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Journal of Clinical Microbiology, November 2006, p. 4142-4148, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.01049-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

FDJS03 Isolates Causing an Outbreak of Aseptic Meningitis in China That Evolved from a Distinct Echovirus 30 Lineage Imported from Countries of the Commonwealth of Independent States

Ya Nan Zhao,^1, David S. Perlin,^2, Steven Park,² Ren Jie Jiang,³ Liang Chen,¹ Yue Chen,⁴ Rebecca Gardiner,² and Qing Wu Jiang^1*

School of Public Health, Fudan University, Shanghai, China,¹ Public Health Research Institute, Newark, New Jersey,² Yancheng Center for Disease Prevention & Control, Yancheng, Jiangsu, China,³ University of Ottawa, Ottawa, Canada,^{4 4}

Received 19 May 2006/ Returned for modification 3 August 2006/ Accepted 30 August 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
We compared echovirus 30 strains (FDJS03) which caused an outbreak of aseptic meningitis in China in 2003 with other human enterovirus B strains. Sequencing of the complete genome of FDJS03_84, a representative strain from this outbreak, revealed a mosaic structure with a putative recombination spot within the 2B gene. It was most similar to a strain of the same serotype, E30-14125-00, in the 5' half of the genome but was almost equidistant to all strains analyzed in most of the 3' half of the genome. Phylogenetic relationships in the 5'-untranslated region and the VP1 gene indicated that the FDJS03 isolates were closely related to a distinct lineage of E30 which circulated in countries of the Commonwealth of Independent States during 1999 and 2000. It is most likely that the ancestor of FDJS03 isolates experienced multiple recombination events in the nonstructural protein coding region, which were partly observed in the phylogenetic analysis of the 3D region.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enteroviruses (EVs) are common human viruses and may infect a billion or more people annually worldwide (14). Several enterovirus serotypes are associated with diseases, such as pandemic acute hemorrhagic conjunctivitis (4), and specific serotypes have emerged to cause outbreaks of major public health concern, including echovirus 30 (E30) (21). E30 is one of the two most widespread serotypes in the United States and is commonly associated with aseptic meningitis outbreaks (2).

E30 is receiving increasing attention in China as well. Zhao et al. reported an outbreak of aseptic meningitis associated with E30 in Jiangsu Province in 2003 (31). E30 was also found to be involved in several outbreaks of aseptic meningitis and hand-foot-and-mouth disease in the neighboring provinces (Zhejiang and Shandong) (6, 29). E30 appears to be widely circulating in these regions, and understanding its pathogenesis and molecular epidemiology will have important public health implications.

Based on VP1 gene analyses, E30 was found to follow a pattern of monophyletic evolution, where lineage displacement correlates with the temporal dynamics of strains of this serotype (15, 20, 25). However, these results are inconclusive due to the geographic limitation of data sources, with only a few Asian sequences available. At present, VP1-based sequence analysis has been adopted as a standard for molecular epidemiologic investigations of both polioviruses and nonpoliovirus enteroviruses (7). However, the prevalence of recombination among enteroviruses and the observed independent evolution of different genomic regions indicate that sequencing of the VP1 region or other limited genomic regions (e.g., capsid P1) may be insufficient to characterize EV strains and that a wider use of full-genome analysis is necessary (11). So far, only a limited number of complete sequences of modern enterovirus isolates have been reported, although sequences are known for all of the prototype strains.

In this study, we sequenced the complete genome of one E30 isolate (FDJS03_84) obtained from the outbreak of aseptic meningitis in Jiangsu Province in 2003 (31). We also determined the partial 5'-untranslated regions (5'UTRs) for another eight isolates. In an earlier report by Zhao et al. (31), a VP1 sequence analysis suggested a distinct lineage of E30 for these strains. However, a full-genome analysis and phylogenetic analyses provided evidence that FDJS03 isolates were most likely descendants of E30 strains which circulated in countries of the Commonwealth of Independent States (CIS) during 1999 and 2000 (10).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viruses. Hospitalized patients with aseptic meningitis during an outbreak in the northern area of Jiangsu Province, China, in 2003 provided clinical specimens, and E30 FDJS03 isolates were recovered for this study. Sample collection, virus isolation, and identification were detailed in a previous report (31). Strain FDJS03_84 was randomly selected for full-genome sequencing.

