Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Clinical Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Virology

Reemergence of Enterovirus 71 in 2008 in Taiwan: Dynamics of Genetic and Antigenic Evolution from 1998 to 2008

Sheng-Wen Huang, Yun-Wei Hsu, Derek J. Smith, David Kiang, Huey-Pin Tsai, Kuei-Hsiang Lin, Shih-Min Wang, Ching-Chung Liu, Ih-Jen Su, Jen-Ren Wang
Sheng-Wen Huang
1The Institute of Basic Medical Sciences
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yun-Wei Hsu
5Division of Infectious Diseases, National Health Research Institutes, Tainan, Taiwan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Derek J. Smith
8Department of Zoology, University of Cambridge, Cambridge, United Kingdom
9Department of Virology, Erasmus Medical Centre, Rotterdam, The Netherlands
10Fogarty International Center, National Institutes of Health, Bethesda, Maryland 20892
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David Kiang
6Viral and Rickettsial Disease Laboratories, California Department of Health Services, Richmond, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Huey-Pin Tsai
4Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kuei-Hsiang Lin
7Department of Clinical Laboratory, Kaoshiung Medical University, Kaoshiung, Taiwan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shih-Min Wang
3Department of Pediatrics, National Cheng Kung University, Tainan, Taiwan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ching-Chung Liu
3Department of Pediatrics, National Cheng Kung University, Tainan, Taiwan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ih-Jen Su
5Division of Infectious Diseases, National Health Research Institutes, Tainan, Taiwan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jen-Ren Wang
1The Institute of Basic Medical Sciences
2Department of Medical Laboratory Science and Biotechnology
4Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan
5Division of Infectious Diseases, National Health Research Institutes, Tainan, Taiwan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: jrwang@mail.ncku.edu.tw
DOI: 10.1128/JCM.00630-09
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

In recent years, enterovirus 71 (EV71) has been a cause of numerous outbreaks of hand-foot-and-mouth disease, with severe neurological complications in the Asia-Pacific region. The reemergence in Taiwan of EV71 genotype B5 in 2008 resulted in the largest outbreak of EV71 in Taiwan in the past 11 years. Phylogenetic analyses indicated that dominant genotype changes from B to C or C to B occurred at least three times between 1986 and 2008. Furthermore, antigenic cartography of EV71 by using neutralization tests revealed that the reemerging EV71 genotype B5 strains formed a separate cluster which was antigenically distinct from the B4 and C genotypes. Moreover, analyses of full-length genomic sequences of EV71 circulating in Taiwan during this period showed the occurrence of intra- and interserotypic recombination. Therefore, continuous surveillance of EV71 including the monitoring of genetic evolution and antigenic changes is recommended and may contribute to the development of a vaccine for EV71.

The genus Enterovirus ([EV] family Picornaviridae) contains numerous viruses that are pathogenic to humans. Human EVs (HEVs) have been classified into four species, HEV-A, HEV-B, HEV-C, and HEV-D, based on their sequence homologies (48). In contrast to other etiological agents of hand-foot-and-mouth disease that tend to cause mild and self-limiting disease, EV71 infection is often associated with other clinical manifestations including acute neurologic symptoms, such as poliomyelitis-like paralysis, encephalitis, aseptic meningitis, shock, and cardiac dysfunction (32).

Since 1969, when EV71 was first isolated in California, EV71-associated outbreaks have been reported worldwide (42). EV71 infection reached epidemic proportions, causing sporadic cases or outbreaks and then becoming prevalent around the Asia-Pacific region including Australia, Malaysia, Singapore, Japan, China, and Taiwan for the past 12 years (1, 16-18, 20, 25, 26, 28, 46, 53). Phylogenetic studies have classified EV71 into genotypes A, B, and C, which can be further subdivided into subgentotypes B1 to B5 and C1 to C5 (7, 8, 17, 20, 22, 25, 28, 41, 45, 52, 53). These reports indicated that the dominant EV71 strains circulating in the Asia-Pacific region varied genetically, suggesting that the virus was evolving.

Intertypic or intratypic recombination of EV71 has been reported to occur frequently in the region encoding the nonstructural proteins and could potentially influence the replication, tissue tropism, and virulence of EV71 (10, 11, 18). These studies emphasized the importance of full-genome sequencing for the surveillance of EV71 evolution. Therefore, to analyze the evolution of EV71, we performed phylogenetic analysis of the Taiwan isolates from 1986 and from 1998 and 2008 based on the complete genomic sequences. In addition, neutralizing activities of human antiserum against the various subgenotypes of EV71 were investigated to evaluate the antigenic changes of EV71. We found evidence for intertypic and intratypic recombination and demonstrated variation in antibody neutralizing activities indicating changes in antigenicity.

MATERIALS AND METHODS

Virus isolation and identification.EV71 isolated in Taiwan in 1986 and from 1998 to 2008 was investigated. The isolates from 1998 to 2008 were isolated from patients at National Cheng Kung University Medical Center in southern Taiwan. Six EV71 isolates from 1986 were isolated by the Virology Laboratory of Kaohsiung Medical University Hospital. Specimens from suspected EV-infected patients were inoculated into appropriate tissue cultures including A549, RD, GMK, and MRC-5 cells. EV71 strains were identified and typed antigenically by either neutralization tests or immunofluorescence tests using monoclonal antibodies (Chemicon International Inc.) (53).

