1 Institute of Poliomyelitis and Viral Encephalitides RAMS, Moscow 142782, Russia
2 Department of Biochemistry and Pharmacy, Åbo Akademi University, PO Box 66, FIN-20521 Turku, Finland
3 Department of Virology, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland
Correspondence
A. N. Lukashev
alexander_lukashev{at}hotmail.com
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ABSTRACT |
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INTRODUCTION |
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Enteroviruses cause a wide range of diseases, ranging from poliomyelitis, myocarditis and sepsis-like disease of newborns to mild common cold-like conditions (Pallansch & Roos, 2001). Most enterovirus infections are subclinical, with only a small fraction of cases accompanied by clinical signs, and even fewer causing severe disease. The role of non-polio enteroviruses in human pathology has been recognized only in recent decades, and many aspects of their biology are not yet completely understood (Muir et al., 1998
).
An important feature of enteroviruses is their high mutation rate (Ward & Flanegan, 1992), which may account for their remarkable genetic variation. Enteroviruses also show a high rate of recombination, studied extensively in poliovirus (reviewed by Agol, 2002
). Recombination has also probably occurred in the phylogenetic history of non-polio enteroviruses (Santti et al., 1999
), and a few studies have demonstrated recombination in circulating enteroviruses (Andersson et al., 2002
; Oprisan et al., 2002
; Santti et al., 2000
). Our previous results from uveitis-causing enteroviruses (Lukashev et al., 2003b
) suggested recombination in the non-structural protein-coding region, and was studied further here.
Enterovirus uveitis (EU), reviewed in more detail by (Lukashev et al., 2002), is a new severe eye disease in infants first identified in 1980. Five outbreaks of enterovirus infection with uveitis, caused by human echovirus 11 (EV11) and human echovirus 19 (EV19) variants, were registered in Russia in the 1980s, in different regions of Siberia (Table 1
). Importantly, EV11/B group uveitis-causing strains were found to be serologically, pathogenetically (Lashkevich et al., 1996
) and phylogenetically (Lukashev et al., 2003b
) very close to EV11 strains isolated from infants with multisystem haemorrhagic (sepsis-like) disease.
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METHODS |
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RNA isolation and sequencing.
RNA isolation and sequencing were done generally as described previously (Lukashev et al., 2003b). Overlapping genome fragments between 600 and 1600 nucleotides in length were amplified by PCR using Taq polymerase. Twenty terminal nucleotides at both ends of the genome were not sequenced, because they are conserved in enteroviruses and were thus of little interest for the current study. The primers used are listed in Table 2
. All nucleotide positions are given relative to the EV11 Gregory genome (Dahllund et al., 1995
). PCR products were sequenced in two directions either directly with PCR primers, or cloned into pSTBlue1 plasmid (Perfectly Blunt kit, Novagen) and sequenced with plasmid primers; this was because direct sequencing of PCR products using highly degenerative primers was not always possible.
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Test strains were also studied for possible gene conversion events using Sawyer's runs test (Sawyer, 1989) as implemented in the GENECONV 1.81 software (www.math.wustl.edu/
sawyer/geneconv/).
Phylogenetic trees were created with either the DNAML module of the PHYLIP package (maximum-likelihood algorithm) (Felsenstein, 1989), or with ClustalX (NJ tree algorithm, Kimura evolution model) using the exclude positions with gaps' and correct for multiple substitutions' options. Only NJ trees are presented here, as those built using both algorithms displayed only minor differences.
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RESULTS |
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All four EV11 strains studied were mosaic compared to the prototype EV11 Gregory (Fig. 1fk). Strain MorM/82 had a 5' NTR that was approximately equally distant from the enteroviruses used for comparison and the structural protein region (VP42A, between nucleotides 840 and 3470) was most similar to the prototype. Most of the non-structural protein (NSP) coding region (between nucleotides 4500 and 6920) of this strain showed more similarity to EV9, and the 3' end of the genome was more similar to EV1 than to any other prototype echovirus strain used for comparison (Fig. 1f
). In addition, strain Kust/86 was more similar to the prototype EV11 only in the structural protein-coding region (between nucleotides 770 and 3540). The 5' NTR of this strain was more similar to EV9, and almost the entire NSP region starting from nucleotide 4050 was more similar to EV1 (Fig. 1g
). Strains Kar/87 and Hun/90, similar in their 5' genome halves to strain Kust/86 (see above), were also similar to EV11 only in the structural protein region (between nucleotides 750 and 3480) and to EV9 in the 5' NTR. In most of the NSP region (between nucleotides 4150 and 6900), however, both these strains had more similarity to EV9, and only the 3'-terminal 500 nucleotides of their genomes were more similar to EV1 (Fig. 1
hk). According to analysis of the informative sites, strains Kust/86 and Kar/87 had the first 3720 nucleotides of their genomes in common, while strains Kust/86 and Hun/90 had the first 4045 nucleotides in common, and strains Kar/87 and Hun/90 shared the first 3745 nucleotides of the genome.
