Recombination in circulating Human enterovirus B: independent evolution of structural and non-structural genome regions

Alexander N. Lukashev1,2, Vasilii A. Lashkevich1, Olga E. Ivanova1, Galina A. Koroleva1, Ari E. Hinkkanen2 and Jorma Ilonen3

1 Institute of Poliomyelitis and Viral Encephalitides RAMS, Moscow, Russia
2 Department of Biochemistry and Pharmacy, Åbo Akademi University, PO Box 66, 20521 Turku, Finland
3 Department of Virology, University of Turku, Turku, Finland

Correspondence
Alexander N. Lukashev
alexander_lukashev{at}hotmail.com


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The complete nucleotide sequences of eight Human enterovirus B (HEV-B) strains were determined, representing five serotypes, E6, E7, E11, CVB3 and CVB5, which were isolated in the former Soviet Union between 1998 and 2002. All strains were mosaic recombinants and only the VP2–VP3–VP1 genome region was similar to that of the corresponding prototype HEV-B strains. In seven of the eight strains studied, the 2C–3D genome region was most similar to the prototype E30, EV74 and EV75 strains, whilst the remaining strain was most similar to the prototype E1 and E9 strains in the non-structural protein genome region. Most viruses also bore marks of additional recombination events in this part of the genome. In the 5' non-translated region, all strains were more similar to the prototype E9 than to other enteroviruses. In most cases, recombination mapped to the VP4 and 2ABC genome regions. This, together with the star-like topology of the phylogenetic trees for these genome regions, identified these genome parts as recombination hot spots. These findings further support the concept of independent evolution of enterovirus genome fragments and indicate a requirement for more advanced typing approaches. A range of available phylogenetic methods was also compared for efficient detection of recombination in enteroviruses.

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AY896760–AY896767.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human enteroviruses, members of the family Picornaviridae, comprise five species, enterovirus A–D and poliovirus. Human enterovirus B (HEV-B) is the largest and the most common species, comprising 28 serotypes of echoviruses (E), six serotypes of coxsackie B virus (CBV), coxsackievirus A9 (CVA9) and a number of newer enteroviruses (http://www.iah.bbsrc.ac.uk/virus/picornaviridae/sequencedatabase/index.html). Most cases of enterovirus infection in humans are asymptomatic or subclinical. Under certain conditions, however, these viruses can cause serious disorders, such as meningitis, myocarditis, encephalitis and haemorrhagic disease of newborns (Pallansch & Roos, 2001). Viruses of the same serotype are known to cause a wide range of clinical manifestations, whilst the same disease can be caused by different serotypes.

Enteroviruses are well known for their ability to undergo extensive recombination. The mechanism of recombination is commonly considered to be ‘copy choice’, i.e. switching of the template RNA molecule by the viral polymerase during the course of minus-strand RNA synthesis (Kirkegaard & Baltimore, 1986). An alternative novel mechanism of non-replicative recombination has recently been suggested (Gmyl et al., 2003). Recombination has been widely reported between three strains of the live polio vaccine after oral administration (Cammack et al., 1988; Macadam et al., 1989; Minor et al., 1986) and between vaccine and/or wild poliovirus strains (Dahourou et al., 2002; Furione et al., 1993; Georgescu et al., 1994, 1995; Guillot et al., 2000; Lipskaya et al., 1991; Macadam et al., 1989). In most cases, recombination was reported in the non-structural protein (NSP) genome region, and intertypic recombination in the structural protein region seems to be the exception rather than the rule (Blomquist et al., 2003). Recombination has also been reported in the non-polio enteroviruses, both among the prototype strains (Brown et al., 2003; Oberste et al., 2004a, c; Santti et al., 1999) and in circulating viruses (Chevaliez et al., 2004; Lindberg et al., 2003; Lukashev et al., 2003, 2004; Oberste et al., 2004d; Oprisan et al., 2002; Santti et al., 2000; reviewed by Lukashev, 2005). Some recent work has suggested a new model of enterovirus genetics, where enteroviruses within species exist not as delimited lineages, but as a pool of independently evolving genome fragments that recombine frequently to give rise to new virus variants (Lukashev et al., 2003; Oberste et al., 2004a; Santti et al., 1999). Currently, sequences are known for all of the prototype enterovirus strains. However, only a limited number of complete sequences of modern enterovirus isolates representing a few serotypes of HEV-B have been reported so far. In this work, we sequenced eight HEV-B strains representing five serotypes that were isolated in the former Soviet Union between 1998 and 2002. These strains proved to be recombinants between the 5' non-translated region (NTR), VP1–2A and 3D genome regions based on analysis of partial sequences (Lukashev et al., 2003). Here, we used full-genome analysis to gain a better understanding of recombination in circulating enteroviruses.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The enterovirus strains used in this work (Table 1) were isolated from clinical (stool samples) and environmental (sewage) specimens collected in accordance with the WHO Polio Eradication Initiative in countries of the former Soviet Union. Virus growth, RNA isolation, PCR and sequencing were carried out as described previously (Lukashev et al., 2003). The primers listed in Table 2 were suitable in the majority of strains (often with various annealing temperatures); however, in several cases, we had to use additional, strain-specific primers based on the actual sequence. All nucleotide positions given in the text are relative to the reference HEV-B sequence (CBV1, GenBank accession no. NC_001472; Iizuka et al., 1987). Sequencing was carried out directly from overlapping PCR products in two directions on a MegaBACE DNA sequencer using a DYEnamic ET terminator kit (Amersham Biosciences). Genome termini (~30 nt) were not sequenced; however, these highly conserved regions carry little phylogenetic information.


