Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes)

A. R. Samuel1 and N. J. Knowles1

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK1

Author for correspondence: Alan Samuel. Fax +44 1483 232448. e-mail alan.samuel{at}bbsrc.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results and Discussion
References
 
Serotype O is the most prevalent of the seven serotypes of foot-and-mouth disease (FMD) virus and occurs in many parts of the world. The UPGMA method was used to construct a phylogenetic tree based on nucleotide sequences at the 3' end of the VP1 gene from 105 FMD type O viruses obtained from samples submitted to the OIE/FAO World Reference Laboratory for FMD. This analysis identified eight major genotypes when a value of 15% nucleotide difference was used as a cut-off. The validity of these groupings was tested on the complete VP1 gene sequences of 23 of these viruses by bootstrap resampling and construction of a neighbour-joining tree. These eight genetic lineages fell within geographical boundaries and we have used the term topotype to describe them. Using a large sequence database, the distribution of viruses belonging to each of the eight topotypes has been determined. These phylogenetically based epidemiological studies have also been used to identify viruses that have transgressed their normal ecological niches. Despite the high rate of mutation during replication of the FMD virus genome, the topotypes appear to represent evolutionary cul-de-sacs.


   Introduction
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Foot-and-mouth disease (FMD) virus (family Picornaviridae, genus Aphthovirus) is a small RNA virus that causes a highly infectious disease affecting up to 70 species of cloven-hoofed mammals (Hedger, 1981 ). In endemically infected countries FMD causes loss of productivity in adult animals and high mortality in young stock. Countries normally free of the disease suffer trade restrictions and severe economic consequences when outbreaks occur.

FMD has occurred in most areas of the world except Greenland, Iceland, New Zealand and the smaller islands of Oceania. Australia has not experienced an outbreak since 1870; the United States of America since 1929; Canada since 1952 and Mexico since 1954. Europe has experienced a number of sporadic outbreaks since the cessation of vaccination on that continent during 1990–91. These outbreaks occurred in Bulgaria (1991, 1993 and 1996), Italy (1993), Greece (1994, 1996 and 2000), Turkish Thrace (1995 and 1996), Albania (1996) and the former Yugoslav Republic of Macedonia (1996).

There are seven immunologically distinct serotypes of FMDV (O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3) and antigenic variation within a serotype can be such that vaccines must be carefully matched to outbreak strains to ensure efficacy. Historically, antigenic relationships between FMDV isolates were determined by serological tests and used to assess which vaccine strains were most appropriate to control outbreaks. Biochemical techniques such as SDS–PAGE analysis of structural proteins and RNase T1 oligonucleotide mapping of the genome have also been used to determine the relationships between isolates of FMDV for epidemiological purposes (King et al., 1981 ; Knowles & Hedger, 1985 ). However, the determination of FMDV nucleotide sequences and phylogenetic analysis is more precise and is now the definitive technique for characterizing individual strains of virus (Knowles & Samuel, 1995 ).

Nucleotide sequencing was first used for the study of the epidemiology of FMD by Beck & Strohmaier (1987) who investigated the origin of outbreaks of types O and A in Europe over a 20 year period. Since then a number of similar studies have been published; serotype O (Knowles et al., 1988 ; Marquardt & Adam, 1990 ; Samuel et al., 1990 , 1993 , 1997 , 1999 ; Krebs et al., 1991b ; Armstrong et al., 1992 ; Marquardt & Krebs, 1992 ; Saiz et al., 1993 ; Stram et al., 1995 ; Singh et al., 1996 ), serotype A (Armstrong et al., 1994 ; Weddell et al., 1985 ; Marquardt & Adam, 1988 ; Samuel et al., 1988 ; Carrillo et al., 1990 ), serotype C (Martínez et al., 1988 ; Piccone et al., 1988 ; Sobrino et al., 1989 ; Knowles & Samuel, 1990 ; Martínez et al., 1992 ), serotype Asia 1 (Ansell et al., 1994 ; Woodbury et al., 1994 ) and SAT 2 (Vosloo et al., 1992 ; Thevasagayam, 1996 ).