RNA extraction and sequencing. Viral RNA was extracted from 200 µl of tissue culture supernatant and then reversely transcribed (15). Overlapping genome fragments were amplified by PCR with several sets of primers designed for conserved regions among enteroviruses and for the VP1 sequences of FDJS03 isolates obtained previously (Table 1). Specific primers were designed from preliminary sequences to close gaps between the original PCR products. PCR products, visualized as single bands in 1% agarose gels, were purified with a Montage PCR₉₆ cleanup kit (Millipore, MA) and subjected to direct sequencing. Otherwise, PCR products were excised from agarose gels, purified with a Montage gel extraction kit (Millipore, MA), cloned into the pCR4-TOPO plasmid vector, and transformed into TOP10 competent cells (Invitrogen, Carlsbad, CA). Plasmid DNA was extracted from positive colonies by using a Fast Plasmid Mini kit (Eppendorf). Automated DNA sequencing was performed using a DTCS Quick Start kit (Beckman Coulter, Fullerton, Calif.) and a CEQ 2000 XL capillary electrophoresis DNA sequencer (Beckman Coulter). A fragment within the 5'UTR amplified by primer pair UG52/UC53 (26) was sequenced for eight FDJS03 strains. Using the same primers, PCR products were sequenced directly in both directions, as the presence of single bands on the gel was observed.

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TABLE 1. Oligonucleotide primers used for amplification of the complete genome of FDJS03_84

Sequence analysis. The resulting DNA sequence fragments were evaluated and assembled manually into a full genome. The full-length genome sequence of FDJS03_84 was aligned by ClustalX (version 1.83) (27) with 39 prototype sequences of human enterovirus B (HEV-B) (16, 17) and 44 complete sequences of field strains of the species (Table 2). The gap opening value and the gap extension value were set to 100 and 10, respectively (12), and the alignments were corrected manually to match the open reading frame. The resulting alignments were analyzed by using SimPlot software, version 2.5 (http://sray.med.som.jhmi.edu/SCRoftware/simplot/), with a sliding window of 400 nucleotides (nt) moving in steps of 50 nt. Bootscanning analysis (22) was conducted with a neighbor-joining (NJ) tree algorithm, the Kimura two-parameter (8) distance model, and 100 pseudoreplicates. Since SimPlot can handle no more than 26 sequences at a time, we used 25 sequences in the analysis every time for comparison with that of FDJS03_84.

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TABLE 2. HEV-B strains used for similarity analysis

Phylogenetic trees for three distinct genomic regions were constructed with the MEGA (version 3.1) software package (NJ algorithm and Kimura evolution model), using alignments produced by ClustalX. We tested the statistical significance of the phylogenies constructed by using bootstrap analysis with 1,000 pseudoreplicate data sets.

Different sets of sequences were included in the analyses of these three regions (Table 3) due to the diverse sequence availability for the target regions. For the VP1 region, a total of 61 E30 sequences were analyzed, including strains reported by Savolainen et al. (25), Palacios et al. (20), Wang et al. (30), and Lukashev et al. (10), 2 Italian sequences (AJ295172 and AJ295185), 5 FDJS03 sequences, and 9 newly announced Chinese E30 sequences (29) (sequences AY695098, AY695101, and AY695102 were submitted directly to GenBank) (Table 3). The nine Chinese strains were isolated from three different regions (Zhejiang, TaiAn, and ZhaoQing in Shandong). E21 was included in the analysis as an outgroup. Besides VP1, two other genomic regions were analyzed as well. Totals of 82 and 44 sequences were used in the analyses for the 5'UTR and 3D regions, respectively. The multiple sequence alignment corresponding to each fragment was constructed and analyzed independently.