Sequencing of EV71.One-tenth of the EV71 isolates were selected for sequencing analyses each month. The EV71 isolates were from a random sampling of patients with diverse clinical presentations, ranging from uncomplicated hand-foot-and-mouth disease to encephalitis and death. Viral genomic RNA was extracted from cell culture isolates by using a Viral RNA Purification Kit II (Geneaid), followed by reverse transcription-PCR and VP1 sequencing as previously described (7). Full-length genome sequences were determined as follows. Sequencing on both the 5′ and the 3′ termini of the genome was performed by 5′ RACE (RACE) and 3′ RACE Systems (Invitrogen) for rapid amplification of cDNA ends, according to manufacturer's instructions, with primers EV71-86 5GSP1, EV71-86 5GSP2, EV71-86 5GSP3, EV71-86 3GSP1, and EV71-86 3GSP2 (see Table S3 in the supplemental material). The amplified products were cloned into pGEM-T Easy (Promega) and sequenced with T7 and SP6 primers. The EV71 full-length genome product was amplified as previously described (5). Briefly, the viral genomic RNA was extracted, and reverse transcription-PCR was performed by using SuperScript II reverse transcriptase (Invitrogen) for reverse transcription and Advantage 2 polymerase (Clontech) for PCR. PCR products were cloned using a TOPO XL PCR cloning kit (Invitrogen) and sequenced. The specific primers used for genome sequencing are indicated in Table S3 in the supplemental material (18). The sequences were assembled with the ContigExpress module of Vector NTI Advance 8 (Invitrogen). Multiple sequence alignments were performed using Clustal X, version 1.83 (50). Some of the EV71 sequences from 1998 to 2005 were from previous studies (18, 52, 53). Accession numbers are given in Table S1 in the supplemental material.

Phylogenetic analyses and estimation of the dN/dS ratio in VP1 sequences.A phylogenetic tree was estimated by the general time reversible model of PAUP*, version 4.0b (35, 54). Statistical robustness of the 1,000 data sets was analyzed, and the estimation of the significance of branch lengths was determined by the maximum-likelihood method. Single-likelihood ancestor counting (SLAC) and fixed-effects likelihood (FEL) methods at the Datamonkey website (http://www.datamonkey.org ) were performed to examine nonsynonymous and synonymous substitution rates (dN and dS, respectively) for different genotypes and the selection signature in the EV71 VP1 protein coding region (33, 34). Selection pressure by the dN/dS ratio for each VP1 codon was measured, and P values were also calculated for these residues. The cutoff P value (<0.1) for the two-tailed extended binominal test was used to classify a site as positively or negatively selected in the SLAC method. In addition, the cutoff P value (<0.1) for the single degree of freedom likelihood ratio test (a chi-squared asymptotic is used) was used to classify a site as positively or negatively selected in the FEL method. The three-dimensional structure of the EV71 VP1 protein was predicted by using the (PS)2 program (Protein Structure Prediction Server; http://ps2.life.nctu.edu.tw/ ) at the Genomic Medicine and Biotechnology Development bioinformatics website (Bioinformatics Core for Genomic Medicine and Biotechnology Development; http://www.tbi.org.tw/about/index.htm ) (12).

Recombination analyses.To analyze EV71 and HEV-A genomes, we used a transition/transversion rate of 10 and a 50% consensus to exclude the poorly conserved sites (10). Resulting alignments were analyzed using bootscan analysis in SimPlot, version 3.5.1, with a neighbor-joining tree algorithm and maximum-likelihood distance model consisting of 1,000 pseudoreplicates (10, 27).

Neutralization test and antigenic cartography.Neutralization tests were performed using antiserum from patients infected by various genotypes of EV71, and samples were assayed in a microneutralization assay with RD cells (29). Titers were determined to 0.5 of a twofold dilution. The tabular neutralization data were analyzed manually and also using antigenic cartography (13, 39, 40, 47). Briefly, antigenic cartography is a way to visualize and increase the resolution of binding assay data, such as microneutralization data. In an antigenic map, the distance between a serum point S and antigen point A corresponds to the difference between the log2 of the maximum titer observed for serum S against any antigen and the log2 of the titer for serum S and antigen A. Thus, each titer in a neutralization assay table can be thought of as specifying a target distance for the points in an antigenic map. Modified multidimensional scaling methods are used to arrange the antigen and serum points in an antigenic map to best satisfy the target distances specified by the neutralization data. The result is a map in which the distance between points represents antigenic distance as measured by the binding assay.

RESULTS

Phylogenetic and epidemiologic analyses of the EV71 VP1 protein coding region.In 2008, a reemergent EV outbreak occurred in Taiwan which resulted in 373 severe cases and numerous fatalities (Taiwan CDC database; http://www.cdc.gov.tw ). The total number of EV71 cases in the National Cheng Kung University Medical Center in the same year also dramatically increased to 367 cases (Table 1). To compare the diversity of EV71 isolates in 2008 with those of other outbreaks, phylogenetic analyses were performed of the VP1 sequences of EV71 strains isolated in Taiwan in 1986 and from 1998 to 2008 and of other EV71 sequences derived from the GenBank (Fig. 1). Phylogenetic analyses revealed that genotypes B1, C2, B4, and C4 were the major genotypes circulating during the outbreaks of 1986, 1998, 2000 to 2001, and 2004 to 2005 in Taiwan, respectively. In 2008, genotype B5 became the dominant genotype, indicating that the major genotypes of EV71 continued to change from 1986 to 2008, alternating between B and C. Genotype B4 (isolate N5101-TW98, where N5101 is the isolate number, TW indicates Taiwan, and 98 indicates 1998) isolates were first detected in 1998 and subsequently became the dominant genotype in the outbreak of 2000 to 2001. Early detection followed by subsequent outbreaks also occurred from 2004 to 2005 and in 2008. Genotype C4 (N3340-TW02) and B5 (N2776-TW03 and N2838-TW03) were first isolated in Taiwan in 2002 and 2003, respectively, and became emergent strains in the outbreaks of 2004 to 2005 and 2008, respectively. Therefore, the epidemiological and phylogenetic results showed that EV71 strains which became dominant genotypes in outbreaks were usually found in circulation prior to the occurrence of the outbreak.

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

Phylogenetic analysis of the VP1 protein coding region of EV71. Phylogenetic analysis of the VP1 protein coding regions of EV71 isolates from 1986 and from 1998 to 2008 from Taiwan and from reference strains from the GenBank based on the VP1 protein coding region (nucleotides 2439 to 3278; 840 bp). The phylogenetic tree was estimated under the general time reversible model with the program PAUP*, version 4.0b. Bootstrap values (percentage of 1,000 pseudoreplicate data sets) of over 75% supporting each cluster are shown at the nodes. CA16 was included as an outgroup. AUS, Australia; SAR, Sarawak, Malaysia; KOR, Korea; SIN, Singapore.

