Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes
© Yoke-Fun and AbuBakar; licensee BioMed Central Ltd. 2006
Received: 22 June 2006
Accepted: 30 August 2006
Published: 30 August 2006
Human enterovirus 71 (EV-71) is a common causative agent of hand, foot and mouth disease (HFMD). In recent years, the virus has caused several outbreaks with high numbers of deaths and severe neurological complications. Several new EV-71 subgenotypes were identified from these outbreaks. The mechanisms that contributed to the emergence of these subgenotypes are unknown.
Six EV-71 isolates from an outbreak in Malaysia, in 1997, were sequenced completely. These isolates were identified as EV-71 subgenotypes, B3, B4 and C2. A phylogenetic tree that correlated well with the present enterovirus classification scheme was established using these full genome sequences and all other available full genome sequences of EV-71 and human enterovirus A (HEV-A). Using the 5' UTR, P2 and P3 genomic regions, however, isolates of EV-71 subgenotypes B3 and C4 segregated away from other EV-71 subgenotypes into a cluster together with coxsackievirus A16 (CV-A16/G10) and EV-71 subgenotype C2 clustered with CV-A8. Results from the similarity plot analyses supported the clustering of these isolates with other HEV-A. In contrast, at the same genomic regions, a CV-A16 isolate, Tainan5079, clustered with EV-71. This suggests that amongst EV-71 and CV-A16, only the structural genes were conserved. The 3' end of the virus genome varied and consisted of sequences highly similar to various HEV-A viruses. Numerous recombination crossover breakpoints were identified within the non-structural genes of some of these newer EV-71 subgenotypes.
Phylogenetic evidence obtained from analyses of the full genome sequence supports the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the most common causal agent of HFMD. It is suggested that these recombination events played important roles in the emergence of the various EV-71 subgenotypes.
More than sixty different types of human enteroviruses have been identified . Species human enterovirus A (HEV-A) which include 11 members of the coxsackievirus A (CV-A) group; CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A7, CV-A8, CV-A10, CV-12, CV-14, CV-A16 and human enterovirus 71 (EV-71) are associated with several human diseases [1, 2]. CV-A2, CV-A6, CV-A8 and CV-A10 are known to cause herpangina ; CV-A2, CV-A4, CV-A7 and CV-A10 cause aseptic meningitis  and CV-A5, CV-A10, CV-A16 and EV-71 usually cause hand, foot and mouth disease (HFMD) . Amongst these HEV-A viruses, only EV-71 is associated with major outbreaks involving infections with severe neurological complications and deaths. Recent outbreaks in Malaysia, Taiwan and Vietnam claimed more than 130 deaths [3, 4]. In these outbreaks, EV-71 was isolated from patients' specimens and post-mortem tissues, suggesting its direct involvement as the etiologic agent [5, 6]. Molecular epidemiological investigations performed using the partial gene sequences of all available EV-71 isolates suggested the presence of several EV-71 lineages co-circulating during most of the Asian outbreaks [3, 6–11]. EV-71 subgenotypes B3, B4, C1 and C2 were reported in Malaysia with subgenotype B3 being the most prevalent in the 1997 outbreak [3, 7–9]. The subgenotype B4 was present initially at a low level but became endemic and eventually replaced subgenotype B3 as the predominant virus in the subsequent outbreaks in Malaysia and the region [7, 9]. Neither the B3 nor B4 subgenotype, however, was associated with any fatal infections outside the Malaysia 1997 outbreak. Similarly, the C2 isolates were identified as the main causal agent of the HFMD-associated severe and fatal infections during the 1998 Taiwan outbreak [6, 10]. These isolates also caused severe infections in the Perth 1999 outbreak [7, 9] but no report of serious infection or death attributed to these isolates was recorded from the Malaysia 1997 outbreak [8, 12]. Recently, two new EV-71 subgenotypes, C3 and C4 were proposed to accommodate EV-71 strains isolated from cases of aseptic meningitis and paralysis in Korea and China, respectively [7, 13, 14]. There were no reports, however, that the subgenotype C4 isolates were associated with severe infections [13, 14]. The emergence of the various EV-71 subgenotypes with varying degree of clinical severity is puzzling. The viruses together with other HEV-A viruses are known to be present in the region and usually cause the typical mild HFMD [7–9, 15]. In the present study, the potential mechanisms that could play important role in the emergence of the different EV-71 subgenotypes are phylogenetically investigated.