Understanding the epidemiology of a disease is essential for the formulation of the most effective control strategies. Determining the source of outbreaks is an important element of epidemiological investigations and for FMD this can be done, as mentioned previously, by nucleotide sequencing. Towards that objective we have established a database of FMDV sequences from isolates collected worldwide by the OIE/FAO World Reference Laboratory for FMD (WRLFMD) since 1924. This includes not only isolates sequenced at Pirbright but also those that have either been published or have been obtained in collaboration with other researchers in the field. Laboratories contributing include: the All-Russian Research Institute for Animal Health (Vladimir, Russian Federation), Indian Veterinary Research Institute (Mukteswar, India), Foot-and-Mouth Disease Centre (Pakchong, Thailand), Onderstepoort Veterinary Institute (Onderstepoort, South Africa), Pan-American Foot-and-Mouth Disease Center (Rio de Janeiro, Brazil), Instituto Nacional de Technologia Agropecuaria (Buenos Aires, Argentina) and Lanzhou Veterinary Research Institute (Lanzhou, People’s Republic of China). The database currently contains approximately 2000 sequences, representing the seven serotypes of FMDV. This includes over 1000 complete or partial VP1 sequences of isolates of serotype O.

Apart from the study of Asia 1 by Ansell et al. (1994) many of the cited molecular epidemiological studies of FMDV have been restricted either geographically or temporally. Samuel et al. (1997) described FMD outbreaks in Saudi Arabia over a 12 year period and concluded that the existence of multiple genetic groups was most likely caused by repeated introductions from neighbouring countries. Additionally, Samuel et al. (1999) described an epidemic of type O FMDV caused by a sublineage that, although present in the Middle East in the late 1980s, suddenly caused extensive outbreaks in this region and spread westwards across North Africa in the early 1990s. This paper is based on an analysis of selected FMDV isolates of serotype O received at the WRLFMD, Pirbright, UK from many different geographical regions. The aim of the study was to determine the extent of genetic diversity within FMDV type O, on a survey of global isolates of the virus. The results presented are the most comprehensive analysis of FMDV serotype O that has been carried out using phylogenetic techniques.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Viruses and cells.
All the virus isolates (Table 1) were obtained from the WRLFMD strain collection either as 10% epithelial suspensions or as cell culture passaged material. They were either used directly or passaged through IB-RS-2 cell monolayers (De Castro, 1964 ) prior to RNA extraction. The nomenclature of the virus isolates is a three-letter code for the country of origin followed by the number of the sample submitted to the WRLFMD and the year of isolation (e.g. SAU/1/88; the first sample received from Saudi Arabia in 1988).


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Table 1. Origin of FMD type O virus isolates and nucleotide sequences examined in this study

 
{blacksquare} Oligonucleotide primers.
Oligonucleotide primers with a Cy5 amidite fluorescent dye for use with the ALFexpress automated sequencer were purchased from Pharmacia Biotech. The sequences of the primers used in this study are detailed in Table 2.


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Table 2. Oligonucleotide primers used for RT–PCR and sequencing of the FMD type O viruses examined

 
Sequences prior to 1993 were obtained by direct sequencing from vRNA templates (Knowles, 1990 ) and more recently by RT–PCR and cycle sequencing (Knowles & Samuel, 1995 ), which is the technique routinely used in the WRLFMD.

{blacksquare} RT–PCR.
This was done using the primer sets L463F/NK61 (2474 bp) or ARS4/NK61 (1301 bp) essentially as described by Knowles & Samuel (1995) .

{blacksquare} Cycle sequencing.
fmol DNA sequencing kits (Promega), which use the cycle sequencing method described by Murray (1989) , were used according to the manufacturer’s protocol with the following amendments: approximately 80 fmol of cDNA template was used in the reactions and 1·5 pmol of Cy5 amidite-labelled primer.