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TABLE 3. HEV-B strains used for partial sequence analyses

Nucleotide sequence accession numbers. The GenBank accession numbers of the sequences reported in this paper are AY948442 and DQ184904 to DQ184911.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sequence analysis of the complete FDJS03_84 genome and other HEV-B genomes. A similarity plot for FDJS03_84 relative to all prototype HEV-B strains showed that this virus was most similar to the prototype E30 strain in the structural region (P1) and was almost equidistant (similarities ranged from 75% to 85%) to all prototype strains in the 5'UTR and nonstructural protein (NSP) regions (Fig. 1A). In a comparison with modern HEV-B strains (Fig. 1B), the similarity plot for FDJS03_84 manifested the highest similarity to a strain of the same serotype, E30-14125-00, in the 5' half of the genome (ending around nt 3800, within the 2B gene). From 2C to the end of the genome, the similarity pattern for FDJS03_84 resembled that observed relative to prototypes, except for a higher-than-average similarity acquired by strain E9-DM in the 3D region. Bootscanning indicated possible recombination events (Fig. 1C). FDJS03_84 was closely related to E30-14125-00 in the first half of the genome, which mirrors the similarity analysis results. In the middle of the 2B region, the bootscan graph showed a sharp drop, with no reliable (bootscan value, >70%) phylogenetic relationship between analyzed strains and FDJS03_84 downstream, until a significant E9-DM (28) similarity started at about nt 6300 in the alignment. The results of both similarity and bootscanning analyses illustrated that FDJS03_84 is a mosaic recombinant. E9-DM could be one of the progenitors in the evolving process of FDJS03_84. However, we do not know the exact ancestors for FDJS03_84 in the NSP region since a sequence might have two or more almost-equidistant relatives in the alignment, such as the case observed in the similarity analysis of the 2C-3C regions.

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FIG. 1. Similarity and bootscanning analyses comparing isolate FDJS03_84 and other HEV-B strains. A sliding window of 400 nucleotides moving in 50-nucleotide steps was used in this analysis. Positions with gaps were excluded from the comparisons. (A) Similarity plot for FDJS03_84 versus HEV-B prototype strains. (B) Similarity plot for FDJS03_84 versus HEV-B modern strains. (C) Bootscan graph for FDJS03_84 versus prototype and modern HEV-B strains. E30, EV73, EV74, CA9, E1, E5, E7, E9, E11, E13, E21, and E27 are abbreviations for prototypes of these serotypes. The NJ algorithm, the Kimura two-parameter model of nucleotide substitution, and 100 pseudoreplicates were used in the analysis. The genomes of the HEV-B strains included here are listed in Table 2. The dotted line shows an arbitrary 70% reliability threshold.

Evolutionary relationship of FDJS03 isolates to recent HEV-B strains. (i) Comparison of VP1 sequences of FDJS03 isolates and other E30 strains. Full-genome analysis showed that a newly sequenced strain, E30-14152-00, was closely related to FDJS03_84 in the 5' half of the genome, with a high degree of similarity (90% to 99%). A reconstruction of the VP1 phylogenetic tree, including newly announced E30 VP1 sequences in GenBank, showed a monophyletic VP1 tree (Fig. 2A) and demonstrated that FDJS03 isolates, together with nine other Chinese E30 isolates, were most likely offspring of seven E30 strains circulating in CIS countries during 1999-2000. All of these strains formed a lineage distinct from any other lineage previously reported (20) (bootstrap value, 100%). Three other CIS strains analyzed (8477-BYE98, 18733-MOL02, and 17891-BYE02) were segregated into lineage F, suggesting that the modern CIS E30 strains evolved in different pathways, one of which may involve FDJS03.