In order to make an additional check for possible recombination events in the phylogenetic history of the studied strains, we used Sawyer's runs test (GENECONV software). All tests were run with a minimum mismatch penalty (/g1 option) to account for the high mutation rate of enteroviruses. First, we limited the search for gene conversion events only to the four EU-causing strains. The only fragment that underwent gene conversion was found between EV11/B subserotype strains Kust/86 and Kar/87 (between nucleotides 50 and 3727), which is well in accordance with bootscanning/informative sites results. Subsequently, all 20 sequences of the alignment were examined and 116 reliable gene conversions were found. The three most reliable ones were between strains Kust/86, Kar/87 and Hun/90, including 5' halves of the genomes (up to nucleotides 3667 to 3709), while others involved all possible combinations of prototype and studied strains. These gene conversions were observed only in the 5' NTR or in the NSP region (2C3D), never in the capsid region.
To further illustrate the mosaic structure of the strains studied, we have built phylogenetic trees for the 5' NTR, VP2VP3VP1, 2C3ABC3D (to nucleotide 6900) and nucleotide 7000 to 3' NTR coding regions, selected in order to stay within areas not affected by recombination (Fig. 2). As could be predicted from bootscan graphs, all four EV11 study strains grouped with the prototype EV11 Gregory only in the structural protein-coding region (Fig. 2b
). On the tree for the 5' NTR EV11 strains K452/81, Kust/86, Kar/87 and Hun/90 grouped with EV9, while strain MorM/82 did not group reliably with any prototype EV strain used for comparison (Fig. 2a
). On the trees for both the NSP region (2C3ABC) and the 3'-terminal 450 nucleotides, all EV11 strains studied grouped together with EV9 and EV1 with very reliable bootstrap values, most strains having sequences closer to EV9 in the NSP region and to EV1 in the 3'-terminal region (Fig. 2
c, d).
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DISCUSSION |
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The comparison of Kust/86, Kar/87 and Hun/90 strains, which belong to the same EV11/B subtype, clearly demonstrates recent recombination events that replaced the 3' part (from nucleotide 3745 or 4095) of the genome of Kust/86 strain (or its close ancestor) with regions from some other enterovirus strains (Fig. 1ce). This recombination did not significantly alter the pathogenetic properties of these strains for monkeys (Koroleva et al., 1998
; Lashkevich et al., 1996
) (Koroleva G. A., unpublished). Thus three viruses, which were very similar clinically, serologically and experimentally and were previously considered to belong to the same subtype, have only the 5' half of genome in common. The 5' halves of the genomes of these strains are very close to each other (over 96·3 % nucleotide sequence similarity in VP1 region). Judging from the reported estimates of evolution rate of other enteroviruses (Brown et al., 1999
; Takeda et al., 1994
), the recombination that resulted in the Kar/87 and Hun/90 strains probably occurred only a few years before their isolation. Putative locations of these recombination events suggest that strain Hun/90 is not a descendant of strain Kar/87, as it shares a larger region with strain Kust/86 than with Kar/87. Thus, the study of full genome sequences revealed additional phylogenetic information, which could not result simply from phylogenetic trees of single regions of the genome.
Our study revealed no genome regions of elevated similarity or gene conversion events between the representatives of the three sero- and genotypes of the EU-causing strains. Therefore, genetic determinants of ophthalmotropism have not been transferred by recombination from one subtype to another, but have either pre-existed in all the strains studied or have emerged three times in Krasnoyarsk in 1980, 1982 and 1986.
A general conclusion that follows from our results is that all enterovirus typing and classification approaches based on the serotype, determined either serologically or by capsid proteins sequencing (Oberste et al., 1999), provide information about merely one-third of the viral genome. It seems that we know very little about the genetics of circulating enteroviruses, and we would not learn much more by routinely sequencing a single coding region for phylogenetic studies.
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ACKNOWLEDGEMENTS |
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Received 30 June 2003;
accepted 20 October 2003.