View this table:
  • View inline
  • View popup
TABLE 1.

Yearly distribution of all EV71 isolates at National Cheng Kung University Medical Center for the period from 1998 to 2008

Antigenic cartography of EV71 by neutralization.Phylogenetic analysis of the VP1 protein coding region showed that EV71 isolates in these outbreaks were clustered into five genotypes, B1 in 1986, C2 in 1998, B4 in 2000 to 2001, C4 in 2004 to 2005, and B5 in 2008. Similar to the immunodominant proteins of EVs (37), genetic evolution of the VP1 protein coding region may contribute to antigenic diversity of the virus. To characterize the antigenic properties of EV71 that circulated in Taiwan, we selected strains representing various genotypes from outbreaks in 1986 and from 1998 to 2008 for microneutralization tests. In our neutralization table, human antiserum from EV71-infected patients during the period of 1998 to 2008 was titrated against isolates clustered in various genotypes (see Table S2 in the supplemental material). These sera obtained from infected individuals were divided into five categories (anti-C2, -B4, -C4, -C5, and -B5) based on the genotype of the infecting virus. A serum sample (sample YFW) from a healthy worker at the National Health Research Institutes was also included. However, the neutralization test table did not clearly show whether the antigenic differences of these EV71 viruses were contributed by different genotypes, and it was difficult to quantitatively analyze and interpret the results of the antigenic evolution of the EV71 isolates. To resolve this issue, antigenic cartography was used to construct an antigenic map from the microneutralization data. In the antigenic map (Fig. 2), the genotype B1 and B4 viruses clustered together while genotype C2 from 1998 was found in another antigenic cluster distinct from genotype B viruses. In comparison to genotype B, genotype C4 viruses were more closely related antigenically to genotype C2. Interestingly, the reemergent genotype B5 viruses in 2003 and 2008 mapped in a separate cluster that was not closer to either genotype B1/B4 or genotype C2/C4 in the antigenic map, indicating that the reemergent EV71 viruses in 2003 and 2008 were antigenically different from the other genotype B and genotype C viruses in the study.

FIG. 2.
  • Open in new tab
  • Download powerpoint
FIG. 2.

Antigenic map of EV71 isolates from 1998 to 2008 isolated in Taiwan. The relative positions of strains (colored shapes) and antisera (uncolored shapes) were adjusted such that the distances between strains and antisera in the map represent the corresponding neutralization assay measurements (see Table S2 in the supplemental material) with the least error. The periphery of each shape denotes a 0.5-unit increase in the total error; thus, size and shape represent a confidence area in the placement of the strain or antiserum. The vertical and horizontal lines represent antigenic distance, and because only the relative positions of antigens and antisera can be determined, the orientation of the map within these axes is free. The spacing between grid lines is 1 unit of antigenic distance, corresponding to a twofold dilution of antiserum in the neutralization assay. Isolate number and genotype of viruses or serum numbers and genotype of infected viruses are indicated.

To determine the amino acids which may contribute to the antigenic mapping characteristics, we aligned and compared the deduced amino acid sequences of selected viruses based on the sequences of their VP1 protein coding regions. The sequence comparisons (Table 2) showed that Glu43, Thr58, Thr184, and Ser240 were specific signature amino acids for genotype B including subgenotypes B1, B4, and B5 while Arg22 and Asp31 were represented in only genotype C2. Moreover, with two exceptions (N2838-TW03 and N2776-TW98), genotype B5 had an aspartic acid at position 164 similar to genotypes C2 and C4. Glu164 was observed in B4, suggesting that this residue may play an important role in the antigenic properties of EV71.

View this table:
  • View inline
  • View popup
TABLE 2.

VP1 amino acid sequence comparison of various EV71 genotypes

Phylogenetic analysis of EV71 VP1.Phylogenetic analyses of the nucleotide sequences of EV71 in the VP1 protein coding region indicated that EV71 continued to evolve from 1986 to 2008. The dominant EV71 strain in every major outbreak was antigenically distinct from the previous outbreak strain, including genotype changes from B1 to C2, C2 to B4, B4 to C4, and C4 to B5. To determine the association between these nucleotide and amino acid sequence mutations, mean dN/dS ratios were calculated by using the SLAC analysis method on the Datamonkey website (Table 3). We divided both the EV71 sequences from our study and the sequences published in the GenBank into genotype B (including B1 to B5) and genotype C (including genotypes C1 to C5) based on phylogenetic analyses. In our results, both genotype B and genotype C showed low mean dN/dS ratios (0.098 and 0.044, respectively), indicating that most of the nucleotide substitutions were synonymous. To further identify the mutations involved in EV71 VP1 evolution, SLAC and FEL analyses were performed to examine the dN/dS ratios of the individual sites in VP1 protein coding regions (Table 3). In genotype C viruses, 24 and 89 negative selection sites were found by using SLAC and FEL methods, respectively; however, neither SLAC nor FEL found evidence of positive selection in the VP1 protein coding region. In contrast, one (SLAC) or two (FEL) positively selected sites were identified in genotype B sequences, and 38 (SLAC) and 84 (FEL) negative selection sites were identified. In SLAC analysis results, codon 145 was determined as a positive selection site, and codons 145 and 98 were determined to be positive selection sites by FEL analysis. The two positive sites were located in the BC (codon 98) and DE (codon 145) loops of EV71, according to the three-dimensional structural prediction of the EV71 VP1 protein by the (PS)2 program (12). These results showed a high frequency of synonymous mutations of EV71 and demonstrated that genotype B but not genotype C was positively selected at codon 145 and/or codon 98 in the VP1 protein.

View this table:
  • View inline
  • View popup
TABLE 3.

