and CF1495-00) within this cluster may represent distinct lineages. Similar observations were made with the C4 cluster. For instance, the sequences of the five 2000 viruses had shorter branches than those determined in the 2005 virus isolates and so the question arises of whether these isolates belong to C4 or a new lineage. Overall, the results were in accordance with earlier findings, which indicated that the E-30 isolates recovered during the 1997–2002 period formed clades defined primarily by the time period of isolation, with some overlap.
Analysis of the 3CD segment
Analysis of the 3CD dataset indicated that the overall amino acid variation observed in the 3CD sequences was twice (3.6 %) that determined with the VP1 sequences, and the 3CD tree displayed a marked star-like shape (Fig. 1c), i.e. few E-30 lineages were related to other lineages with high bootstrap support. The genetic clusterings determined with the 3CD sequences were clearly different from those determined with the VP1 sequences. All but two of the clusters (C2 and C3) identified with the VP1 sequences split into two or three subgroups, resulting in a total of 10 lineages with the 3CD sequences. In the 3CD tree topology, the clusterings of the E-30 sequences were also clearly correlated with time of virus isolation, as were groupings observed in the other topologies. The C0 sequences segregated into two lineages (designated C0a and C0b), whilst the C1 sequences split into three, including the C1c lineage, which was related only distantly to the C1a and C1b lineages. Whereas the C2 and C3 viruses remained closely related in both the VP1 and 3CD tree topologies, the viruses in the C4 cluster split into three lineages when the 3CD sequences were compared. The C4a lineage grouped together sequences of viruses collected in 2000 and 2001 (PB, 100 %), the C4b lineage included the sequences of isolates recovered in 2002 and 2005, and the C4c lineage comprised those of three other 2005 viruses. It was interesting to note that the outgroup virus CF298-81 was related closely to the C0a viruses, whilst the other two outgroup isolates formed two distinct lineages. Finally, a phylogenetic analysis was also performed including 38 sequences representing recent or circulating HEV-B viruses for which the full-length genome sequences were also available. The resulting phylogenetic tree displayed a pronounced star-like shape with multiple supplementary clades (data not shown). Despite the fact that none of the 10 E-30 lineages showed close genetic relatedness with the new clades, which could have indicated a common origin, the analysis suggested the difficulty of making an assessment of the complete set of 3CD clades within the HEV-B species.
Overall, clustering of the E-30 isolates was ordered temporally with the four genomic segments analysed and, interestingly, subclustering determined in the 3CD topology was also apparent in the VP1 topology. Most of the E-30 lineages included viruses recovered during short periods of 1–5 years, with the exception of cluster C1, which included viruses collected over 8 years.
Analysis of a 4878 nt long genomic segment in 13 viruses representing 10 lineages
To investigate whether recombination might have produced the VP1 and 3CD tree topologies in E-30, a subgenomic segment was analysed in 13 viruses that represented 10 lineages observed in the 3CD phylogeny (Fig. 1c). In the similarity analyses performed along an alignment of all of the sequences, a specific pattern was obtained when each sequence was compared with the others. Only seven sample patterns are presented in Fig. 2. Most of the similarity plots displayed a biphasic profile and the main differences between the plots were the different locations of the breakpoints between a high-nucleotide-similarity segment (90–98 %) and a segment of lower similarity (70–84 %). In most instances, the breakpoints fell within the 2A, 2B and 2C genes. The other distinguishing feature in some similarity plots was the observation of high similarity values within the 3C and 3D genes (Fig. 2b, d–g).
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), were located by the bootscanning method and results are summarized in Fig. 3. The bootscanning plots for the CF298-81 strain, representing an old clade, and strains representing the C0a, C0b, C1a, C2 and C4b lineages exhibited a typical mosaic pattern, i.e. across the genome of a strain in a given lineage, the presence of successive segments that can cluster with significant bootstrap values (>90 %) with homologous segments of other strains from different genetic origins. For instance, from Fig. 3(c), it was clear that the C0b strain was monophyletic with CF298-81 in the genomic segment comprising phylogenetically informative sites 1386–2057, and with the C3 strain in the segment including sites 3250–3609. Bootscanning plots also clearly showed the boundaries of the putative recombinant pieces in a number of other lineages (Fig. 3a, b, d, e, g; Table 1). However, with the exception of the genomic segments indicated in Table 1, the exact genetic origins of other parts within the genomes remained undetermined for the 1981, C0a, C0b, C2 and C4b lineages because there was no apparent parental lineage represented in the dataset studied, i.e. no known lineage that was more closely related phylogenetically to the strains over these segments. The bootscanning plot of the C1c lineage (Fig. 3e) was given as an example of the plots determined for the C1b, C3, C4a and C4c lineages (data not shown), which displayed multiple short segments with bootstrap values of between 70 and 90 %. In addition, these segments were interspersed with stretches of unknown phylogenetic origin. Hence, without a parental clade available, the C1b, C1c, C3, C4a and C4c lineages did not show close genetic affinity with any other lineages and no clear-cut indication about their evolutionary origins was obtained from the bootscanning analysis. Overall, these observations indicated a mosaic structure of genomes in the above lineages – different portions along the genome within a given lineage may exhibit different genetic origins.