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Table 1. Virus strains used in this study

 

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Table 2. Oligonucleotides used in this study

 
The resulting DNA sequences (GenBank accession nos AY896760–AY896767) were aligned with the complete prototype HEV-B genome sequences reported or referred to by Oberste et al. (2004a). EV74 and EV75 sequences (GenBank nos AY556057 and AY556070, respectively) have been described by Oberste et al. (2004b). Additionally, we used the sequences of three E11 strains isolated in 1989–1991 in Europe (GenBank nos AJ577589, AJ577590 and AJ577594; Chevaliez et al., 2004), four E11 strains and one E19 strain isolated in Russia and Europe in 1982–1989 (AY167103–AY167107; Lukashev et al., 2004), one E3 sequence (AJ849942) and one E9 sequence (AF524867; Paananen et al., 2003). Strains Hun90 (GenBank no. AY167103) and HUN-1108 (AJ577589) were almost identical and therefore the latter was omitted. Only two of 12 available CA-V9 sequences were used (GenBank nos AY466022 and AY466030), as the other CA-V9 sequences were very similar to each other. Poliovirus type 1 (GenBank no. NC_002058) was included in the alignment to root the phylogenetic trees. The sequences were aligned by the neighbour-joining algorithm as implemented in CLUSTAL_X (Thompson et al., 1997), with manual corrections to match the open reading frame. The resulting alignments were analysed by using SimPlot version 2.5 (http://sray.med.som.jhmi.edu/SCRoftware/SimPlot). Similarity plots were built with a window size of 500 nt, which provided optimal balance between lowering noise and keeping significant data. Bootscan analysis (Salminen et al., 1995) was run with a neighbour-joining tree algorithm (Kimura distance model) and 100 pseudoreplicates.

To verify our results, we used the probabilistic divergence measures (PDM) method (Husmeier & Wright, 2001) as implemented in TOPALi version 0.22 (Milne et al., 2004). PDM recombination points were identified as positions with highest local divergence, in all cases with significance of >99 %. We also tried a number of other phylogenetic approaches: Sawyer's runs test (Sawyer, 1989) as implemented in GENECONV version 1.81 (www.math.wustl.edu/~sawyer/geneconv/), the difference of sum squares method (McGuire & Wright, 2000) implemented in TOPALi, and the informative sites test (Robertson et al., 1995) implemented as the FindSites feature in SimPlot.