Evolutionary pattern of EV71 in VP1 protein coding region

Intratypic and intertypic recombination between EV71 strains in Taiwan and other HEV-A viruses.In addition to the analysis of the capsid protein, which contains determinants for cell binding, we also analyzed the untranslated region and nonstructural protein coding regions of EV71 in 1986 and between 1998 and 2008. The complete genome sequences were aligned with other HEV-A sequences to examine the genetic evolution of EV71 isolates. Similarity plot analysis was first performed using a 50% consensus sequence of each virus in 1998 or of individual viruses isolated from 1999 to 2008.

In 1998, genotype C2 caused a large outbreak associated with severe encephalitis. By bootscan analysis, there were two recombination crossover points with a high χ2 value, which supported the presence of EV71 subgenotype C and coxsackievirus A8 (CA8) genome sequences within the genome of subgenotype C2 isolates (Fig. 3A). These results showed a possible recombination event between sequences of the CA8 nonstructural protein coding region and the EV71 structural protein coding region. Isolates of subgenotype B4, which were initially identified in 1998 in Taiwan, caused large outbreaks from 2000 to 2001 and continued to circulate in 2003. Genotype B4 of EV71 circulating in Taiwan from 1999 to 2002 (N7008-TW99, as a representative) were phylogenetically close to genotype B3 in the 5′ untranslated region and P1 polyprotein coding regions and similar to genotype B2 in the P3 polyprotein coding region, each with a high bootstrap value (Fig. 3B). These results revealed the possible evolution of genotype B4 from genotypes B3 and B2.

  • Open in new tab
  • Download powerpoint
  • Open in new tab
  • Download powerpoint
FIG. 3.

Dynamic analysis of EV71 genetic recombination from 1998 to 2008. Complete genome sequences of EV71 isolates of various genotypes were analyzed by bootscan analysis, including 1998 viruses (A), N7008-TW99 (B), N2838-TW03 (C), and S0584-TW04 (D). This analysis was calculated by SimPlot, version 3.5.1, using the neighbor-joining tree algorithm (Kimura distance model) in a sliding window of 500 bp with a 20-bp step. The EV71 genetic map is shown at the top of each panel.

After the occurrence of genotype B4 for 5 years (1998 to 2003), only some sporadic genotype B4 and B5 viruses were isolated in 2003 in Taiwan. We also analyzed the full genome sequence of one genotype B5 strain (N2838-TW03) from 2003 (Fig. 3C). Both similarity plot and bootscan analysis showed that the genotype B5 genome was more similar to genotype B4 (92.9 to 97.8%) than to other genotypes, especially in the nonstructural region. These results were supported by a high bootstrap value. In 2004 and 2005, a subgenotype shift was observed from genotype B4 to genotype C4, which emerged and became predominant in Taiwan. Bootscan analysis revealed that genotype C4 was similar to EV71 genotype C in the P1 polyprotein coding region and to genotype B from the 2B to 3B protein coding region (with a high bootstrap value of up to 95%) (Fig. 3D). The patterns from similarity plots and bootscan analysis performed with strains from 2004 and 2005 were similar to those reported with N3340-TW02 in a previous study (18). These results suggested that the recombination of the subgenotype C structural protein coding region with the genotype B nonstructural protein coding region resulted in the subgenotype C4 virus responsible for the 2004 and 2005 outbreaks. In 2008, the results showed that genotype B5 exhibited phylogenetic similarity to genotype B4 in the nonstructural region (data not shown), which was identical to N2838-TW03 in 2003.

DISCUSSION

Several molecular epidemiology studies of EV71 have revealed the genetic evolution of EV71 isolates by using partial- or whole-genome sequencing (7, 8, 15, 17, 20, 22, 23, 25, 28, 32, 45, 53); however, to our knowledge, little has been reported about the antigenic evolution of EV71. A previous genetic and antigenic study, which examined the neutralization antibody titer of antiserum from EV71-infected rabbits and patients, suggested cross-neutralization activities of the antiserum against either homogenous or heterogeneous EV71 genotype viruses (22). In our neutralization data, human antiserum showed various neutralization antibody titers against different viruses, indicating antigenic variation among viruses. In this study, we report an antigenic map of EV71 which showed that genotypes B1/B4, B5, and C2/C4 split into different groups in the antigenic map, and the reemerging B5 exhibited different antigenic properties from other genotypes. In comparisons of amino acid sequences of viruses belonging to different genotypes, the VP1 residues at positions 43, 58, 184, and 240 were conserved in homogeneous genotypes but diverged in heterogeneous genotypes. Nonetheless, the reemerging B5 isolates that clustered in an individual group on the antigenic map had an Asp164 residue in VP1, which was identical with genotype C but not genotype B viruses. Therefore, it appeared that residue 164 of the capsid protein, VP1, might contribute to the distinct antigenicity of genotype B5 strains resulting from selective pressures in the environment. In addition, the evolutionary pattern of EV71 indicated that two positive selection sites including codons 98 and 145 of genotype B viruses were exposed regions of VP1. These two positively selected positions were also reported to be in the BC loop which contains neutralizing antigenic sites and is located in the canyon rim, a region involved in the receptor binding for EV71 (31, 43). Moreover, two additional VP1 protein sites at positions 58 and 241 may also be influenced by positive selection pressure, based on a comparison of EV71 sequences from Australia since 1999 and from the United States from 1980 to 1989, respectively (43). EV71 isolates from 1998 to 2008 in Taiwan did not show positive selection at position 58 and 241 but presented polymorphisms at these sites (43). Furthermore, in the mouse model study of EV71, codon 145 in VP1 was also found as the virulence determinant in an NOD/SCID mouse model and affected the binding of EV71 to RD cells (4). Amino acid residues 98 and 145 in VP1 affected EV71 infectivity in a macrophage cell line (data not shown). However, the effect of these residues still needs to be examined by genomic investigations, and the effects of other residues in VP1, VP2, or VP3 proteins, which can potentially cocontribute to the neutralizing determinants, need to be investigated further.