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). The ML trees revealed clear, discordant phylogenetic clusterings for the C1b, C1c, C3, C4a and C4c lineages, supporting the hypothesis of their putative recombinant origin. For instance, the C1b and C1c lineages were related closely in the 1D and 2A genes, but exhibited different origins in the other genomic regions. Within the C4 clade, the C4a and C4c lineages displayed major changes in their phylogenetic clustering patterns when the different tree topologies were considered. In the VP1 and 2A trees, all of the C4 sequences clustered in a monophyletic group, but in the 2A tree, they grouped at the tip of a distinct, long and deeply rooted branch. In the 2BC genes, the C4c lineage underwent a major splitting process from the other C4 lineages, but it was again associated with the C4b lineage in the 3AB genes. The above results showed a number of significant differences in branch order, which further substantiated the hypothesis that serial recombination events occurred during the intricate transmissions of these lineages. However, the borders of the recombinant segments could not be identified.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In the present study, we examined the viruses recovered over an extended time period and characterized one additional variant, i.e. viruses recovered in the early 1990s. Moreover, although the isolates collected in 2005 grouped within the previously described C4 cluster, they formed a separate subcluster; this indicates significant evolution in the most recent viruses, from a common ancestor. Overall, five E-30 variants were detected in only 15 years, which raises the question of the evolutionary processes associated with their emergence. From an epidemiological point of view, two processes may be responsible for the presence of several virus variants of a given enterovirus serotype in a given geographical area: periodic introduction of new viruses or genetic evolution of endemic virus populations. Earlier, we found evidence that the local C3 viruses had close relatedness with viruses first observed in the Netherlands in 1994 (Savolainen et al., 2001; Bailly et al., 2002). The present study also shows phylogenetic relationships that suggest recent emergence processes, whether or not they took place locally. In particular, clusterings determined with the sequences in the 5' part of the genome show clearly that the C4 virus populations are derived from C3 viruses. In fact, C4 viruses were present as early as April 2000 in north-western France, 3 months before they were detected locally (J.-L. Bailly, unpublished results). It should also be noted that viruses related to lineages other than C4 were not observed locally in 2005, indicating that E-30 lineages detected earlier in the 1990s may be extinct. The number of distinct lineages and their succession over 15 years show that frequent emergence of new lineages, selection of short-lived variants and rapid spread are major evolutionary factors in E-30.
Genetic processes involved in the multiplicity of E-30 lineages
The congruence between phylogenetic groupings is almost complete between three segments located in the 5' third of the virus genome, which suggests that the 5' UTR, partial VP0 and complete VP1 sequences evolved only by fixation of single mutations and that no recombination event occurred in this part of the genome. This is in agreement with earlier findings in other enterovirus serotypes, for instance coxsackieviruses A9 and B4 (Mulders et al., 2000; Santti et al., 2000), and provides further evidence that only rare recombination events can occur in the genome region that encodes the capsid proteins in enteroviruses (Blomqvist et al., 2003). More specifically, our observations also mean that nucleotide changes associated with lineage differentiation are fixed by genetic drift in the 5' part of the E-30 genome and, as anticipated (Bailly et al., 2002), that fixation of non-synonymous substitutions accounts for little evolution of the coding sequences.
Compared with the phylogenetic links determined with the VP1 sequence, the E-30 isolates exhibited very different relationships with the 3CD sequence. Discrepancy between the phylogenetic groupings determined with both sequences substantiated different modes of evolution in the two parts of the virus genome, which was suggested earlier by the study of prototype strains (Santti et al., 1999; Oberste et al., 2004a) and circulating strains (Lukashev et al., 2005). The high number of lineages and the increased genetic distances observed with the 3CD sequence indicate a distinctly greater diversity in this sequence, which encodes non-structural proteins. This finding is consistent with previous observations in other HEV-B serotypes (Santti et al., 2000; Lindberg et al., 2003; Lukashev et al., 2003; Oberste et al., 2004b). Comparative analysis of 13 subcomplete genomes representing 10 lineages showed discrete breakpoints in nucleotide similarity between lineages, and bootscanning patterns were compatible with the existence of mosaic genomes. Although they were more frequent within the 2C gene, the breakpoints in the similarity profiles were also detected in the 2A and 2B virus genes. They may be ascribed to the occurrence of genetic recombination events involving genome portions of different length at multiple points in the history of lineages.