Phylogenetic relationships between EV-71 and other HEV-A viruses
The complete genome sequences of all the EV-71 isolates were aligned with other available HEV-A complete genome sequences obtained from the Genbank. The total length of the genomes varied between 7395–7434 nucleotides and they encode for 2189–2201 amino acids. The differences in genome length were caused by the presence of deletions or insertions within the 5' UTR, capsid gene and 3' UTR sequences. The capsid genome region of EV-71 was longer (1 – 4 amino acids) or of the same length with other HEV-A viruses, with the exception of CV-A4 and CV-A6 which have eight extra amino acid insertions. No deletions and insertions were observed in the P2 and P3 genomic regions, hence, the gene length within these genomic regions remained constant for all the HEV-A viruses.
At the P2 genomic region, EV-71 BrCr clustered with CV-A2, CV-A3, CV-A6, CV-A10 and CV-A12, while the EV-71 subgenotype C2 isolates were phylogenetically closer to CV-A8 (Figure 2c). EV-71 isolates of subgenotype B2 and C4 segregated from all other EV-71 and formed a cluster together with CV-A4, CV-A5, CV-A14 and CV-A16/G10 (Figure 2c). CV-A16 Tainan5079 segregated away from the prototype CV-A16/G10 and grouped with EV-71 BrCr, CV-A2, CV-A3, CV-A6, CV-A10 and CV-A12 (100% bootstrap support, Figure 2c). At the P3 genomic region, the different EV-71 lineages formed four distinct clusters together with other HEV-A viruses. Isolates of subgenotype C2 were phylogenetically closer to CV-A8 and this was supported by a high bootstrap value (100%). Isolates of subgenotype B3 and C4 isolates clustered with CV-A4, CV-A14 and CV-A16 and support for the clustering was also significant (100% bootstrap). EV-71 isolates of subgenotype B2 and B4 were phylogenetically closer to CV-A5, although this did not receive high bootstrap support (65%). The genotype A isolate BrCr segregated from all the recent EV-71 subgenotypes and clustered with CV-A2, CV-A3, CV-A6, CV-A10 and CV-A12 (Figure 2d). At the P3 genomic region, isolate CV-A16 Tainan5079 segregated away from CV-A16/G10 and grouped with EV-71 BrCr, CV-A2, CV-A3, CV-A6, CV-A10 and CV-A12 (Figure 2d).
An unrooted phylogenetic tree drawn using only the 3' UTR showed clustering of EV-71 isolates of subgenotype of B3 and C4 with CV-A4, CV-A14 and CV-A16 (95% bootstrap support, not shown). No significant segregation (< 30% bootstrap support), however, was observed for the remaining isolates and this was perhaps due to the short sequence length of the 3' UTR. The clustering of isolates of subgenotype B3 and C4 with CV-A4, CV-A14 and CV-A16 at the 3' UTR genomic region was consistent with the previous clustering at the P2 and P3 genomic regions. Based on these results, it appeared that sequences of genes at the 3' half of the EV-71 genome contributed to the multiple and diverse EV-71 subgenotypes and these genes showed high similarity to different HEV-A viruses.
Inter-typic recombination between EV-71 and other HEV-A viruses
Support for inter-typic recombination involving CV-A16 and EV-71 were also obtained from the phylogenetic investigation of the CV-A16 Tainan5079. Several recombination breakpoints were revealed when the CV-A16 Tainan5079 whole genome sequence was queried against several potential parental genome sequences (Figure 4c). A major recombination breakpoint was identified between nucleotides 3616–3781 within the 2A gene (≥ 84% sequence similarity), hence switching the CV-A16 Tainan5079 genome sequences to that of the non-structural gene sequences of EV-71 BrCr (nucleotides 3781–7409). Two additional breakpoints were noted between nucleotides 168–193 and 513–584 of the 5' UTR sequences switching the genome sequence from CV-A16-like to that of EV-71 subgenotype C2 (Figure 4c). Acquisition of the EV-71 subgenotype C2 sequences also resulted in the CV-A16 Tainan5079 isolate acquiring the EV-71-like 5' UTR RNA stem loop structure (not shown). When the amino acid sequences of the non-structural genes were examined, at least 45 amino acids were found to be similar to that encoded by the ancestral EV-71 BrCr and not to CV-A16 G/10. Of these amino acids, 21 amino acid differences occurred within the 3D RNA polymerase gene alone, suggesting that the gene was very much EV-71-like than CV-A16 (not shown). These results revealed that the recently isolated CV-A16 had most of its genome 5' half including all the structural genes except the 5' UTR consisting of CV-A16-like sequences and the remaining 3' half of the genome consisting of the supposedly no longer circulating EV-71 genotype A BrCr sequence.