The reactions were heated to 95 °C for 2 min and subjected to 30 cycles of the following programme on a thermal heating block (Omnigene, Hybaid UK): 95 °C for 30 s, 42 °C for 30 s and 70 °C for 1 min. The reactions were terminated by adding 4 µl of Cy5 sequencing stop solution (Pharmacia Biotech) and cooled to 4 °C. The reactions were heated to 95 °C for 3 min prior to loading on an ALFexpress DNA Sequencer (Pharmacia Biotech). The software AM V3.01 (Pharmacia Biotech) was used to process the data which was then exported as an ASCII text file, aligned manually and analysed using the EpiSeq v2.0 suite of computer programs (N. J. Knowles, unpublished).

{blacksquare} Computer analysis for genetic relationships.
Nucleotide sequences were analysed on an IBM compatible personal computer using programs written by one of the authors (N.J.K.). All pairwise comparisons were performed by giving each base substitution equal statistical weight (ambiguities were ignored). A binary tree was constructed according to sequence relatedness across the interval of nucleotides 469 to 639 of the 1D (VP1) gene using the unweighted pair group mean average (UPGMA) method as implemented in the computer program NEIGHBOR and a phylogenetic tree plotted using the program DRAWGRAM, both from the PHYLIP 3.5c phylogeny package (Felsenstein, 1993 ). The UPGMA method constructs a tree by successive (agglomerative) clustering using an average-linkage method. UPGMA assumes a clock but the branch lengths are not optimized by the least squares criterion. This makes the method very fast and thus able to accommodate large data sets.

To confirm the findings based on the partial VP1 sequence data, we used the complete VP1 sequences of 23 of the virus strains. This data were analysed using the neighbour-joining algorithm (Saitou & Nei, 1987 ) as implemented in the program CLUSTAL X (Thompson et al., 1997 ). Distances were corrected for multiple substitutions according to the method of Kimura (1983) . Confidence limits were placed on the tree branches using the bootstrap resampling method (Felsenstein, 1985 ; 1000 replicates), which is part of the CLUSTAL X program. The subsequent unrooted tree was plotted using TreeView v1.6 (Page, 1996 ).


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Nucleotide sequence analysis
The significance of percentage differences in nucleotide sequence comparisons is not clearly understood. However, it has been suggested by researchers studying the epidemiology of picornavirus infections that approximately 85% identity is a realistic cut-off to differentiate major genotypes (Rico-Hesse et al., 1987 ; Vosloo et al., 1992 ). From the UPGMA dendrogram shown in Fig. 1 it can be seen that if a value of approximately 15% nucleotide sequence difference is used to differentiate genotypes, eight distinct genetic lineages, which fall into geographically distinct regions, are apparent (Fig. 2). These have been designated topotypes and named Europe–South America (Euro-SA), Middle East–South Asia (ME-SA), South-East Asia (SEA), Cathay (an ancient and poetic name for China and east Tartary), West Africa (WA), East Africa (EA), Indonesia-1 (ISA-1) and Indonesia-2 (ISA-2). This discrimination is indicative of genetic lineages that have evolved independently (Knowles & Samuel, 1993 ).



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Fig. 1. UPGMA tree showing the relationships between the FMDV type O isolates studied and their division into eight topotypes based on a comparison of the 3' 171 nucleotides of the VP1 gene.

 


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Fig. 2. Distribution of FMDV type O topotypes. (a) Distribution prior to 1993; (b) distribution since 1993.

 
Within each of the topotypes it can be seen that there are distinct sublineages. It can be considered that isolates showing <5% nucleotide difference are closely related and could either be from the same outbreak or are from viruses closely related temporally (Samuel et al., 1997 , 1999 ).

To confirm the conclusions drawn from the analysis of the partial VP1 data, we compared the complete VP1 sequences of 23 of the 105 viruses, including multiple representatives of all eight topotypes, using the neighbour-joining method with bootstrap resampling. The resulting unrooted tree (Fig. 3) clearly shows eight distinct genetic groups, with high bootstrap confidence limits. These were generally >80%, but as would be expected, some of the shorter branches had lower bootstrap values.



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Fig. 3. Unrooted neighbour-joining tree showing the relationships between the FMDV type O isolates studied and their division into eight topotypes based on a comparison of complete VP1 gene sequences (639–642 nucleotides).

