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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.
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).
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.
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).
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 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.
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 (Table1).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. (Fig.1).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.
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. (Fig.2),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.
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 (Table2)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.
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 (Table3).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 (Table3).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.
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. (Fig.3A).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. (Fig.3B).3B). These results revealed the possible evolution of genotype B4 from genotypes B3 and B2.
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. (Fig.3C).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. (Fig.3D).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.
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.
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).
Published ahead of print on 23 September 2009.
†Supplemental material for this article may be found at http://jcm.asm.org/.