Results from the full genome sequence similarity plot analyses and phylogenetic investigations presented here support the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the common causal agent of HFMD. All the EV-71 isolates examined had conserved 5' half of the genome, which consisted of mainly the structural genes including VP1, VP2, VP3 and VP4. These structural gene sequences particularly VP1 and VP4 when used to construct the phylogenetic trees clustered all EV-71 isolates into a unique clade of its own, away from other HEV-A viruses. This justifies usage of these genes for the molecular identification of EV isolates [19, 20]. It is possible that the structural genes especially the virus capsid gene, carries genetic restrictions required to ensure virus receptor recognition, binding and entry into host cells, and hence remain highly conserved. CV-A16-like sequences were present within the genome 3' end of EV-71 subgenotype B3 and C4. Conversely, EV-71-like sequences were also found within the genome 3' end of CV-A16 Tainan5079. These highlight the possible occurrence of recombination events in the wild between the two common causal agents of HFMD. This possibility is not difficult to envisage considering that both EV-71 and CV-A16 viruses are known to co-circulate within the same population and cause a common disease. It is particularly true in parts of the world where the level of hygiene practices and sanitation are lacking or less developed, as in these circumstances the possibility of co-infection is expected to be higher.
Results from the present study also suggest that inter-typic recombination events occurred between EV-71, CV-A16 and other HEV-A viruses. The incongruent phylogeny patterns observed suggest the possible occurrence of recombination in EV-71 in a similar manner to those previously reported for a number of enteroviruses [21–27]. It is reported that most of the current circulating HEV-B viruses are actually recombinants relative to its prototype strains isolated in the 1950s [23, 27]. Recombination amongst HEV-A viruses has also been described in CV-A6, CV-A10 and CV-A12  and a novel group of HEV-A consisting of EV-76, EV-89, EV-90 and EV-91 . The P1 genomic region of EV-70, a virus that causes acute haemorrhagic conjunctivitis is highly similar to EV and rhinovirus. In addition, the P2 and P3 genomic regions are phylogenetically closer to CV-B and swine vesicular disease virus . Another EV, CV-B5, has its major capsid proteins that are most closely related to swine vesicular disease virus, with the 5' and 3' UTR and the P3 genomic region that are highly similar to the corresponding regions of other CV-B viruses . Other example of EV with highly diverse genome sequence origin includes the prototype EV-73 isolated in California in 1988. This EV has P1 genomic region that is highly similar to echovirus 6, echovirus 29, echovirus 21 and echovirus 30, with the P2 and P3 genomic regions that share high similarity to CV-B3 . Together, these findings reiterate the suggestion that recombination is one of the potential mechanisms by which genetic diversity, is generated in EV-71 and other HEV-A viruses especially within the non-structural genes where recombination hot spots are mainly located [21, 23, 32–35]. While recombination could occur within the structural gene region, it perhaps yielded nonviable or unstable progeny viruses that did not survive and this could be due to generation of less fit variants with deleterious mutations . Selection of a better fit virus is likely to have contributed towards the demise of the recombinant EV-71 B3 virus once found prevalent in Peninsula Malaysia and Sarawak during the 1997 outbreak, Singapore and Japan in 1997 [7, 8] and Perth in 1999 . This virus was shown to replicate less efficiently in comparison to EV-71 B4 viruses , hence it could be the reason why it was rapidly replaced with EV-71 B4 viruses which remained endemic in the region for at least up to 2002, causing infections in Sarawak, Singapore, Taiwan and Japan [7, 8]. The acquisition of a segment of genes from CV-A16 by EV-71 B3 virus [21, 37] could have rendered the virus less fit to adapt to a new environment especially the host immunity.
Phylogenetic evidence of possible inter-typic recombination amongst HEV-A viruses including between the two causal agents of HFMD, EV-71 and CV-A16, were obtained from analyses using the virus isolates' full genome sequences. The finding highlights the potential role of recombination in the emergence of the newer EV-71 lineages including those perhaps with varied virulence and clinical manifestations. Active molecular surveillance of EV-71 and CV-A16 would help to ensure rapid identification of emergence of any potentially highly virulent EV-71 or CV-A16 strains. This is particularly pertinent in regions where recurring HFMD outbreaks involving the different HEV-A have occurred.
RNA isolation and sequencing
EV-71 and HEV-A sequences used for sequence analysis and construction of phylogenetic trees.