 
Europe–South America (Euro-SA) topotype
This topotype includes viruses which occurred in Europe until the cessation of vaccination in 1990–91. European viruses within this topotype are represented by isolates from 1924 to 1988. FMD was first introduced into South America in 1870 when immigrants from Europe settled with their livestock in Argentina. This explains the genetic link between viruses from Europe and South America. There has been exchange of FMD viruses in the intervening years in both directions but particularly in the 1960s when outbreaks in Europe were attributed to imported meat-on-the-bone from South America.

Included in the analysis is a virus from the United Kingdom from 1924 which can be grouped with more recent isolates from South America. European strains have been used extensively in vaccines and it is probable that some of these strains were introduced into the field by improperly inactivated vaccines. This would account for the lack of observable variation in many of the European virus isolates. This topotype has also been found in Africa, represented in these results by an isolate from Angola (O/ANG/1/75). The introduction of this strain has been attributed to the importation of animals from South America during the 1970s and is consistent with the sequencing results showing that O/ANG/1/75 has a closer relationship to an isolate from Brazil in 1970 (O1/RS/BRA/70; ~9%) than to the European strains (~12·5%).

Cathay topotype
This topotype contains viruses which, until recently, were principally found in Hong Kong and China. However, it has only been possible to include a single sequence of a Chinese isolate belonging to this lineage in the study (O/GD/CHA/86). At present the WRLFMD database has 60 sequences of viruses from Hong Kong and, apart from a small number of isolates obtained from imported cattle, they belong exclusively to this group (Knowles et al., 2001 ). The viruses in this topotype are very highly adapted to pigs. A virus belonging to this topotype was introduced into the Philippines in 1994 and became endemic on Luzon Island. Previous outbreaks of type O in the Philippines were in cattle and water buffalo and caused by viruses belonging to the ME-SA topotype.

A strain from the Cathay topotype was also responsible for outbreaks of FMD that occurred in Thalheim, Austria in 1981 (Fig. 1) and Wuppertal, Germany in 1982 (data not shown). The viruses that caused these outbreaks were antigenically distinct from strains circulating in Europe and the Middle East but it was only with the advent of nucleotide sequencing and phylogenetic analysis that their relationship to viruses from the Far East became apparent. Similarly, an outbreak close to Moscow in 1995 was caused by a virus belonging to the Cathay topotype. It was thought that this virus was introduced into Russia in pig meat imported from China (V. V. Drygin, personal communication, 1995).

Although little is published about the FMD situation in China it is probable that outbreaks of FMD in Hong Kong are due to viruses which have evolved in China. Hong Kong has a pig population of about 470000, but imports approximately 2 million pigs annually from mainland China. In 1997, isolates from Taiwan Province of China (which had been FMD-free since 1929) were also found to be related to viruses belonging to the Cathay topotype. Type O viruses isolated from pigs in Vietnam during 1997 and 1999 belong to this topotype, but viruses isolated from cattle in the same country belonged to either the SEA (1997 and 1999) or the ME-SA (1999) topotypes. The reasons for the recent spread of viruses from this topotype are not clear but may be a reflection of a higher incidence of FMD type O in China, or changes in import/export policies in the region.

Interestingly, the Chinese isolate O/Akesu/CHA/58 is related to viruses in the ME-SA topotype and so probably is a representative of another lineage present in China which has its origins in southern Asia.

West Africa (WA) topotype
Isolates belonging to this topotype originate from Ghana, Ivory Coast and Guinea. Outbreaks caused by this lineage have recently also occurred in Algeria, Tunisia and Morocco (Fig. 1). It is more usual for viruses from the ME-SA topotype to be introduced into North Africa, facilitated by the seasonal demand for sheep during specific religious festivals (Samuel et al., 1999 ). The occurrence of the West African topotype in North Africa may be explained by the illegal movement of Zebu cattle into Algeria from West Africa that took place in 1999 (Anon., 1999 ).