Phylogenetic trees were created with CLUSTAL_X (neighbour-joining algorithm, Kimura evolution model) using the ‘exclude positions with gaps' and ‘correct for multiple substitutions' options. We used 500 nt alignment fragments for all genome regions except the 5' NTR and VP4 regions to obtain comparable phylogenetic trees. Trees were drawn with the Ngraph module of CLUSTAL_X and the in-tree comments were added in CorelDraw 12.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We determined the nucleotide sequences of eight circulating HEV-B strains and compared them with the sequences of all prototype strains and a number of circulating strains available in GenBank. Several approaches were used to screen for recombination. Similarity plots for sequenced strains (e.g. Fig. 1a and b) revealed several obvious cases of recombination and gave an idea of nucleotide sequence distances in different genome regions. Similarity plots for most strains studied (for example, for strain E7-15936-01, Fig. 1a) compared with the prototype HEV-B strains showed increased similarity to the prototype strain in the structural region and to the prototype E30, EV74 and EV75 strains in most of the NSP genome region. Similarity-plot analysis of the strains studied compared with modern HEV-B strains (e.g. E6-14103-00, Fig. 1b) showed increased similarity to strains of the same serotype in the capsid-encoding genome region in all cases and, in most cases, increased similarity to other modern strains studied in this work and to the prototype E30, EV74 and EV75 strains. Strain E30-8477-98 was most similar in the NSP genome region to most modern HEV-B strains sequenced in other reports (E11, CAV9, E3 modern strains) and to the prototype E1 and E9 strains. As similarity plots do not provide any statistical support, our main tool was bootscanning. First, we sought to compare the studied strains with the prototype HEV-B strains. As SimPlot can handle no more than 26 sequences at a time, we used only 25 prototype strains for the analysis. Sequences for the bootscanning analysis were chosen to include those most similar to our strains based on preliminary phylogenetic trees for the 5' NTR, VP1, 2C and 3D genome regions (data not shown). According to the similarity plots, in the NSP region, most viruses sequenced here were similar to the prototype E30, EV74 and EV75 strains (Fig. 1a); therefore, a consensus sequence of these three prototype strains was used for bootscanning. In addition, prototypes E1 and E9 were combined into a consensus sequence, as strain E30-8477-98 was similar to both of these viruses in the NSP region.



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Fig. 1. (a, b) Similarity plots comparing strain E7-15936-01 with the prototype HEV-B strains (a) and strain E6-14103-00 with circulating and some prototype HEV-B strains (b). Window 500 nt, step 20 nt. (c–k) Bootscan graphs of the studied strains compared with the prototype HEV-B strains by using the neighbour-joining tree algorithm (Kimura distance model). Window 500 nt, step 20 nt. The dotted line shows an arbitrary 70 % reliability threshold.

 
According to the bootscan graphs (Fig. 1c–k), all strains studied were mosaic recombinants related to the prototype HEV-B strains, as was expected from our previous results (Lukashev et al., 2003). It turned out that all modern strains had only the P1 region that encodes capsid proteins (VP4)–VP2–VP3–VP1, similar to the corresponding prototype virus. For all eight strains, this region of highest similarity to their prototype strain (bootstrap values >70 % with window size 500 nt) started at nt 690–1140 and ended at nt 3260–3530 (Table 3). The PDM method run with four ‘ancestor’ sequences (usually the query strain, corresponding prototype strain and the prototype E30 and CVB1 strains) identified by bootscanning confirmed the results of the bootscanning (Table 3). As only a few sequences could be used for PDM analysis, there was an apparent increase in the resulting length of related regions between different viruses. The calculated recombination points lay in or very close to the VP4 region, which spans nt 742–948, and in the 2A region, which covers nt 3286–3735.


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Table 3. Estimated recombination points delimiting P1 to prototype and NSP to E30 similarity by using different phylogenetic methods

The bootscan crossing point is the position where the bootstrap value with the 500 nt window reached 70 %. The PDM crossing points indicate maximum local divergence. NA, Not applicable.

 
In the NSP region, all strains studied except for E30-8477-98 were most similar to the consensus E30/EV74/EV75 sequence. This region of E30/EV74/EV75 similarity started in all strains at position nt 4220–4500, approximately in the middle of the 2C genome region (Table 3). Importantly, the same picture was observed for the E30 strain E30-14125-00 (Fig. 1g). This virus turned to be most similar to the prototype E30 strain Bastianni in the genome regions P1 (as in E30 strains) and in 2C–3D (as in most modern strains), but not in the 2AB(C) region. In the 5' NTR, most strains studied grouped with the E1/E9 consensus sequence. We did not notice any correlation between phylogenetic relationships in the 5' NTR and the 2C–3D genome regions of the strains studied, which excludes the possibility that these parts of the genome have co-evolved.