A previous report of EV71 molecular surveillance in the United Kingdom showed that genotype C was the only genotype found in the study period (6); however, in Taiwan the EV71 genotype continued to change between outbreaks: B1 in 1986, C2 in 1998, B4 in 2000 and 2001, C4 in 2004 and 2005, and B5 in 2008. Our phylogenetic analyses revealed that the genotypes which resulted in outbreaks were usually in circulation 2 to 5 years before the outbreaks occurred. For instance, approximately 10% of EV71 isolates in 1998 were genotype B4, and this genotype later became the predominant strain in the 2000 to 2001 outbreak. In addition, N3340-TW02 (genotype C4) was first isolated in 2002, and C4 became the outbreak genotype from 2004 to 2005. Finally, genotype B5 viruses were initially isolated in 2003 and reemerged in the 2008 outbreak. It was also shown that every large EV71 epidemic was associated with a genotypic change (genotype B1 to C2, C2 to B4, B4 to C4, and C4 to B5) from the previous outbreak. The shift between the different types may reflect the ability of different strains to spread efficiently as a result of a lack of immunity within the community. Resulting antigenic changes can potentially help EV71 escape from herd immunity and circulate in the human population. Therefore, antigenic properties provide another possible predictor for future outbreaks, and continual surveillance is important. In our surveillance results, the genotype C5 isolate N1859-TW05 from a child with severe illness was identified in 2005. Subsequently, genotype C5 strains were identified in low numbers in 2006 and 2007 in Taiwan (19). Genotype C5 EV71 was first described in Japan and then isolated in Vietnam in 2005 with high prevalence and mortality (30, 51). Whether this new genotype C5 of EV71 may cause a future outbreak in Taiwan should be monitored.

In addition to analysis of the antigenic evolution in the capsid protein region, we also examined the dynamics of EV71 evolution in Taiwan through complete genome sequencing and phylogenetic analysis. The EV71 strain isolated from 1998 belonged to genotype C2 and included the untranslated regions and a nonstructural protein coding region derived from CA8, which suggested that this EV71-CA8 recombinant caused the large epidemic in 1998 in Taiwan. Partial EV71-CA8 gene sequences were also reported by Chan et al. (10); in addition, they found genotype B3 EV71 recombined with coxsakievirus A16 (CA16) in the P3 polyprotein coding region (11). However, unlike CA16, the resulting genotype B3/CA16 recombinant EV71 in the P3 polyprotein coding region did not increase neurovirulence of EV71 in neonatal mice (9). Moreover, the investigators reported that genotype B4 and C4 viruses were found to be recombinants between genotypes B3 and B2 and between genotypes C and B, respectively. In our study, recombinants of genotypes B3 and B2 as well as those with genotype B4 viruses from 1998 to 2002 and genotype C4 from 2002 to 2005 (C/B recombinants) in Taiwan suggest an epidemiological link between EV71s from Taiwan and Malaysia. In the investigation of Chan et al., the authors also found that the P3 polyprotein coding region of genotype C4 from China (SHZH-98 and SHZH-98) recombined between genotype C and CA16-like viruses (10). In addition to EV71, other recombinant EVs such as poliovirus have been reported in recent years (2, 3, 21, 36, 38, 44, 49, 55). In outbreaks of circulating vaccine-derived polioviruses (cVDPVs), polioviruses that were recombinants of vaccine polioviruses and HEV-C became dominant and pathogenic (2, 3, 14, 21, 24, 36, 38, 44, 49, 55). It has also been shown that recombination between vaccine poliovirus strains and HEV-C resulted in cVDPVs which displayed increased neurovirulence, as in transgenic poliovirus-receptor mice in vivo (21). Similar to cVDPV outbreaks, recombination of EV71 viruses with other HEV-A viruses or various lineages of EV71 resulted in strains which became predominant in these large outbreaks around the Asia-Pacific region and caused many severe cases and even deaths. These results suggest that recombination between EV71 subgenotypes or other EVs might promote the reemergence of EV71.

In summary, genotype B5 caused the reemergence of EV71 in 2008 with genetic and antigenic diversity from other genotypes. Dynamics of genetic and antigenic evolution of EV71 in the recent decade showed that genotype shift with antigenic property changes and genome recombination contributed to the emergence and the reemergence of EV71 in the Asia-Pacific region. Therefore, continuous surveillance of the genetic evolution of EV71 viruses with antigenic investigations is needed to monitor the evolution of EV71 and would help in EV71 vaccine strain selection.

ACKNOWLEDGMENTS

We thank the members of Virology Laboratory at National Cheng Kung University for isolation of EV71 for this study.

This study was supported by National Health Research Institutes grants, National Science Council grant NSC 97-3112-B-006, and Department of Health, Taiwan Centers for Disease Control grant CB097135. D.J.S. was supported by a Director's Pioneer Award from the U.S. NIH (grant DP1-OD000490-01).

FOOTNOTES

    • Received 28 March 2009.
    • Returned for modification 3 June 2009.
    • Accepted 12 September 2009.
  • Copyright © 2009 American Society for Microbiology