East Africa (EA) topotype
This topotype is represented by viruses isolated in Kenya and Uganda. Virus isolates from other East African countries i.e. Tanzania, Eritrea and Ethiopia, are all members of the ME-SA topotype. It is quite possible that the East African topotype has a wider distribution than Kenya and Uganda but very few samples are routinely submitted to the WRLFMD from East African countries.

Middle East–South Asian (ME-SA) topotype
The Indian subcontinent and the Middle East represent a very complex epidemiological situation. FMDV type O is endemic in all countries of the region and the trading relationships both legal and illegal, are reflected in the movement of individual strains. The movement of animals within and between countries is largely uncontrolled due to poor veterinary infrastructure and open borders. Many outbreaks go unreported and attempts to limit the spread of disease are generally unsuccessful. Outbreaks of FMD caused by type O in Bulgaria (1991, 1993 and 1996), Italy (1993), Greece (1994 and 1996) and European Turkey (1995–96) were closely related genetically to viruses circulating in the Middle East (Samuel et al., 1993 ; A. R. Samuel & N. J. Knowles, unpublished data).

The genetic sublineages present in India are diverse and none are confined to any particular area. This situation is almost certainly due to the virtually uncontrolled movements of animals within the country. However, there is no evidence that vaccine strains have been the origin of outbreaks in the field (Pattnaik et al., 1998 ).

In 1995 a virus belonging to the ME-SA topotype was found in Malaysia (data not shown). This virus was found to be related to viruses from India and the Middle East and its presence may have been due to the importation of animals from India. In 1999 and 2000, the same strain re-entered South-East Asia, probably from China, and has swept through the region. This same virus strain has also been responsible for causing recent outbreaks in the Taiwan POC, Japan, Republic of South Korea, eastern Russia and Mongolia (Knowles et al., 2000 ).

South-East Asia (SEA) topotype
Viruses belonging to the SEA topotype appear to have evolved independently from those in the rest of Asia and, until recently, only viruses belonging to this topotype were seen in South-East Asia (Myanmar, Thailand, Cambodia, Malaysia, Laos and Vietnam). However, as described above, there has been a recent incursion of a virus strain belonging to the ME-SA topotype into many South-East Asian countries (Knowles et al., 2000 ). It remains to be seen if this virus strain will become endemic in the region as it has in the Middle East (Samuel et al., 1997 ) or whether it will eventually disappear.

Two Indonesian topotypes
Two topotypes were found amongst isolates from Indonesia, which we have named Indonesia-1 (ISA-1) and Indonesia-2 (ISA-2). The ISA-1 topotype comprises three viruses isolated in 1962, 1974 and 1983, while the ISA-2 topotype contains two very closely related viruses from 1972 and 1974. Although not evident from the phylogenetic trees, members of these two topotypes may have a common ancestor since all the Indonesian isolates have an insertion of a single amino acid between residues 45 and 46. These residues are located close to the 5-fold axis of the virus capsid in the VP1 {beta}B–{beta}C loop which forms part of antigenic site 3 (Acharya et al., 1989 ). FMD has not been reported in Indonesia since 1983 and the viruses belonging to either of these topotypes have not been detected in other Asian countries; therefore it is probable that these topotypes are now extinct. It is possible that other FMD outbreaks in South-East Asia during the same period may have been caused by viruses related to this topotype but which have not been sequenced.

General discussion
Phylogenetic analyses of the outer capsid-coding genes of the seven FMDV serotypes show seven distinct genetic lineages which correlate with serotype as exemplified by a comparison of the 3' end of the VP1 gene (Fig. 4). Analysis of genome regions either side of the capsid-coding region do not correlate with serotype (N. J. Knowles, unpublished data). We have shown that within FMDV serotype O genetic lineages fall into geographically distinct groups which we have called topotypes. Martínez et al. (1992) postulated that if a constant evolutionary rate is assumed then an early diversification event gave rise to the three South African serotypes (SAT 1, SAT 2 and SAT 3) as well as a precursor of the present day O, A, C and Asia 1 serotypes. A further diversification event gave rise more recently to the Eurasian serotypes. These two events appear to have taken place within a relatively short space of time (Domingo et al., 1995 ). Furthermore, phylogenetic trees based on either the P1 or VP1-coding regions of isolates of serotype C also show good correlation between genetic distances and serological subtype classification (Martínez et al., 1992 ).