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FIG.2. Phylogenetic trees depicting genetic relationships between FDJS03 isolates and other HEV-B strains. The NJ trees were constructed using the MEGA, version 3.1, software package, with the Kimura two-parameter model as the nucleotide substitution model. Numbers at nodes correspond to bootstrap support (>70% bootstrap values) for the clusters to the right of the nodes. The trees are based on sequence alignments for (A) VP1 (nt 2448 to 3323), (B) the 5'UTR (nt 336 to 549), and (C) 3D (nt 6447 to 6898). All nucleotide positions are relative to FDJS03_84 sequence numbering. •, FDJS03 isolates; , HEV-B prototypes, except for E30 prototype (); , CIS E30 strains; and , Chinese E30 strains.

(ii) Phylogenetic analyses of the 5'UTR. The phylogenetic tree for the partial 5'UTR (nt 336 to 549) (Fig. 2B) demonstrated low bootstrap values and showed no correlation between serotypes and genetic groupings of modern HEV-B strains. A notable cluster with a robust bootstrap value (78%) was observed; however, it comprised all FDJS03 isolates and a small group of four recent CIS E30 strains. None of these strains grouped with the E30 prototype strain. Together with the results of VP1 analysis and the complete genome analysis, it is evident that the common ancestors of FDJS03 isolates evolved from a recent CIS lineage of E30 strains.

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FIG. 2— Continued.

(iii) Phylogenetic relationship in the 3D genomic region. A fragment of the 3D region (nt 6447 to 6898) was extracted from the FDJS03_84 genome and compared with available HEV-B sequences of the corresponding genome region. For this region, the analyzed HEV-B strains clustered into three groups with confidence (bootstrap values, >70%) (Fig. 2C). Group 1 was present in most prototype strains and a few modern strains, including FDJS03_84. Three E11 strains circulating in Russia in the late 1990s and E9-DM were the closest relatives to FDJS03_84 in this group. Two other groups comprised a majority of modern HEV-B strains. Members of group 2 were E1 prototype and E1-like modern strains, most of which were isolated in the 1980s. Group 3 harbored the prototype strains of E30, EV74, and EV75 and similar modern strains that originated predominantly in the 1990s. The putative ancestor of FDJS03_84 in the first half of the genome in the bootscanning analysis, E30-14125-00, resided in group 3, hardly contributing to the 3D evolution of FDJS03_84.

Top Abstract Introduction Materials and Methods Results Discussion References

 
FDJS03_84, a representative E30 isolate which caused an outbreak of aseptic meningitis in China in 2003, was compared with the genomes of other HEV-B strains and demonstrated a mosaic genomic structure. A putative parental sequence, E30-14125-00, in the 5' half of the genome suggested that recombination played a role in the evolution of FDJS03_84. Previous analysis of full-genome sequences of all HEV-B prototype strains provided multiple lines of evidence for ubiquitous recombination within this species (16). Other studies have demonstrated that most of the circulating HEV-B viruses are recombinants (3, 9-12, 18, 19, 23), with most strains showing multiple traces of recombination in the NSP region. Lukashev et al. identified 2ABC genome parts as recombination hot spots (11). Our study showed similar results, and a distinct recombination site was mapped to the 2B region. Upstream of this location, a modern E30 strain circulating in Ukraine in 2000 (E30-14125-00) showed a high level of similarity to FDJS03_84, suggesting a derivative relationship between these two strains. The bootscanning profile downstream of the putative recombination point in 2B was different from that observed for the neighboring genome part. No strain studied, either a prototype or a modern strain, showed significant similarity to FDJS03_84 in the 2C-3C region. This observation differs from those reported for modern HEV-B strains, which were found to be most similar to the prototype E30, EV74, and EV75 strains in the 2C-3D region compared with all HEV-B prototypes (11).