In the second part of our study, we compared the sequenced strains with each other and with the modern HEV-B sequences available in GenBank. Here, the results were not as clear-cut as with the prototype strains. Modern isolates tend to be more similar to each other than to the prototype strains and, if a sequence has two or more almost-equidistant relatives in the alignment (i.e. strains of the same serotype for the P1 genome region or E30-like strains for the 2C–3D region), recombination analysis is complicated. Therefore, analysis of strains E30-8477-98, CBV3-11059-99, E7-15936-01 and E11-18744-02 did not produce an informative output (see Fig. 2a and b). In contrast, analysis of strains E6-10887-99, E6-14103-00, E30-14125-00 and CBV3-18219-02 clearly detected additional recombination events in the 2C–3D genome region involving the modern strains (e.g. Fig. 2c and d). In two cases, CBV3-11059-99 (Fig. 2a) and CVB3-18219-02 (Fig. 2b), we observed what could be traces of recombination in the P1 genome region between strains of the same serotype. However, only PDM with four involved strains reliably detected recombination in the VP2–VP3–VP1 region of these strains. In contrast, these cases did not get reliable support in bootscanning and similarity plots, even when only the four strains involved were used for analysis (not shown).



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Fig. 2. Bootscan graphs comparing strains CBV3-11059-99 (a), E11-18744-02 (b), E6-10887-99 (c) and CBV3-18219-02 (d) with other circulating and several prototype strains by using the neighbour-joining tree algorithm (Kimura distance model). Window 500 nt, step 20 nt. The dotted line shows an arbitrary 70 % reliability threshold.

 
In most cases, bootscan graphs showed a sharp drop in the VP4 and 2AB(C) genome regions (Figs 1 and 2). Only a few of the strains studied had a close ‘relative’ here. To clarify the situation in these genome regions and to strengthen our results further for other genome regions, we produced phylogenetic trees for the 5' NTR (nt 1–741), VP4 (nt 742–948), VP2 (nt 949–1448), VP1 (nt 2452–2952), 2AB (nt 3486–3985), 2C (two trees using 5' and 3' halves of the region, nt 4033–4532 and nt 4519–5019) and 3D (nt 6001–6500) regions (Fig. 3). We used 500 nt fragments where possible in order to get comparable results. Phylogenetic trees for the VP1 and 3D regions confirmed the results of the similarity plots and results reported previously (Lukashev et al., 2003). In VP1, all strains grouped reliably with their prototype viruses. In the 3D genome region, most of the strains studied here grouped with the prototype E30, EV74 and EV75 strains, and strain E30-8477-98 grouped with the prototype E1 and E9 strains and with most modern HEV-B strains sequenced elsewhere. In the VP4 region, phylogenetic grouping correlated completely with the serotype only for E30 strains. Clustering of all of the other strains studied did not resemble that for the adjacent 5' NTR and VP2 genome regions. In the VP2 genome region, the strains studied grouped with their corresponding prototype strains, although less reliably than in VP1. In the 2AB genome region, the tree was extremely star-like and only a few reliable groups were observed. Comparison of the phylogenetic trees for the halves of the 2C genome region supported the results from bootscanning and PDM (Table 3), indicating that the common E30-like fragments of the modern HEV-B strains studied here start near the middle of 2C, as the E30/EV74/EV75-like group was clearly observed only in the phylogenetic tree for the 3' half of 2C. It is important to note that the number of groups with a bootstrap value of >70 % was highest in the 3D region (20 groups), VP1 region (21 groups) and in the second half of the 2C region (17 groups). Fewer groups were observed in other genome regions, with only six in the 2AB region and seven in a short VP4 region. The variability of enterovirus amino acid sequences is lowest in the 3D, 3C and 2C genome regions and highest in VP4 and 2A (Gromeier et al., 1999); therefore, one could expect pseudoreversions in the conserved 2C–3D part of the genome to weaken the phylogenetic signal. However, the opposite was observed in practice (Fig. 3), which can readily be explained by a high recombination incidence in the VP4 and 2A genome regions, resulting in loss of phylogenetic signal. The star-like topology of the phylogenetic trees for the 2AB and, to a lesser extent, the VP4 genome regions, as well as observations from bootscanning (see above), highlight the role of 2AB and VP4 as recombination hot spots.