To what extent do the variations observed in different genomic regions interlace to produce new lineages?
Strict temporal phylogenetic groupings were determined with all of the analysed sequences. This indicates that changes observed in the 5' and 3' parts of the genome are related closely to the period of virus circulation. The five E-30 lineages were derived from a common ancestor related to the C0 viruses and their emergence is connected closely with genetic recombination. It is plausible that the emergence of the common ancestor itself results from a recombination event, as the genomic 3' part in the closely related C0a sequence was also observed in an outgroup isolate recovered in 1981. In the evolutionary process from the common ancestor, two virus branches evolved after different recombination events: one led to C1-like viruses and the other to C3-like viruses. According to the VP1 ML tree and bootscanning analysis, the C1 (particularly C1a) and C2 viruses evolved from a common putative ancestor virus and their evolutionary pathways are associated with different crossover sites. In the 2A phylogenetic tree, the location of the C4 viruses on a long branch, which clearly distinguishes these viruses from the others, may be evidence of a past recombination event or result from fixation of mutations caused by a high substitution rate and long evolutionary time. The 5' boundary of a similarity breakpoint site was observed clearly close to the VP1/2A junction in all C4 viruses. All of these observations are in favour of genetic recombination, rather than evolution through mutation fixation.
Overall, the close relatedness between the virus isolates allowed the accurate mapping of crossover sites for some lineages (C0a, C0b, C1a, C2 and C4b), which showed the involvement of recombination in their emergence pathway, something not observed before in a non-polio enterovirus. In other lineages (C1b, C1c, C3, C4a and C4c), the identities of parental sequences integrated within the E-30 lineages are not known and mapping of recombinant genomic pieces was not possible by the bootscanning method. Bootscanning plots determined for these lineages (particularly C1c and C4a) corresponded to highly divergent genomes that putatively resulted from a complex succession of recombination events during their history.
Evidence of genetic recombination was obtained in many enteroviruses, including the prototype strains (Santti et al., 1999; Oberste et al., 2004a) and recent clinical isolates (Oprisan et al., 2002; Lindberg et al., 2003; Chevaliez et al., 2004; Lukashev et al., 2004, 2005; Oberste et al., 2004b). In a recent study, mosaic genomes were described in type 1 polioviruses that circulated in China in the 1990s, indicating frequent recombination events (Liu et al., 2003). All of the E-30 lineages described in this study are virtually of recombinant origin, which suggests the huge involvement of genetic recombination in the evolutionary process of E-30. The main findings to emerge from the study of this serotype – mosaic structure of E-30 genomes and rapid turnover of recombinant viruses – are likely to be valid in a number of other HEV-B serotypes whose genetic diversity is similar to that of E-30, e.g. C-A9 (Santti et al., 2000), C-B4 (Mulders et al., 2000; Lindberg et al., 2003), E-11 (Oberste et al., 2003) and E-13 (Archimbaud et al., 2003; Avellón et al., 2003; Kaida et al., 2004; Mullins et al., 2004). Finally, the time-correlated recombination frequencies between the VP1 and 3CD sequences in the E-30 genomes are in agreement with the results of a recent investigation of the dynamics of recombination in different serotypes within the HEV-A and -B species (Simmonds & Welch, 2006) and support the hypothesis of the involvement of genetic recombination, rather than mutation, as the main driving force in the emergence process of new circulating enterovirus variants.
Although intraspecific recombination appears widespread in E-30, more precise estimates of recombination rates are required to achieve a complete understanding of its role in the evolution, epidemiology and pathogenicity of the virus. The biological significance of genetic recombination and, more specifically, the relative importance of recombination versus the accumulation of point mutations for evolutionary change are still open questions in enteroviruses. In this respect, rapid lineage extinction indicates that stochastic processes also play a role in shaping virus genetic diversity, for instance through random expansion of a given lineage during an outbreak.
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ACKNOWLEDGEMENTS |
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Received 18 April 2006;
accepted 20 September 2006.
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