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Fig. 4. Phylogenetic (UPGMA) tree depicting the genetic relationships between representative strains of all seven serotypes of FMDV. Sequence database accession numbers are indicated.

 
Studies of FMDV type C in the Philippines and type A in Turkey, where it is known when particular lineages, were introduced have shown that the rate of evolution is approximately 1% per year (10-2 substitutions per site per year; N. J. Knowles & A. R. Samuel, unpublished data). If the concept of a constant evolutionary rate is accepted and there are no constraints on virus evolution then it would expected that new topotypes could arise in approximately 15 years. In reality, this extent of evolution probably takes much longer. For example, FMD viruses belonging to the Asia 1 serotype, first identified in samples from Pakistan in 1954 (Brooksby & Rogers, 1957 ), have not yet exceeded 15% nucleotide difference and therefore all belong to a single topotype (Ansell et al., 1994 ; N. J. Knowles & A. R. Samuel, unpublished data). Obviously, fundamental structural constraints limit variation not only between serotypes but also within serotypes. Therefore, it is highly probable that these constraints allow the genetic lineages to enter evolutionary ‘cul-de-sacs’, which is consistent with the concept of the topotypes described in this paper.

Some viruses belonging to the Euro-SA topotype are less diverse than others, for example the O1/Campos/BRA/58, O1/BFS 1860/UK/67 and O1/Burgwedel/FRG/87 isolates are virtually identical (Fig. 1; Knowles et al., 1988 ). This is almost certainly due to the fact that many of these strains have been employed as vaccines and have on occasions been re-introduced into the field (Beck & Strohmaier, 1987 ; Carrillo et al., 1990 ). Although this study and those of serotype A (Armstrong et al., 1991) and serotype C (Martínez et al., 1992 ) have shown that genetic diversity is usual, it has been reported that long-term genetic conservation has occurred in type C in South America (Piccone et al., 1988 ). However, it is much more likely that this could also be explained as a consequence of the accidental re-introduction into the field of vaccine virus strains.

Although eight geographically distinct genetic lineages (topotypes) have been identified, and generally exist within regional boundaries, it can be seen that more than one topotype may be present in a particular geographical region. This is exemplified in Africa where a distinct African topotype is evident in East Africa (Kenya and Uganda) but viruses belonging to the ME-SA topotype have also been isolated from Ethiopia, Sudan and North Africa and, as mentioned previously, a virus related to the Euro-SA topotype has been isolated from Angola.

The UPGMA dendrogram (Fig. 1) was constructed mainly from sequences obtained from recent isolates of viruses from the countries represented. This helps the analysis due to the fact that the UPGMA method treats all the viruses as contemporaneous. The issue of the ancestry of the different sublineages is difficult to address using the UPGMA method; however, using the neighbour-joining method, the branch linking the two African topotypes to the main tree (Fig. 3) is well supported (82·4%), suggesting that they could have had a common ancestor. Some of the genetic lineages or sublineages may in fact be extinct and so any relationships inferred from comparisons with contemporary strains may be misleading in these instances. The effects of competition between viruses of the same serotype and the possibility of recombination could obviously cause quite marked effects on apparent genetic relationships. However, although recombination has been documented in studies of FMDV (King et al., 1982) in vitro, its occurrence in the field has only been documented in regions of the genome encoding non-structural proteins (Krebs & Marquardt, 1993 ). However, recombination has been shown to play a significant role in the long-term evolution of both plant and animal positive-strand RNA viruses (for review see Dolja & Carrington, 1992 ).


   Acknowledgments
 
We thank Drs David Rowlands and Berwyn Clarke for the use of unpublished sequences. We also thank Paul R. Davies and David M. Ansell for valuable technical assistance. This work was supported, in part, by the Ministry of Agriculture, Fisheries and Food, UK.


   References
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 26 July 2000; accepted 28 November 2000.