REFERENCES

  1. 1.↵
    AbuBakar, S., H. Y. Chee, M. F. Al-Kobaisi, J. Xiaoshan, K. B. Chua, and S. K. Lam. 1999. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res.61:1-9.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Adu, F., J. Iber, D. Bukbuk, N. Gumede, S. J. Yang, J. Jorba, R. Campagnoli, W. F. Sule, C. F. Yang, C. Burns, M. Pallansch, T. Harry, and O. Kew. 2007. Isolation of recombinant type 2 vaccine-derived poliovirus (VDPV) from a Nigerian child. Virus Res.127:17-25.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    Agol, V. I. 2006. Molecular mechanisms of poliovirus variation and evolution. Curr. Top. Microbiol. Immunol.299:211-259.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    Arita, M., Y. Ami, T. Wakita, and H. Shimizu. 2008. Cooperative effect of the attenuation determinants derived from poliovirus Sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model. J. Virol.82:1787-1797.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Arita, M., N. Nagata, N. Iwata, Y. Ami, Y. Suzaki, K. Mizuta, T. Iwasaki, T. Sata, T. Wakita, and H. Shimizu. 2007. An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J. Virol.81:9386-9395.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Bible, J. M., M. Iturriza-Gomara, B. Megson, D. Brown, P. Pantelidis, P. Earl, J. Bendig, and C. Y. Tong. 2008. Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006. J. Clin. Microbiol.46:3192-3200.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Brown, B. A., M. S. Oberste, J. P. Alexander, Jr., M. L. Kennett, and M. A. Pallansch. 1999. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J. Virol.73:9969-9975.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    Cardosa, M. J., D. Perera, B. A. Brown, D. Cheon, H. M. Chan, K. P. Chan, H. Cho, and P. McMinn. 2003. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg. Infect. Dis.9:461-468.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Chan, Y. F., and S. AbuBakar. 2005. Human enterovirus 71 subgenotype B3 lacks coxsackievirus A16-like neurovirulence in mice infection. Virol. J.2:74.
    OpenUrlCrossRefPubMed
  10. 10.↵
    Chan, Y. F., and S. AbuBakar. 2006. Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes. BMC Microbiol.6:74.
    OpenUrlCrossRefPubMed
  11. 11.↵
    Chan, Y. F., and S. AbuBaker. 2004. Recombinant human enterovirus 71 in hand, foot and mouth disease patients. Emerg. Infect. Dis.10:1468-1470.
    OpenUrlCrossRefPubMed
  12. 12.↵
    Chen, C. C., J. K. Hwang, and J. M. Yang. 2006. (PS)2: protein structure prediction server. Nucleic Acids Res.34:W152-W157.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    de Jong, J. C., D. J. Smith, A. S. Lapedes, I. Donatelli, L. Campitelli, G. Barigazzi, K. Van Reeth, T. C. Jones, G. F. Rimmelzwaan, A. D. Osterhaus, and R. A. Fouchier. 2007. Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe. J. Virol.81:4315-4322.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    Estivariz, C. F., M. A. Watkins, D. Handoko, R. Rusipah, J. Deshpande, B. J. Rana, E. Irawan, D. Widhiastuti, M. A. Pallansch, A. Thapa, and S. Imari. 2008. A large vaccine-derived poliovirus outbreak on Madura Island-Indonesia, 2005. J. Infect. Dis.197:347-354.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    Herrero, L. J., C. S. Lee, R. J. Hurrelbrink, B. H. Chua, K. B. Chua, and P. C. McMinn. 2003. Molecular epidemiology of enterovirus 71 in peninsular Malaysia, 1997-2000. Arch. Virol.148:1369-1385.
    OpenUrlCrossRefPubMedWeb of Science
  16. 16.↵
    Ho, M., E. R. Chen, K. H. Hsu, S. J. Twu, K. T. Chen, S. F. Tsai, J. R. Wang, and S. R. Shih for the Taiwan Enterovirus Epidemic Working Group. 1999. An epidemic of enterovirus 71 infection in Taiwan. N. Engl. J. Med.341:929-935.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    Hosoya, M., Y. Kawasaki, M. Sato, K. Honzumi, A. Kato, T. Hiroshima, H. Ishiko, and H. Suzuki. 2006. Genetic diversity of enterovirus 71 associated with hand, foot and mouth disease epidemics in Japan from 1983 to 2003. Pediatr. Infect. Dis. J.25:691-694.
    OpenUrlCrossRefPubMed
  18. 18.↵
    Huang, S. C., Y. W. Hsu, H. C. Wang, S. W. Huang, D. Kiang, H. P. Tsai, S. M. Wang, C. C. Liu, K. H. Lin, I. J. Su, and J. R. Wang. 2008. Appearance of intratypic recombination of enterovirus 71 in Taiwan from 2002 to 2005. Virus Res.131:250-259.
    OpenUrlCrossRefPubMed
  19. 19.↵
    Huang, Y. P., T. L. Lin, C. Y. Kuo, M. W. Lin, C. Y. Yao, H. W. Liao, L. C. Hsu, C. F. Yang, J. Y. Yang, P. J. Chen, and H. S. Wu. 2008. The circulation of subgenogroups B5 and C5 of enterovirus 71 in Taiwan from 2006 to 2007. Virus Res.
  20. 20.↵
    Jee, Y. M., D. S. Cheon, K. Kim, J. H. Cho, Y. S. Chung, J. Lee, S. H. Lee, K. S. Park, J. H. Lee, E. C. Kim, H. J. Chung, D. S. Kim, J. D. Yoon, and H. W. Cho. 2003. Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Korea during 2000. Arch. Virol.148:1735-1746.
    OpenUrlCrossRefPubMed
  21. 21.↵
    Jegouic, S., M. L. Joffret, C. Blanchard, F. B. Riquet, C. Perret, I. Pelletier, F. Colbere-Garapin, M. Rakoto-Andrianarivelo, and F. Delpeyroux. 2009. Recombination between polioviruses and co-circulating coxsackie A viruses: role in the emergence of pathogenic vaccine-derived polioviruses. PLoS Pathog.5:e1000412.
    OpenUrlCrossRefPubMed
  22. 22.↵
    Kung, S. H., S. F. Wang, C. W. Huang, C. C. Hsu, H. F. Liu, and J. Y. Yang. 2007. Genetic and antigenic analyses of enterovirus 71 isolates in Taiwan during 1998-2005. Clin. Microbiol. Infect.13:782-787.
    OpenUrlCrossRefPubMed
  23. 23.↵
    Li, L., Y. He, H. Yang, J. Zhu, X. Xu, J. Dong, Y. Zhu, and Q. Jin. 2005. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People's Republic of China. J. Clin. Microbiol.43:3835-3839.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    Liang, X., Y. Zhang, W. Xu, N. Wen, S. Zuo, L. A. Lee, and J. Yu. 2006. An outbreak of poliomyelitis caused by type 1 vaccine-derived poliovirus in China. J. Infect. Dis.194:545-551.
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    Lin, K. H., K. P. Hwang, G. M. Ke, C. F. Wang, L. Y. Ke, Y. T. Hsu, Y. C. Tung, P. Y. Chu, B. H. Chen, H. L. Chen, C. L. Kao, J. R. Wang, H. L. Eng, S. Y. Wang, L. C. Hsu, and H. Y. Chen. 2006. Evolution of EV71 genogroup in Taiwan from 1998 to 2005: an emerging of subgenogroup C4 of EV71. J. Med. Virol.78:254-262.
    OpenUrlCrossRefPubMedWeb of Science
  26. 26.↵
    Lin, T. Y., L. Y. Chang, S. H. Hsia, Y. C. Huang, C. H. Chiu, C. Hsueh, S. R. Shih, C. C. Liu, and M. H. Wu. 2002. The 1998 enterovirus 71 outbreak in Taiwan: pathogenesis and management. Clin. Infect. Dis.34(Suppl. 2):S52-S57.
    OpenUrlCrossRefPubMedWeb of Science
  27. 27.↵
    Lole, K. S., R. C. Bollinger, R. S. Paranjape, D. Gadkari, S. S. Kulkarni, N. G. Novak, R. Ingersoll, H. W. Sheppard, and S. C. Ray. 1999. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol.73:152-160.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    McMinn, P., K. Lindsay, D. Perera, H. M. Chan, K. P. Chan, and M. J. Cardosa. 2001. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. J. Virol.75:7732-7738.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    Melnick, J. L., and I. L. Wimberly. 1985. Lyophilized combination pools of enterovirus equine antisera: new LBM pools prepared from reserves of antisera stored frozen for two decades. Bull. W. H. O.63:543-550.
    OpenUrlPubMedWeb of Science
  30. 30.↵