The VP1 region has been an important target for E30 molecular epidemiology because its serotype is determined by major antigenic sites in the region. Using genetic typing derived from the entire VP1 gene, FDJS03 isolates were identified as being E30 strains (31). Based on the full-genome analysis in this study and updated VP1 sequences in GenBank, the donors for the 5' halves of the genomes of FDJS03 isolates surfaced. The reconstructed VP1 tree showed that all Chinese E30 isolates aggregated into a tight cluster which was derivative from E30 strains that circulated in four CIS countries in 1999-2000. The nucleotide sequence identities between FDJS03 isolates and the CIS E30 strains in the VP1 region were 95% to 97%. Given that enteroviruses incorporate mutations at a rate of about 1% per year (5), it is reasonable to speculate that the VP1 sequences of FDJS03 isolates evolved from the CIS strains. We also found that FDJS03 isolates, which were previously identified as a distinct lineage, were actually the latest offspring of the whole lineage. This lineage was divergent from any reported lineages of E30 in previous studies (15, 20, 25).

We observed a reliable grouping of FDJS03 isolates and four CIS E30 strains in the phylogenetic tree for the partial 5'UTR. The bootstrap values for the whole tree were low, which was likely due to the highly conserved quality of this genome part and the result of ubiquitous recombination in the 5'UTR within HEV-B (10). In addition, the similarity and bootscanning profiles of this genome part provided further supports for the notion that the ancestor(s) of the 5' genome halves of FDJS03 isolates originated from CIS countries. There is a new model of enterovirus genetics considering that enterovirus species exist as a pool of independent evolving genome fragments that recombine frequently to give rise to new virus variants (10, 16, 24). In this work, we did not find any correlation between phylogenetic relationships in the 3D region and those in two other genomic regions of FDJS03_84, which supports the notion of independent evolution of different genome fragments. All strains analyzed in this region split into three reliable groups, namely, most of the prototype strains, prototype E1 plus E1-like modern strains, and prototype E30, EV74, and EV75 strains plus E30/EV74/EV75-like modern strains. Previous studies have documented such clustering of 3D sequences of HEV-B strains (10, 11, 13), with a grouping tendency for modern strains. Since the analyzed region in this work is in the middle of the 3D gene, in contrast to that studied by Lukashev et al., who aimed at the 5' end of 3D (or 3CD), we constructed a phylogenetic tree for the same region (data not shown). For 3CD, a similar tree topology was observed, and FDJS03_84 was still distant from most modern strains; however, the group harboring FDJS03_84 and most of the prototype strains was not supported by an appreciable bootstrap value. A possible reason for this observation is that recombination sites might be included in this region, as seen in the bootscan graph. The phylogenetic relationship of the 3D region demonstrated that the closest relatives of FDJS03_84 were three E11 modern strains isolated in Russia during 1996-1997 and E9-DM, which was recovered from a child with diabetes (28). The representative sequence of the FDJS03 isolates' ancestor in the 5' half of the genome, E30-14125-00, was seen to group with the E30 prototype strain in this region, which underscored the observation that recombination occurred in the evolving pathway of FDJS03 isolates.

In summary, our work revealed the evolutionary history of FDJS03 strains, the cause of an outbreak of aseptic meningitis in China in 2003, and represented a starting point for studies to examine the effects of genetic features on various properties of E30 strains in China. Given the propensity of these viruses for outbreaks, better local and global monitoring is warranted.

 
We thank the prefectural hospitals in Yancheng City, Jiangsu Province, China, for providing clinical samples.

 
* Corresponding author. Mailing address: Department of Epidemiology, School of Public Health, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China. Phone: 86-21-54237435. Fax: 86-21-64037350. E-mail: epicorrespond{at}yahoo.com.cn.

Published ahead of print on 6 September 2006.

Ya Nan Zhao and David S. Perlin contributed equally to this paper.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, November 2006, p. 4142-4148, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.01049-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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