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Fig. 3. Neighbour-joining phylogenetic trees (CLUSTAL_X) for different genome fragments of the studied HEV-B strains. Strains sequenced in this study are marked in bold. Trees were rooted with poliovirus type 1 (not shown). Bootstrap values <70 % were mostly omitted. Braces show the E30/EV74/EV75-like group of modern strains in the 2C and 3D genome regions. Bars indicate nucleotide sequence distance.

 
We also considered other methods of recombination analysis. A Sawyer's runs test failed to detect recombination events that were obvious from other methods. This probably happened because even the lowest possible mismatch penalty (/g1 option) could not compensate for the high mutation rate of enteroviruses. The difference of sum squares method was also of little use, probably because there were only a few sharp recombination positions and relatively longer regions of high incidence of recombination. Therefore, a step size of 100 nt had to be used to get output comparable to that from other methods, which resulted in very imprecise results. The informative sites test was also attempted with little success, due to high phylogenetic noise and the absence of close ancestors of the strains being analysed.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this work, we studied recombination in eight complete sequences of circulating HEV-B strains. Our results further support our previous conclusion on independent evolution of different fragments of the enterovirus genome (Lukashev et al., 2003). However, with the full sequences, it is now possible to identify recombination crossover regions, borders of independently evolving fragments and recombination hot spots. All of the strains studied were most similar to their serotype prototype strains only in the VP2–VP3–VP1 genome region, with recombination mapping most often to the VP4 and 2AB(C) regions. Recombination has often been reported in the 2AB genome region. The fact that VP4 turned out to be a recombination hot spot was somewhat unexpected, but not surprising. This protein is not exposed on the capsid surface and probably does not require strict and precise interactions with other capsid proteins, which might increase its evolutionary flexibility. There was no trace of intertypic recombination in the VP2–VP1 genome region; however, intratypic recombination could not be excluded. Similar to our results, somewhat uncertain indications of intertypic recombination have been obtained for circulating CBV strains (Oberste et al., 2004d). Currently, only a few modern strains have been sequenced completely, which gives little chance of detecting intratypic recombination. We can expect the situation to be clarified when additional full genomes of circulating enteroviruses are sequenced. Based on our results, we suggest that the common rule of ‘(almost) no recombination in P1’ can be changed to ‘(almost) no intertypic recombination in VP2–VP3–VP1’.

It was shown previously, based on a partial 3D region sequence, that many modern HEV-B strains had 3D regions related closely to that of the prototype E30 strain Bastianni (Lukashev et al., 2003; Oprisan et al., 2002). In other studies, the NSP regions of most modern HEV-B strains were found to be more similar to the prototype E1 and E9 strains (Lukashev et al., 2004) or to either E30 or E1/E9 (Lindberg et al., 1999; Oberste et al., 2004d). Importantly, the modern strains grouped specifically with E30/EV74/EV75 or with E1/E9 on the properly rooted (by the addition of poliovirus type 1) phylogenetic trees for the 3D genome region (Fig. 3), thus indicating that a majority of modern HEV-B isolates are phylogenetically similar to the prototype HEV-B strains in the NSP genome region. A possible explanation of what we observed in the phylogenetic trees (Fig. 3) may be that most E30/EV74/EV75-like strains were predominantly isolated in the late 1990s, whilst E1/E9-like strains mostly originated in the 1980s. However, the number of strains sequenced so far and the limited geographical coverage are insufficient to allow any firm conclusions to be drawn.

In our work, similar genome regions between the strains from the 1990s (with the exception of E30-8477-98) and the prototype E30/EV74/EV75 strains spanned the 2C–3D region in all cases (Fig. 2c–k). In contrast, we noticed multiple recombination events in the 2C–3D genome region when comparing the circulating strains studied here with each other (Fig. 3c and d). In fact, the NSP regions of most, if not all, strains studied underwent additional recombination events after diverging from a putative E30-like ancestor. This observation further underlines the ubiquitous prevalence of recombination among enteroviruses and urges a wider use of full-genome analysis of circulating strains. Tracking recombination events in complete enterovirus genomes might help to resolve the complicated phylogenetic relationships of enterovirus strains.