    Mizuta, K., C. Abiko, T. Murata, Y. Matsuzaki, T. Itagaki, K. Sanjoh, M. Sakamoto, S. Hongo, S. Murayama, and K. Hayasaka. 2005. Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J. Clin. Microbiol.43:6171-6175.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    Oberste, M. S., K. Maher, D. R. Kilpatrick, and M. A. Pallansch. 1999. Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J. Virol.73:1941-1948.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    Ooi, M. H., S. C. Wong, Y. Podin, W. Akin, S. del Sel, A. Mohan, C. H. Chieng, D. Perera, D. Clear, D. Wong, E. Blake, J. Cardosa, and T. Solomon. 2007. Human enterovirus 71 disease in Sarawak, Malaysia: a prospective clinical, virological, and molecular epidemiological study. Clin. Infect. Dis.44:646-656.
    OpenUrlCrossRefPubMedWeb of Science
  33. 33.↵
    Pond, S. L., and S. D. Frost. 2005. Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics21:2531-2533.
    OpenUrlCrossRefPubMedWeb of Science
  34. 34.↵
    Poon, A. F., S. D. Frost, and S. L. Pond. 2009. Detecting signatures of selection from DNA sequences using Datamonkey. Methods Mol. Biol.537:163-183.
    OpenUrlCrossRefPubMedWeb of Science
  35. 35.↵
    Posada, D. 2003. Using MODELTEST and PAUP* to select a model of nucleotide substitution. Curr. Protoc. Bioinformatics, chapter 6, unit 6.5. http://www.currentprotocols.com/protocol/bi0605 .
  36. 36.↵
    Rakoto-Andrianarivelo, M., N. Gumede, S. Jegouic, J. Balanant, S. N. Andriamamonjy, S. Rabemanantsoa, M. Birmingham, B. Randriamanalina, L. Nkolomoni, M. Venter, B. D. Schoub, F. Delpeyroux, and J. M. Reynes. 2008. Reemergence of recombinant vaccine-derived poliovirus outbreak in Madagascar. J. Infect. Dis.197:1427-1435.
    OpenUrlCrossRefPubMedWeb of Science
  37. 37.↵
    Rossmann, M. G., E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H. J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, et al. 1985. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature317:145-153.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    Rousset, D., M. Rakoto-Andrianarivelo, R. Razafindratsimandresy, B. Randriamanalina, S. Guillot, J. Balanant, P. Mauclere, and F. Delpeyroux. 2003. Recombinant vaccine-derived poliovirus in Madagascar. Emerg. Infect. Dis.9:885-887.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    Russell, C. A., T. C. Jones, I. G. Barr, N. J. Cox, R. J. Garten, V. Gregory, I. D. Gust, A. W. Hampson, A. J. Hay, A. C. Hurt, J. C. de Jong, A. Kelso, A. I. Klimov, T. Kageyama, N. Komadina, A. S. Lapedes, Y. P. Lin, A. Mosterin, M. Obuchi, T. Odagiri, A. D. Osterhaus, G. F. Rimmelzwaan, M. W. Shaw, E. Skepner, K. Stohr, M. Tashiro, R. A. Fouchier, and D. J. Smith. 2008. The global circulation of seasonal influenza A (H3N2) viruses. Science320:340-346.
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    Russell, C. A., J. T., I. G. Barr, N. J. Cox, R. J. Garten, V. Gregory, et al. 2008. Influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses. Vaccine26:D31-D34.
    OpenUrlCrossRefPubMedWeb of Science
  41. 41.↵
    Sanders, S. A., L. J. Herrero, K. McPhie, S. S. Chow, M. E. Craig, D. E. Dwyer, W. Rawlinson, and P. C. McMinn. 2006. Molecular epidemiology of enterovirus 71 over two decades in an Australian urban community. Arch. Virol.151:1003-1013.
    OpenUrlCrossRefPubMed
  42. 42.↵
    Schmidt, N. J., E. H. Lennette, and H. H. Ho. 1974. An apparently new enterovirus isolated from patients with disease of the central nervous system. J. Infect. Dis.129:304-309.
    OpenUrlCrossRefPubMedWeb of Science
  43. 43.↵
    Shi, W.-F., Z. Zhang, A.-S. Dun, Y.-Z. Zhang, G.-F. Yu, D.-M. Zhuang, and C.-D. Zhu. 2009. Positive selection analysis of VP1 genes of worldwide human enterovirus 71 viruses. Virol. Sinica24:59-64.
    OpenUrlCrossRef
  44. 44.↵
    Shimizu, H., B. Thorley, F. J. Paladin, K. A. Brussen, V. Stambos, L. Yuen, A. Utama, Y. Tano, M. Arita, H. Yoshida, T. Yoneyama, A. Benegas, S. Roesel, M. Pallansch, O. Kew, and T. Miyamura. 2004. Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001. J. Virol.78:13512-13521.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    Shimizu, H., A. Utama, N. Onnimala, C. Li, Z. Li-Bi, M. Yu-Jie, Y. Pongsuwanna, and T. Miyamura. 2004. Molecular epidemiology of enterovirus 71 infection in the Western Pacific Region. Pediatr. Int.46:231-235.
    OpenUrlCrossRefPubMedWeb of Science
  46. 46.↵
    Shimizu, H., A. Utama, K. Yoshii, H. Yoshida, T. Yoneyama, M. Sinniah, M. A. Yusof, Y. Okuno, N. Okabe, S. R. Shih, H. Y. Chen, G. R. Wang, C. L. Kao, K. S. Chang, T. Miyamura, and A. Hagiwara. 1999. Enterovirus 71 from fatal and nonfatal cases of hand, foot and mouth disease epidemics in Malaysia, Japan and Taiwan in 1997-1998. Jpn. J. Infect. Dis.52:12-15.
    OpenUrlPubMed
  47. 47.↵
    Smith, D. J., A. S. Lapedes, J. C. de Jong, T. M. Bestebroer, G. F. Rimmelzwaan, A. D. Osterhaus, and R. A. Fouchier. 2004. Mapping the antigenic and genetic evolution of influenza virus. Science305:371-376.
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    Stanway, G., F. Brown, P. Christian, T. Hovi, T. Hyypiä, A. M. Q. King, N. J. Knowles, S. M. Lemon, P. D. Minor, M. A. Pallansch, A. C. Palmenberg, and T. Skern. 2005. Family Picornaviridae, p. 757-778. In C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (ed.), Virus taxonomy: classification and nomenclature of viruses. Eighth report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, San Diego, CA.
  49. 49.↵
    Tapparel, C., T. Junier, D. Gerlach, S. Van-Belle, L. Turin, S. Cordey, K. Muhlemann, N. Regamey, J. D. Aubert, P. M. Soccal, P. Eigenmann, E. Zdobnov, and L. Kaiser. 2009. New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses. Emerg. Infect. Dis.15:719-726.
    OpenUrlCrossRefPubMedWeb of Science
  50. 50.↵
    Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res.25:4876-4882.
    OpenUrlCrossRefPubMedWeb of Science
  51. 51.↵
    Tu, P. V., N. T. Thao, D. Perera, T. K. Huu, N. T. Tien, T. C. Thuong, O. M. How, M. J. Cardosa, and P. C. McMinn. 2007. Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam, 2005. Emerg. Infect. Dis.13:1733-1741.
    OpenUrlCrossRefPubMed
  52. 52.↵
    Wang, J. R., H. P. Tsai, P. F. Chen, Y. J. Lai, J. J. Yan, D. Kiang, K. H. Lin, C. C. Liu, and I. J. Su. 2000. An outbreak of enterovirus 71 infection in Taiwan, 1998. II. Laboratory diagnosis and genetic analysis. J. Clin. Virol.17:91-99.
    OpenUrlCrossRefPubMed
  53. 53.↵
    Wang, J. R., Y. C. Tuan, H. P. Tsai, J. J. Yan, C. C. Liu, and I. J. Su. 2002. Change of major genotype of enterovirus 71 in outbreaks of hand-foot-and-mouth disease in Taiwan between 1998 and 2000. J. Clin. Microbiol.40:10-15.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    Wilgenbusch, J. C., and D. Swofford. 2003. Inferring evolutionary trees with PAUP*. Curr. Protoc Bioinformatics, chapter 6, unit 6.4. http://www.currentprotocols.com/protocol/bi0604 .
  55. 55.↵
    Yang, C. F., T. Naguib, S. J. Yang, E. Nasr, J. Jorba, N. Ahmed, R. Campagnoli, H. van der Avoort, H. Shimizu, T. Yoneyama, T. Miyamura, M. Pallansch, and O. Kew. 2003. Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993. J. Virol.77:8366-8377.
    OpenUrlAbstract/FREE Full Text
PreviousNext
Back to top
Download PDF
Citation Tools
Reemergence of Enterovirus 71 in 2008 in Taiwan: Dynamics of Genetic and Antigenic Evolution from 1998 to 2008
Sheng-Wen Huang, Yun-Wei Hsu, Derek J. Smith, David Kiang, Huey-Pin Tsai, Kuei-Hsiang Lin, Shih-Min Wang, Ching-Chung Liu, Ih-Jen Su, Jen-Ren Wang
Journal of Clinical Microbiology Oct 2009, 47 (11) 3653-3662; DOI: 10.1128/JCM.00630-09

