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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AY896760AY896767.
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INTRODUCTION |
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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), VP12A 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.
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METHODS |
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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.
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RESULTS |
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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 2C3D 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 2C3D 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 VP2VP3VP1 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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DISCUSSION |
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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 2C3D region in all cases (Fig. 2ck). In contrast, we noticed multiple recombination events in the 2C3D 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 12 % 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.
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ACKNOWLEDGEMENTS |
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Received 15 June 2005;
accepted 9 September 2005.
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