As reported previously, most of the prototype HEV-B strains showed complex network-like phylogenetic relationships in the NSP genome regions back in the 1950s, indicating frequent recombination events (Oberste et al., 2004a). Some prototype strains, however, then became ‘outcasts' of the ubiquitous recombination, being fairly distant from other prototype strains and lacking apparent traces of recombination. The most prominent example of this is E30 Bastianni. In our study, we observed that the E30-like NSP region has spread to many other serotypes and has then restored a mosaic complexity through additional recombination events. One would assume that an enormous selection pressure has driven these events, as they occurred in the majority of circulating HEV-B strains representing 11 serotypes (Lukashev et al., 2003). Unfortunately, based on our current knowledge of enteroviruses, it is not possible to say whether this assumed selection pressure was due to higher ‘efficiency’ of the encoded proteins or a lower herd immunity to the former ‘outcast’ proteins.

Our results, as well as multiple recent reports on ubiquitous recombination in enteroviruses (Chevaliez et al., 2004; Oberste et al., 2004d; Santti et al., 1999), may explain the failure of the current enterovirus typing approaches based on partial VP1 sequencing or the neutralization test to identify patterns of virulence in non-polio enteroviruses. Indeed, our results indicate that knowing the serotype of an enterovirus only indicates that roughly one-third of its genome is >70 % similar to the prototype strain. Therefore, if certain virulence determinants are located outside the structural genome regions and can easily assume a different serotype, standard typing would produce misleading results. The observed independent evolution of different genome regions and the assumed selection pressure on the NSPs underlines their importance for understanding the biology of enteroviruses. It also indicates that sequencing of several genome regions of circulating enteroviruses will not be always sufficient. For example, strain E30-14125-00 could easily be mistaken for an E30 descendant based on partial analysis of the VP1 and 3D genome regions. The full-genome analysis of this strain, however, demonstrated that it had sequence fragments rather distant from those of the prototype E30 in the 2AB(C) and 5' NTR regions.

We tried to compare a number of phylogenetic methods to analyse recombination in enteroviruses. As suggested from the similarity plots (Fig. 1a and b), proper detection of recombination events in enteroviruses is not a trivial task. These viruses accumulate mutations very quickly, at a rate of about 1–2 % nucleotide substitutions per year (Gavrilin et al., 2000). However, the protein sequences in the NSP genome region are highly conserved and the maximum difference in amino acid sequence is limited to only about 5 %. Therefore, the maximum difference in RNA sequence cannot exceed 25 % in the NSP genome region. This results in fast accumulation of phylogenetic noise due to random pseudoreversions, and some methods, such as the informative sites test or Sawyer's runs test, which are able to detect only very recent recombination events in enteroviruses (Lukashev et al., 2004), failed in this study. Another complication in searching for recombination in enteroviruses comes from the generally low phylogenetic signal in the VP4 and 2AB genome regions, which is probably due to multiple recombination events. Therefore, there is often no sharp change in the phylogenetic relationship of the recombinant strains (Fig. 1c–k), but rather a transition from one tree topology to a region of inconclusive phylogeny, as seen in the VP4 and 2AB genome regions, and then to another tree topology, which additionally complicates the use of the informative sites test and the difference of sum squares method. Ultimately, phylogenetic trees and their derivative, bootscanning, are able to handle large numbers of sequences and seem to be the most reliable methods to study recombination among enteroviruses, with other approaches playing a supportive role.


   ACKNOWLEDGEMENTS
 
This work was supported in part by the Sigrid Juselius Foundation. The strains sequenced were isolated as part of the WHO Polio Eradication Programme in the European Region. We would like to acknowledge Dr V. Gidirim (National Centre of Preventive Medicine, Kishinev, Moldova), Dr L. Yektova (Centre for State Sanitary Epidemiological Surveillance, Donetsk, Ukraine), Dr M. Bichourina (Pasteur Institute, St Petersburg, Russian Federation) and Dr E. Romanenko (State Center of Sanitary-Epidemiological Surveillance, Stavropol, Russian Federation) for providing the strains. We are grateful to Jussi Mantere for technical assistance with the DNA sequencer.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 15 June 2005; accepted 9 September 2005.



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