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Clinical Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Reemergence of Enterovirus 71 in 2008 in Taiwan: Dynamics of Genetic and Antigenic Evolution from 1998 to 2008
(Your Name) has forwarded a page to you from Journal of Clinical Microbiology
(Your Name) thought you would be interested in this article in Journal of Clinical Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Reemergence of Enterovirus 71 in 2008 in Taiwan: Dynamics of Genetic and Antigenic Evolution from 1998 to 2008
Sheng-Wen Huang, Yun-Wei Hsu, Derek J. Smith, David Kiang, Huey-Pin Tsai, Kuei-Hsiang Lin, Shih-Min Wang, Ching-Chung Liu, Ih-Jen Su, Jen-Ren Wang
Journal of Clinical Microbiology Oct 2009, 47 (11) 3653-3662; DOI: 10.1128/JCM.00630-09
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Antigens, Viral
Disease Outbreaks
Enterovirus A, Human
Enterovirus Infections
Evolution, Molecular
RNA, Viral

Related Articles

Cited By...

About

  • About JCM
  • Editor in Chief
  • Board of Editors
  • Editor Conflicts of Interest
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Resources for Clinical Microbiologists
  • Ethics
  • Contact Us

Follow #JClinMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

 

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0095-1137; Online ISSN: 1098-660X