Paralytic illness +
Not known +
Bulbar poliomyelitis +
Sore throat, nuchal rigidity, muscle weakness +
Flies in polio community +
Oligonucleotide primers for cDNA synthesis and PCR.
Nucleotide sequence (5' to 3')
Nucleotide position *
TAA AAC AGC YKK KGG KTK KYA CC
TGM ACR TGR ATG CAR AAC C
CCD GAT GTR YTR ACW GAA ACC
ATC ACA TCT GCC ACY CTR TCY
GGR GCR CCY AAY ACW GCY TAY ATW WTR GC
CYT GRG CRG TRG TWG AHG AHA CKA RB
CAR GTY TCH GTB CCR TTY ATG TCA CC
GCT GTY TTB GMY TTR AYC CAV GC
YAA CTC WMD SAG AAA RCA CTA YCC
GWS GAK GCA ATS ACA AAC TTD GAK G
CCW GAY CAY TTT GAY GGR TAY MAR C
AHC CRT ACT GVV CVA CRT CHY C
GTT GAG CGH CAC YTS AAY AGA GY
GYA RRT CYA ACC CAT ACT TRT CC
CAT YAA GAA RAR RGA CAT YYT BG
TTG CTA TTC TGG TTA TAA CAA ATT TAC CC
Overlapping DNA sequences with at least 85% sequence homology and a minimum of 20 overlaps were assembled into contigs to generate consensus sequences using Sequencher version 4.0.5 (Gene Codes Corporation, USA). The consensus sequences were aligned against other EV-71 and HEV-A complete genome sequences retrieved from the GenBank (Table 1) using Clustal X version 1.81 . All completely sequenced EV-71 isolates available in the GenBank and the six isolates obtained from the present study were used for construction of phylogenetic trees. The genetic distance was determined by a pairwise estimation of the sequences percent divergence. Positions with gaps were included, transition and transversion ratio was fixed at 10 and corrections for multiple substitutions were made. These options were chosen to take into account the ambiguous part of the alignments and to correct for more than one substitutions happening at many sites that may underestimate the actual genetic distances . Phylogenetic trees presented here were constructed using the neighbour-joining method . All the unrooted trees were displayed using TreeView version 1.66 . The strength of the phylogenetic trees was estimated by bootstrap analyses using 1000 random samplings. A bootstrap value of ≥ 70% indicates a strong support for the tree topology . Amino acid sequences were examined after stripping the 5' UTR and 3' UTR sequences and consensus sequences for each EV-71 genotype were established.
Potential recombination amongst the isolates was determined as previously described . Initially, the neighbour-joining trees were constructed using the various gene regions of the complete genome. The trees were manually inspected to determine if there was any shifting of the tree topology in comparison to that constructed using the whole genome sequence. The putative recombinant isolate, determined from the shifting of the tree topology, was then queried against all the existing HEV-A virus genomes. Initially, all the 31 manually corrected and aligned complete nucleotide sequences were used. Isolates that shared similar patterns were grouped together to generate consensus sequences. Recombination was identified when conflicting genome sequence profiles appeared suggesting acquisition of sequences from a different parental genotype. Phylogenetic trees were constructed for each of the putative recombinant sequences using the neighbour-joining tree algorithm and maximum likelihood distance model and support for the tree topology was determined by bootscanning analyses that utilized the bootstrapping procedures with 100 resamplings [42, 43]. Bootstrap values of > 70% were used to indicate robust support for the tree topology . The crossover breakpoints were identified when χ2 values were at the maximum .
For the analysis of EV-71 and HEV-A viruses, all gaps were stripped and the sequences were corrected for multiple substitutions, transition to tranversion ratio of ten was used and 50% consensus files were used to exclude the poorly conserved sites. A sliding window of 1000 nucleotides was moved in increments of 20 nucleotides at a time and the resulting similarity values were plotted along the virus genome. Transition and transversion ratio of 10 was chosen based on that used for HEV-A viruses to avoid underestimation of actual genetic distances . Both the similarity plot and bootscanning analysis sliding windows were based on that used for HEV-B to increase the phylogenetic signals and reduce the noise of unrelated phylogeny . However, for the analysis amongst EV-71, the sequences were not corrected for multiple substitutions, and a transition to tranversion ratio of two and a sliding window of 400 nucleotides was used to map the possible recombination breakpoints .
- HFMD Hand:
foot and mouth disease
This study is funded in parts by a grant from the Ministry of Science, Technology and Innovation, Malaysia # 06-02-09-001-BTK/TD/002.
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